Biological Innovations that Built the World: A Four-billion-year Journey through Life and Earth History 9783030160562, 9783030160579

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Roberto Ligrone

Biological Innovations that Built the World A Four-billion-year Journey through Life and Earth History

Biological Innovations that Built the World

Roberto Ligrone

Biological Innovations that Built the World A Four-billion-year Journey through Life and Earth History

Roberto Ligrone Department of Environmental, Biological and Pharmaceutical Sciences and Technologies University of Campania “Luigi Vanvitelli” Caserta, Italy

ISBN 978-3-030-16056-2 ISBN 978-3-030-16057-9 https://doi.org/10.1007/978-3-030-16057-9

(eBook)

© Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Front cover credit: By NASA/Goddard Space Flight Center/Arizona State University – http://www. nasa.gov/image-feature/goddard/lro-earthrise-2015, Public Domain, https://commons.wikimedia. org/w/index.php?curid=45729896

Nothing in science is accepted on the grounds of authority, revelation, faith, or longevity. Heresy is not hostile to science; it is the very process of science. Paul M. Bingham and Joanne Souza (2009)

To Gabriella and Stefano, the best part of my life, with love

Preface

All organisms living on Earth, however different, share hundreds of complex traits inherited from a common ancestor. The grand unification of biological sciences performed by the molecular revolution in the last decades has crystallized this notion, pointing to life as a unitary phenomenon. Quite paradoxically, in the same time interval, the level of specialization of biological sciences has risen to the point where researchers working in different areas have few occasions for cultural interaction and, sadly, know little biology outside their own field. This is a particularly serious problem for human biology, which has primarily become a domain of medicine, while biological anthropology has disappeared from Departments of Anthropology at many universities. The main goal of this book is to guide the reader toward an understanding of the continuity of life across diversity and of its deep links with Earth. The route chosen is an analysis of ten fundamental transitions from the very beginning to the present, each pivotal to further change. Life most likely appeared on Earth around 4 billion years ago. Resuming the main events of this kind of time scale in a book not so big as to discourage hurried readers, but informative enough to meet the expectations of more demanding ones, is no easy task. The first, perhaps greatest, difficulty arises from our incomplete understanding of key events such as the origin of life itself, the genesis of the eukaryotic cell, and evolutionary interrelationships of major taxonomic lineages. As I used to say to my first-year undergraduate students while introducing them to the beautiful intricacy of biology, the challenge is not in complex topics but in those yet to be fully understood. Rather surprisingly, considering the theme of the book, a second difficulty comes from the fact that life, while adapting to Earth, also produced dramatic changes in the geochemistry and morphology of the planet, at least in the superficial part now known as the biosphere. The global network of interactions between life and Earth resembles the system of internal regulation of a living organism. This idea underpins the Gaia model proposed by British chemist James Lovelock in 1972, according to xi

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which Earth and its living communities form a highly integrated system with feedback mechanisms that maintain conditions suitable to life across time. Pruned from teleological nuances, the Gaia model is now widely accepted as a property spontaneously emerging from interaction of life and environment. Thus, even if restricted to major events, a history of life needs to include essential facts about the planet. A third difficulty, of a more contingent nature, lies in the necessity of using terms and concepts that cannot always be exhaustively explained in the text, given the range of the topics covered. Thus, the book requires a basic knowledge of chemistry, physics, and biology and includes a glossary for less common technical terms. Choosing fundamental transitions in the history of life is not as straightforward as one might initially believe. Some transitions are obligate; for example, nobody would deny that the appearance of life was by itself a fundamental biological transition. The same probably applies to the evolution of photosynthesis and of the eukaryotic cell. The history of life, however, encompasses so large a number of longranging innovations that there is plenty of space for subjective choice. The criterion adopted in this book is environmental impact, with the goal to produce an integrated account of major biological changes that contributed to create the world in which the humans evolved. By attempting to bridge the gap between topics traditionally treated separately, this book admittedly follows the Big History approach advocated by Fred Spier in his fascinating book Big History and the Future of Humanity (2010). Independently of the methodological approach, the present work distinguishes itself from Nick Lane’s beautiful book Life Ascending: The Ten Great Inventions of Evolution (2012) in the choice of the events and constant reference to phylogeny. I have chosen a number of transitions not covered in Lane’s book, namely, the oxygenation of the planet, the transfer of photosynthesis from bacteria to eukaryotes, the appearance of animals and land plants, and the emergence of humans, whereas others such as the evolution of movement, sight, warm blood, or awareness have been omitted as secondary manifestations of the “animal innovation.” A second book with a similar title is the scholarly masterpiece The Major Transitions in Evolution published by John Maynard Smith and Eörs Szathmáry in 1995 and, in a simplified version, in 2000 (The Origins of Life: From the Birth of Life to the Origin of Language). The main focus of The Major Transitions in Evolution is on changes and recurrent patterns in the way biological information is transferred across generations. Although sharing four topics with The Major Transitions in Evolution (the origin of life, eukaryotic cells, sex, and multicellularity), the present book could not be more divergent in style, contents, and objectives. Not the least, it incorporates a large body of information not available in 1995, especially from genomics and phylogenomics. Thus, despite similar titles, the three books deal with different topics or offer different perspectives on shared topics, being in several respects complementary to each other. The present book was primarily planned as a textbook for graduate courses of Integrative Biology and as a reference text for university lecturers and high-school teachers; the book, however, is accessible to anyone with an undergraduate training in biology. Inevitably, some readers will be more familiar with certain topics than

Preface

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with others. They can obviously skip parts, each chapter being self-standing, with references to other parts of the book helping trace connected topics. The reader will probably find the book style somewhat mingled, combining obvious, almost trivial information with cutting-edge data sometimes of considerable complexity; this approach has an explanation in the need to reframe long-established notions into a more widely integrated perspective. A list of most relevant publications is included at the end of each chapter for further coverage. In order to keep the number of references within limits, reviews have generally been preferred to research papers, and, with some exceptions, priority has been given to most recent publications; because of this, references to specific primary work are often retrievable only indirectly. Topics relevant to but not quite homogeneous with the main text have been treated in separate boxes. A constant effort was made to cover essential points. I will be indebted to anybody for suggestions and for signalling errors or omissions, a likely contingency in view of the vast range of this work. I thank Dr. Marco Vigliotti (Department of Civil Engineering, Design, Building and Environment, University of Campania Luigi Vanvitelli, Italy) for the help in the choice and preparation of the figures and three anonymous referees for their insightful comments. I also express appreciation to the Department of Environmental Sciences of the Second University of Naples (now a part of the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies at the University of Campania Luigi Vanvitelli) for encouraging me to take charge, in the last 15 years of my university career, of courses not strictly related to my own academic area, an experience that enabled me to see connections I had long overlooked before. Roberto Ligrone

Contents

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 19

2

The Origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Birth of the Solar System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 The Earth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 The Activation of Tectonics . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Tectonic Processes Are Essential to Life . . . . . . . . . . . . . . . . . . 2.5.1 Tectonics Drives a Powerful Global Thermostat . . . . . . . 2.5.2 Tectonics Recycles Bioelements . . . . . . . . . . . . . . . . . . 2.6 Birth of the Atmosphere-Ocean-Continental Crust System . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 23 24 28 32 35 36 39 40 50

3

The Birth of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Seafloor Hydrothermal Vents as Settings for the Emergence of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 A Primordial Role for RNA? . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 A RNA-Protein World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Biological Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 From Geochemistry to Biochemistry: The Emergence of an Autonomous Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 DNA Replaced RNA as the Repository of Biological Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 The Bacterial-Archaeal Divide: Ancestral or Derived? . . . . . . . . 3.10 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 54 57 62 65 68 71 82 86 87 90 92

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4

5

6

7

Contents

Moving to the Light: The Evolution of Photosynthesis . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Light, Pigments and Photosystems . . . . . . . . . . . . . . . . . . . . . 4.3 Accessory Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 The Photochemical Pathway in Anoxygenic Bacteria . . . . . . . . 4.5 Evolutionary Interrelationships of Type-1 and Type-2 Photosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Oxygenic Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Pathways of Carbon Photosynthetic Fixation . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

99 100 106 112 113

. . . .

115 116 121 125

The Great Oxygenation Event . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Planetary Oxygen Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Methane Was Probably as a Key Driver of Planetary Oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 The GOE Was Associated with a Long Phase of Climatic Instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 After the GOE, the Earth Stabilized in a Low-Oxygen State for over 1 Billion Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 The Earth Entered a High-Oxygen Phase About 800 Million Years Ago . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 The Impact of Oxygen on Biological Evolution . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

129 130 134

Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Rise and Fall of the Archaezoan Model of Eukaryogenesis . . . 6.3 Post-archaezoan Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 The Neomuran Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 The Origin of the Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Was the Host a Primitive Eukaryote or a Complex Archaeon? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 The Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1 Facts About Mitochondria . . . . . . . . . . . . . . . . . . . . . 6.8 The Last Eukaryote Common Ancestor (LECA) Possessed a Full Set of Fundamental Eukaryotic Traits . . . . . . . . . . . . . . 6.9 Eukaryote Phylogeny and Systematics . . . . . . . . . . . . . . . . . . 6.10 Dating Eukaryote Appearance and Diversification . . . . . . . . . . 6.11 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sexual Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Origin of Meiotic Sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 The Case of Parthenogenetic Rotifers . . . . . . . . . . . . . . . . . .

. . . .

. . . . . .

137 141 144 147 149 152 155 156 164 166 169 173

. 175 . 180 . 184 . . . . .

188 190 211 222 223

. . . .

233 233 236 241

Contents

Sexual Reproduction Sets Strong Species Boundaries in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Species Boundary in Prokaryotes Is Conventional . . . . . . . . . . 7.6 Gamete Differentiation and Genders . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7.4

. . . .

242 244 245 247

Multicellularity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Cellular Specialization and Levels of Organization in Multicellular Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Stem Cells and Germ Line . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

253 262 265 266

9

The Chloroplast and Photosynthetic Eukaryotes . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Birth of the Primary Chloroplast . . . . . . . . . . . . . . . . . . . . . . 9.3 The Plants (Archaeplastida) . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Secondary Chloroplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Phylogeny of Eukaryotes with Secondary Chloroplasts . . . . . . 9.6 Chloroplast Thieves and Other Stories . . . . . . . . . . . . . . . . . . 9.7 Plastid Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8 Storage Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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269 270 272 281 285 292 296 301 302 306

10

The Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Embryo Development and Body Simmetry . . . . . . . . . . . . . . 10.3 Animal Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Cellular Junctions and Epithelia . . . . . . . . . . . . . . . . . . . . . . 10.5 The Intestine and Animal Evolution . . . . . . . . . . . . . . . . . . . 10.6 Chordates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 The Impact of Animals on the Global Environment . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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311 312 315 319 324 331 333 342 343

11

Land Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 An Early Step in Plant Terrestrialization Was a Change in the Ancestral Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Early Land Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Stomata and Homeohydry . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Land Plant Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 The Xylem: A Lignified Water-Conducting System . . . . . . . . 11.7 Multiple Evolution of Leaves and Roots . . . . . . . . . . . . . . . . 11.8 Land Plants Co-evolved with Symbiotic Fungi . . . . . . . . . . .

. 347 . 348

8

. 251 . 252

. . . . . . .

350 354 359 364 371 381 385

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11.9 The Impact of Land Plants on the Global Environment . . . . . . 388 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 12

13

The Emergence of Humanity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 The Evolution of Bipedality . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Australopithecines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 The Genus Homo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Origin and Diffusion of Modern Humans . . . . . . . . . . . . . . . 12.6 Death from a Distance: High-Speed Throwing . . . . . . . . . . . 12.7 Why Don’t Humans Have Fur? . . . . . . . . . . . . . . . . . . . . . . 12.8 Reproductive Biology of Humans . . . . . . . . . . . . . . . . . . . . . 12.9 The Evolution of Language . . . . . . . . . . . . . . . . . . . . . . . . . 12.10 The Cognitive Revolution: Interplay of Genetics and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.11 Cooperation and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.12 The Transition to Food Production . . . . . . . . . . . . . . . . . . . . 12.13 The Evolution of Social Organization . . . . . . . . . . . . . . . . . . 12.14 The Search for “Human” Genes . . . . . . . . . . . . . . . . . . . . . . 12.15 Is Humankind Compatible with the Global Ecosystem? . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

399 400 406 408 410 419 425 427 430 436

. . . . . . .

439 443 447 451 456 460 462

Synopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

Abbreviations

AOC CD DT ECM Gb GY GYA HGT Kb KY KYA LECA LUCA Mb MY MYA PAL PMF ppm RNP

Atmosphere-Ocean-Continental Crust Carbon Dioxide Desiccation Tolerance Extracellular Matrix Billion Base Pairs Billion Years Billion Years Ago Horizontal Gene Transfer Thousand Base Pairs Thousand Years Thousand Years Ago Last Eukaryotic Common Ancestor Last Universal Common Ancestor Million Base Pairs Million Years Million Years Ago Present Atmospheric Level Proton Motive Force Parts Per Million Ribonucleoprotein

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Chapter 1

Introduction

Biology is an historical science. The further life diverges, the more its direction is determined by the accumulated quirks of past history. Lineweaver and Chopra (2012)

Abstract After a brief introduction of the fundamental properties of life and of major paradigms of current evolutionary theory, this chapter presents the organization and main topics of the book. Box 1.1 introduces biological systematics in the historical context and illustrates the “molecular clock” approach.

For exploring the history of life from its origins, we need a clear boundary between the living and non-living world. Defining life is no easy task (Jortner 2006). Let us start with an operational approach. Living organisms have two fundamental properties, autopoiesis and reproduction. Autopoiesis (from the Greek autos, itself, and poiein, to make, literally “to make itself”) is the ability to grow by using raw materials and energy from the outside; reproduction is the ability to multiply by producing copies of itself. From a functional perspective, the terms autopoiesis and reproduction are equivalent to metabolism and genetics, respectively. Through metabolism, living systems obtain the materials and energy they need to grow and to maintain themselves in a condition far removed from thermodynamic equilibrium. Genetics provides the instructions necessary for metabolic activity and controls the replication of information during reproduction. The above properties are far more complex than they might appear superficially, thus they require a complex chemistry, with molecules functioning as templates for self-replication and others that recognize specific targets and selectively accelerate chemical transformations that otherwise would be too slow. This is possible only with big molecules; it is not by chance, therefore, that the chemistry of life is based on carbon, the fourth most abundant element in the Universe and the only one that © Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_1

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1 Introduction

Fig. 1.1 The chemical elements utilized in living organisms, or “bioelements”, are sorted into four classes: fundamental elements, macroelements, universal microelements, and specific microelements. The universal microelements are necessary in all living beings, from bacteria to plants and animals; the specific microelements are required in some but not all organisms; for example, the elements marked in yellow are microelements with possible functions in mammals. (Figure downloaded from “CMB Timeline300 no WMAP” by NASA/WMAP Science Team – Original version: NASA; modified by Ryan Kaldari. Licensed under Public Domain via Commons https:// commons.wikimedia.org/wiki/File:CMB_Timeline300_no_WMAP.jpg#/media/File:CMB_Time line300_no_WMAP.jpg)

forms long chains sufficiently stable and at the same time flexible enough to produce the multiform and versatile molecular architectures necessary for building living systems. Carbon is so unique among the existing elements that it is most likely at the base of any form of chemical life that might have appeared in the Universe, besides the Earth (Lineweaver and Chopra 2012). Life uses numerous other elements in addition to carbon, the most important being oxygen, hydrogen and nitrogen. Along with carbon, these are the fundamental bioelements and account for about 97% dry weight of total biomass. Phosphorus and sulphur make up another 1% and the remaining 2% is dominated by potassium, sodium, calcium, magnesium and chlorine, collectively indicated as the macroelements. Last come the microelements, elements indispensable to life but used in very tiny amounts (Frausto da Silva and Williams 2001; Chopra and Lineweaver 2008; Lineweaver and Chopra 2012; Chopra et al. 2010; Fig. 1.1). We have now to consider a second fundamental property of life, evolution. The word evolution means “gradual change”; in this sense, many things evolve and the term is commonly used for changes in non-living entities such as astrophysical and geological processes. Biological evolution, however, has special properties that arise

1 Introduction

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from the capacity of living systems to reproduce. Although faithful, reproduction is prone to spontaneous, random errors, or mutations, that are inherited by the following generations. Within their ecological niche, the organisms compete with conspecifics for the resources available. The appearance of variant individuals in a population in consequence of mutation activates a mechanism that Darwin called natural selection: with time the relative abundance, or frequency, of some variants increases whilst others become rarer and eventually disappear. Natural selection has no pre-determined end or direction; it is a contingent process that rewards fitness, the ability of an organism to reproduce in competition with others for the same resources. Individual variations caused by random mutation may disappear or persist and increase in frequency in the population according to the fit they have to the current environment. Biological evolution operates over very large time scales; nevertheless, it is the maker of all the amazing adaptations present in living beings, from organs and behaviours to molecules. Indeed, biological adaptation is often so extraordinary that it is hard to believe that it comes out of mere chance. Chance is actually only responsible for errors during the replication of biological information. Much of the evolutionary process is rigorous necessity arising from competition for resources. Because of its opportunistic character, natural selection generates new forms in response to environmental pressures, thus functioning as an inexhaustible source of biological diversity (Monod 1971). The concept of biological evolution by natural selection is sometimes challenged by supporters of “intelligent design”, a religion-based idea maintaining that complex structures present in living beings - where any alteration of a single component, however small, may compromise the functionality of the system – could not have evolved gradually, thus they must arise from a transcendent plan or, in more explicit terms, a “divine project”. Progress in biological knowledge continues to produce robust evidence that, given the time and conditions necessary, evolution can proceed spontaneously and gradually at all levels of complexity, from molecules to organs and behaviours. As Robert Hazen (2012) observes, “if God created life, she is smart enough to use evolution” (with the female pronoun giving the sentence further wit). Intrinsically linked to the binomial “reproduction and random change”, Darwinian evolution is a fundamental and unique property of life. We can define life, therefore, as “a chemical system that maintains itself in a condition of non-equilibrium and is able to evolve with a Darwinian mechanism”. There are other systems that owe their existence to thermodynamic disequilibrium such as the stars, volcanoes, hurricanes or fires, but none of these complies with the definition above. Life variants based on physical rather than chemical processes are theoretically possible, for example software that is capable not only of self-replicating but also of producing the necessary hardware from raw material; more difficult is to imagine how such systems might emerge spontaneously in our Universe. Anyhow, the association of “non-equilibrium” and “Darwinian evolution” appear to be an essential requisite for any form of life. From early in school, we learn that the instructions necessary for the functioning of living organisms are stored in DNA, a molecule whose double-filament structure

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immediately suggests its replication mechanism. Life, as we know it, is founded on the ability to convert digital information stored in DNA (genotype) into threedimensional protein structures with catalytic or structural functions. This process generates the phenotype, i.e. the set of perceivable characters of an organism. Biological information consists of DNA sequences called “genes”, a term introduced by Danish botanist Wilhelm Johannsen in 1909. Since natural selection requires more than one generation for producing effects, individuals cannot evolve (at least not in the biological sense). Evolution by natural selection is possible only if reproduction is faithful, namely if the number of changes at each generation or reproductive cycle is low relative to genotype size (expressed as the number of DNA base-pairs). Too high a mutation rate would cause a rapid dissolution of the information stored in DNA. We will see in Chap. 7 that sex is essential to compensate for the effect of spontaneous mutation and to preserve genetic information across time. The combination of Darwin theory with population genetics and subsequent incorporation of novel insight from embryology to sociobiology and molecular biology in the process known as the “modern synthesis” produced Neodarwinism, a continuously refining paradigm defined as the state of the art in evolutionary biology at any chosen time from the 1890s to the present. In contemporary evolutionary theory, biological evolution is defined as change in the frequency of DNA sequences (i.e. genes and associated regulatory regions) in a population, from one generation to the next (Ridley 2004). Four processes are currently recognized as the major drivers of biological evolution: mutation and recombination, genetic drift (the random loss of sequences) and natural selection, the first two producing genetic variation, the latter two sorting it (Lynch 2007). Of these, only natural selection is adaptive, in the sense that it is a function of the fitness properties of individuals, the other three being non-adaptive random processes. The recognition of non-adaptive processes as fundamental drivers of genetic change led to the formulation of the “neutral theory of molecular evolution”, an important addition to the paradigm of modern synthesis that emphasizes how neutral mutations dominate DNA evolution (Kimura 1968). Most mutations are deleterious and rapidly disappear by the action of natural selection, thus making no significant contributions to variation within and between species. Neutral mutations change the DNA sequence without affecting the organism fitness, therefore their frequency in populations does not change over the time; neutral mutations in encoding sequences mainly depend on genetic code degeneracy and produce neutral alleles (Glossary). The neutral theory emphasizes how most DNA variation within and between species, including gene polymorphism (Glossary) is merely a produce of neutral mutation, the fate of neutral alleles essentially depending on genetic drift. Neutral mutations underpin the molecular clock principle (Box 1.1). Many biological structures and processes are by far more complex and redundant than apparently necessary. Gratuitous complexity is a common occurrence in biology that emerges when random neutral change is fixed due to a ratchet-like process known as constructive neutral evolution. Examples include cellular properties such as genomic organization, RNA splicing and ribosome structure in eukaryotes (Lynch

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2007; Gray et al. 2010; Lukeš et al. 2011). At the organismal level, Maynard Smith and Szathmary (1995) showed how a ratchet mechanism, which they called “contingent irreversibility”, might cause previously independent evolutionary units to become mutually dependent. An example is the emergence of mutual interdependence between the mitochondria or plastids and their host cells (Chaps. 6 and 9). Box 1.1: Taxonomy, Systematics, Phylogeny From the phylogenetic standpoint, the organisms that we perceive are not steps in evolution, but rather dead-end states, transient manifestations of particular lines of descent. (Pace 2009)

From the beginning, humans worked hard to analyse and codify the surrounding world. By observing the diversity of living forms, they learnt that some were dangerous and had to be avoided carefully; others instead were harmless or could even be used as a source of food or for other purposes. By giving different forms a name that bore information on certain properties, and by transferring this expanding body of knowledge to the following generation, humans founded biological taxonomy. With time, humans noticed that living forms have varying degrees of similarity. The snakes, for example, may be sharply different from each other in size, colour and dangerousness, some being harmless, others extremely poisonous; all snakes, however, are limbless animals sharing a distinctive type of locomotion. By creating categories based on affinities and divergences, humans have invented biological systematics, a major branch of science whose objective is to organize biological knowledge. For example, saying that an animal is a “primate” implies a sizable amount of information including the possession of opposable thumbs, eyes in a frontal position for stereoscopic vision, and unique skin sensorial organs known as the Meissner corpuscles. Carl Nilsson Linnaeus, a Swedish physician and naturalist, created the first universal biological system, encompassing all living forms known in the eighteenth century. The Linnean system had a hierarchical structure, in the sense that living forms clustered together in progressively smaller categories based on increasing affinity. Many principles and categories introduced by Linnaeus are still in use, notably the binomial denomination of species. Over time, the number of known species has grown to over 1.75 million living species and about 250,000 extinct ones, and it is estimated that several millions are yet to be described (http://www.catalogueoflife.org/col/browse/tree). The classification of such a huge number of species is a work of great complexity; over time, two alternative methods have consolidated in use: phenetic systematics (or simply phenetics) and phylogenetic systematics (or phylogenetics). (continued)

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Box 1.1 (continued) Phenetic systematics classifies living forms based on their perceivable (phenotypic) characters: if two forms are more similar to each other than each is to a third form, they are set together in a group that excludes the latter. The degree of affinity may be expressed quantitatively by assigning each character a numerical value, generally 1 if present or 0 if absent. The resulting classifications have a hierarchical structure, with the degree of within-group affinity increasing from higher to lower ranks. Phylogenetic systematics, instead, classifies life forms based on genealogy, namely it groups together the descendants of a common ancestor whilst separates forms with different origins. This method aims to build a genealogic tree such as the shared progenitor becomes more and more ancient when passing from terminal to larger branches and eventually to the main trunk. In most cases, phenetic systematics and phylogenetic systematics give much the same results. Nevertheless, the two approaches are deeply different and in certain cases they may produce sharply divergent classifications. Phenetic systematics does not need to know anything about evolution: it uses any observable character, independently of its evolutionary history. This implies that when two forms are similar not out of a common ancestry but because they have evolved under similar environmental constraints, they tend to be linked together by phenetic systematics whereas they are set separately by phylogenetic systematics. Similar characters evolved independently in phylogenetically separate forms are named homoplasies, and the process that generates them is known as evolutionary convergence or parallel evolution. Parallel evolution occurs when different lineages fill similar niches in separate ecosystems around the world; because natural selection works in the same way in these environments, the same types of adaptations are selected for. A remarkable example of parallel evolution is found in some species of Euphorbiaceae and Cactaceae, which have evolved closely similar forms by adapting to dry, sunny environments (Fig. 1.2). Homoplasies give affinity signals that may be in sharp contrast with other characters. In the example given in Fig. 1.2, flower anatomy clearly dictates classification of succulent spurges and cactuses into separate groups. In order to prevent this kind of conflict, phenetic systematics needs to analyse a great number of characters and expresses the results in quantitative terms. This, however, does not completely suppress the necessity of subjective choice; for example one can choose to use a set of absolute values close to each other (nearest neighbour) or the average values (average neighbour) and this may profoundly affect the final result. Phenetic systematics has been popular until the 1950s, but is now mainly used for species identification, not to solve evolutionary relationships. (continued)

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Box 1.1 (continued)

Fig. 1.2 A classic example of evolutionary convergence in the Euphorbiaceae (a Euphorbia caput-medusae) and Cactaceae (b Melocactus dumortieri). In both lines, the leaves have been converted into spines and the photosynthetic function has been transferred to the stem; in addition, the stem has developed abundant water-storing parenchyma. The convergence of the two forms also includes a special metabolic variant of photosynthesis known as “crassulacean acid metabolism”, which increases plant efficiency in water use

The Linnean system was essentially phenetic (not by chance, in fact, it treated sponges as plants). The acceptance of Darwinian evolution in the second half of nineteenth century favoured the establishment of phylogenetic systems of classification; particularly influential was the work of Ernst Haeckel, whose three-kingdom system (Fig. 1.3) has been popular for over a century. Like phenetics, phylogenetic systematics classifies living forms based on their affinities, and uses both morphological (macro- and microanatomy and embryology) and molecular characters (DNA, RNA and protein sequences), but it rigorously excludes homoplasies from analysis. Phylogenetic systematics is intrinsically objective (there can be only one correct genealogic tree) and it is the only approach currently in use to resolve evolutionary relationships. (continued)

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Box 1.1 (continued) However, phylogenetic systematics also faces practical and theoretical difficulties that may seriously hinder phylogenetic reconstruction, notably signal erosion by mutation and horizontal gene transfer (Schuh and Brower 2009).

Fig. 1.3 The tree of life published by Ernst Haeckel in 1866 had three major branches, or “kingdoms”, namely Protista, Plantae and Animalia. For a historical review of the Tree of Life, see Kutschera (2011)

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Box 1.1 (continued) An important methodological innovation in phylogenetic systematics stemmed from the work of German entomologist Willi Hennig in the second half of the past century. Hennig’s ideas have been pivotal to the emergence of cladistic systematics or simply cladistics, a novel form of phylogenetic systematics based on the concept of monophyletic group, or clade (http://www. ucmp.berkeley.edu/clad/clad1.html). A clade is a group encompassing an ancestor and all its descendants, to the exclusion of any other organism. Cladistic systematics uses clades in phylogenetic analysis, whereas it rejects paraphyletic groups (encompassing the ancestor and only a part of its descent) and polyphyletic groups (encompassing organisms derived from separate ancestors) (Fig. 1.4). A second founding principle of cladistic systematics is parsimony, by which the phylogenetic tree that explains the data with a minimum number of branches or steps is to be preferred.

Fig. 1.4 Cladistic systematics reconstructs phylogeny by identifying the bifurcations, or nodes, where lines of descent have separated from a common ancestor. A phyletic tree built with cladistic criteria is called “cladogram”. Well-resolved cladograms have a dichotomous structure, i.e. consist of sequentially bifurcating branches. This structure reflects the very process of replication of genetic information (Puigbò et al. 2013) and, at the population level, the pattern of divergence of novel evolutionary lineages. If the data are insufficient, more than two lineages will depart from the same nodes, meaning that analysis failed to resolve the divergence succession at these points. Cladistic systematics only accepts monophyletic groups, for example the group encompassing lineages E to H and their common ancestor in (A), whereas it excludes paraphyletic (B) and polyphyletic groups (C). The essential difference between paraphyletic and polyphyletic groups is that the former include the common ancestor, the latter do not. Clades diverging from the same node, thus deriving from the same ancestor, are referred to as “sister clades” or “sister groups”. For example, the clades (a, b) and (c, d), or the clades (a–d) and (e–h) in figure (A) are sister to each other. The phylogenetic sequence can be directly converted into a taxonomic hierarchy, for example as shown in figure (D). Cladograms have a temporal dimension (“time”); when the length of the branches is calibrated for time, this is explicitly indicated

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Box 1.1 (continued) By applying the above principles, cladistic systematics rejects as paraphyletic a number of groups firmly established in traditional systems and common language, such as “fishes” and “reptiles” (Benton 2000; Fig. 1.5). Not surprisingly, cladistics has initially met with considerable opposition. Among its critics is Oxford Professor Thomas Cavalier-Smith, whom we will encounter again several times in this book. Cavalier-Smith distinguishes monophyletic and holophyletic groups as the equivalents of Hennig’s paraphyletic and monophyletic groups, respectively, and uses either as convenient (Cavalier-Smith 2010; Ruggiero et al. 2015). Despite initial criticism, the cladistic method is today universally adopted in phylogenetic analysis, classifications being labelled either as phylogenetic or evolutionary, depending mainly upon whether or not they reject paraphyletic groups.

Fig. 1.5 Phyletic tree of amniotes (terrestrial vertebrates with an amnios 10.4). Reptiles (including turtles, tuatara, amphisbaenia, snakes, lizards and crocodiles) is clearly a paraphyletic group because the ancestor of crocodiles also generated the birds, which are not reptiles. In contrast, the Sauropsids (reptiles + birds) is monophyletic. Because terrestrial vertebrates share a common ancestor with a line of fish, Pisces (Fishes) also is a paraphyletic group. (Redrawn from Hickman et al. 2007)

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Box 1.1 (continued) Cladistic systematics distinguishes two types of homologies: those appeared in the most recent common ancestor (apomorphies) and those present in more remote ancestors, thus also shared with organisms outside of the clade considered (plesiomorphies). Apomorphies are innovative characters that mark the divergence of a certain clade and distinguish it from the others, whereas plesiomorphies are antecedent to the separation event. The apomorphic or plesiomorphic status of a character depends on the level considered. For example, opposable thumbs, flat nails and Meissner corpuscles are apomorphies of primates. Hairs, instead, are a plesiomorphy in primates because they also occur in non-primate mammals; in fact, hairs appeared in the common ancestor of mammals and are an apomorphy of all present-living mammals. Likewise, the vertebral column is an apomorphy of Vertebrates but a plesiomorphy in Mammals. Often, the name assigned to a certain group recalls one of its apomorphies, for example Cnidaria (from cnidocytes, specialized poisonous cells of jellyfish and other members of the group), Hexapoda (six legs), Mammals (mammary glands), Spermatophytes (seeds), Angiosperms (fruits). Being firmly rooted in phylogeny, cladistic systematics tends to recognize a number of hierarchical levels widely exceeding the nomenclature introduced by Linnaeus (species, genera, families, etcetera). To solve the problem, it has been proposed to use instead a numerical nomenclature or denominations devoid of any hierarchical implications. Currently, there are three separate codes that govern the assignment and use of scientific names, each with different requirements and terminology: the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code), the International Code of Zoological Nomenclature (ICN) for the animals and protozoa (grossly, the unicellular eukaryotes), and the International Code of Nomenclature of Bacteria (ICNB) for prokaryotes other than cyanobacteria. Figure 1.6 resumes major changes in biological systematics from Linnaeus time to the end of twentieth century. With the improvement of techniques of molecular analysis, whole-genome sequencing has become much faster and cheaper than in the recent past, thus the number of species for which the whole genome sequence has been determined grows continuously. This has given extraordinary impetus to phylogenetic analysis based on molecular sequences, which are much more suitable to phylogenetic inference than morphological traits. In recent years, genomics and molecular phylogeny have merged into phylogenomics, which is currently one of the most dynamic areas in biological research. The development of reliable trees based on molecular data makes it possible to infer the evolution of morphological characters from molecular trees, thus reversing the traditional process of evolutionary analysis, and sometimes (continued)

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Box 1.1 (continued) confuting long-established assumptions. A major difficulty of molecular phylogeny is in the necessity of distinguishing sequences vertically inherited from a common ancestor, which contain phylogenetic information, from sequences acquired by horizontal gene transfer (HGT) from distantly related groups, which blur the phylogenetic signal (Daubin and Szöllősi 2016). Important characters such as photosynthesis or aerobic respiration have been extensively transferred by HGT, thus they may have little phylogenetic value despite their great ecological relevance. HGT is a serious, possibly insuperable problem in prokaryote phylogeny, but may also be a severe hindrance in eukaryote systematics. The systematics of bacteria and archaea is still poorly resolved not only because of rampant HGT but also for difficulties in the isolation of pure cultures from the environment. Meta-phylogenomics, phylogenetic analysis of raw DNA extracted directly from the environment, is currently opening new perspectives in microbial systematics (Rinke et al. 2013; Spang et al. 2015; Schulz et al. 2017).

Fig. 1.6 Major innovations in biological systematics from the eighteenth to twentieth century. Following the revolutionary work by Linnaeus, the principal breakthroughs have been (1) the formal recognition of organisms not belonging to plants or animals, named Protista by Haeckel (1866); (2) the recognition of the fundamental divide between prokaryotes and eukaryotes; (3) the creation of the kingdom Monera, or Bacteria; (4) the introduction of a five-kingdom system (see historical review by Hagen 2012); (5) the recognition of Archaebacteria as a novel prokaryotic kingdom (Woese and Fox 1977); (6) the separation of life forms into three domains, Archaea, Bacteria and Eukarya (Woese et al. 1990). Further major innovations, especially in eukaryote phylogeny and systematics (Chap. 6), have come from molecular research in the last 30 years. The term “protists” is now informally used to denote unicellular eukaryotes. For a historical review of the prokaryote/eukaryote dichotomy, see Sapp (2005). (Figure adapted from New World Encyclopedia: http://www.newworldencyclopedia.org/p/index.php?title¼New_World_Encyclo pedia:Terms_of_Use&oldid¼943147 (last visited October 27, 2018))

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Box 1.1 (continued) In the last 20 years, phylogenomics has revolutionised the systematics of eukaryotes, although a definitive assessment is still not at hand. The genome of eukaryotes consists by about 50% of unique “eukaryotic” sequences, the rest being sequences sharing a common ancestry with archaeal or bacterial sequences, which makes the reconstruction of evolutionary interrelationships of the three domains a most complex puzzle (Koonin and Wolf 2009). A further outcome of the “molecular revolution” is the introduction of DNA barcoding, a method that uses short sequences in DNA from an unknown sample to identify it as belonging to a particular species or group. The markers used range from highly variable (e.g. mitochondrial DNA sequences) to highly conservative (e.g. ribosomal DNA sequences), depending on the taxonomic level of identification desired. The markers also vary according to the type of organism. For example, a segment of approximately 600 base pairs of the mitochondrial gene for cytochrome oxidase I (COI or COX1) is commonly employed for animals, an internal transcribed spacer (ITS2) between rRNA genes is utilized for fungi, and sequences in rbcL and matK chloroplast genes are applied to plant identification (Hebert et al. 2003; Kress et al. 2005). The technique has turned out to be particularly useful for estimating the taxonomic diversity of samples from the environment, an approach known as metabarcoding. Metabarcoding of samples from marine, fresh-water and terrestrial habitats has revealed that genetic diversity in protists (¼ unicellular eukaryotes) greatly exceeds that found in plants, fungi and animals (Pawlowski et al. 2012; de Vargas et al. 2015). The taxonomic entities detected by metabarcoding are referred to as Operational Taxonomic Units (OTUs). The number of different OTUs present in a sample is an index of biological diversity, but there is currently no agreement about how to interpret OTUs in terms of species. Ruggiero et al. (2015) have produced a high-level, non-phylogenetic classification of all organisms; for a comprehensive phylogenetic classification of eukaryotes, see Adl et al. (2012, 2018). Molecular Clocks The molecular-clock hypothesis, viz. the idea that molecular evolution occurs at an approximately uniform rate over time, was first formulated by Emile Zuckerkandl and Linus Pauling in 1962, based on the observation that amino acid differences in the hemoglobins of different animal species increase linearly with the time of divergence estimated from fossils. This suggests that neutral mutations, which do not significantly affect the organism fitness, tend to accumulate in the genome at a constant rate. Known the mutation rate, therefore, it would be possible to estimate the age of a group from sequence analysis of living representatives without the need for paleontological records (Kumar 2005; Fig. 1.7). (continued)

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Box 1.1 (continued)

Fig. 1.7 The rationale of molecular-clock analysis is the relation e ¼ n/2t, where e is the mutation rate, n the number of mutations occurred after divergence, and t the divergence time. The factor 2 is added because n is the sum of mutations from the common ancestor to both descendants. As an example, if the fossil record indicates that clade B and clade C diverged from a common ancestor around 9 MYA, and homologous sequences in B and C differ for a total of 12 nucleotides, then e ¼ 0.67. This value permits an estimation of the age of clade A in the absence of palaeontological evidence. For example, a difference of 35 nucleotides between A and B or A and C suggests that the clade A and the clade B+C diverged about 26 MYA

The implementation of rapid and reliable methods for genome sequencing has given great impetus to molecular-clock studies. The interpretation of the data, however, turned out not to be as simple as initially thought. It was discovered, in fact, that the mutation rate could vary remarkably in different taxa, even in homologous genome sequences. In addition, the chances of multiple mutations affecting the same site and of retro-mutation, namely a second mutation that cancel a former one, are concrete problems when considering ancient groups. When enough time has elapsed, many sites may have undergone multiple changes, but only one of these is detectable. Consequently, the observed number of changes is no longer linear with time, but instead flattens out. A careful choice of the sequences analysed and an accurate calibration of the molecular clock may attenuate these difficulties. For dating relatively recent evolutionary events, for example species divergence within the same genus or even genetic isolation of specific populations (a common issue in anthropological research) it is necessary to compare fast-evolving sequences. (continued)

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Box 1.1 (continued) In contrast, for dating ancient evolutionary events, for example the divergence of major eukaryotic lineages or even of the three domains of life, it is necessary to analyse conservative sequences. Non-coding sequences, for example non-coding mitochondrial DNA, tend to rapidly change with time as they are not subject to evolutionary constraints; in contrast, ribosomal RNA is extremely conservative because few changes are tolerated by natural selection. Molecular clock analysis is feasible only with a correct calibration, generally based on fossil evidence (in special cases such as molecular dating of virus strains, fossil-independent calibration methods can be used). The accuracy of estimates varies with the organisms analysed, it is better for recent than for ancient groups, and improves when the database expands. In addition, more cautious, “relaxed” procedures have been introduced to correct potential variation in evolutionary rates across phylogeny (Christin et al. 2014; Donoghue and Yang 2016). A poor palaeontological record and difficulties in the identification of orthologous sequences (Glossary) make it particularly problematic the dating of ancient groups. Molecular dating tends to produce older estimates than fossils. In some measure, this is explainable: molecular dating gives, or should give, absolute age estimates whereas fossils only give a minimum age, as it is not possible to exclude that a significant time length elapsed between the first appearance of the organism and recorded fossilization. Nevertheless, persistent divergences of tens or hundreds of million years between the two value sets point to the need for a further refinement of molecular-clock techniques (Wray 2015). Following the definition of the mechanism by which genetic information is expressed into biologically active proteins, the discovery of the lac operon by Jacob and Monod (1961) revealed that genes could reversibly be switched on and off. This gave the start to research directed at understanding how genes cooperate in interactive networks. Perhaps the most important insight from this effort has been the discovery that cellular differentiation and organ development in multicellular organisms are under the control of genes encoding for transcription factors that generate temporal and spatial patterns. This advance led to the emergence of evolutionary developmental biology, informally “evo-devo” (Carroll 2005; Ioannidis 2008), a field of biological research that investigates molecular mechanisms underpinning developmental processes to reconstruct evolution and infer phyletic relationships (Box 1.1). A further breakthrough in our understanding of how the genetic system works was the discovery that changes in gene expression may be transmitted to next generation without modifying the DNA sequence. These are known as epigenetic changes and the branch of genetics that studies them is called epigenetics from the Greek prefix “epi”, over or additional (Morris 2001). Epigenetic events are

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modulated in response to endogenous or exogenous signals and can be transmitted to the progeny as pre-adaptations to stressing conditions, which is in some way a form of Lamarckian inheritance (Galton 2016). Epigenetic changes are also involved in cellular differentiation in multicellular organisms, for example in enabling cellular lineages to maintain their identity through multiple cycles of cell division (Carey 2011; Baulcombe and Dean 2014). A different form of epigenetic transmission is ecological inheritance, an idea introduced by John Odling-Smee in 1988 and subsequently expanded in collaboration with Kevin Laland and Marcus Feldman into a theoretical framework currently known as the “niche construction theory” (Odling-Smee et al. 2003; Matthews et al. 2014; Laland et al. 2016). According to the proponents, niche construction is the capacity of organisms to change their environment and transmit the modified environment to next generations, thereby acting as co-directors of their own and other species’ evolution. The niche-construction theory interprets the adaptive complementarity of organisms and environment in terms of a dynamic, reciprocal interaction between natural selection and life-driven environmental change. In this perspective, natural selection shapes life to fit pre-existing environments, yet life changes the environments, thus establishing an endless cycle of causation and feedback (Laland and Brown 2006). Necessary conditions for a process to be “niche construction” are that an organism substantially modifies ecological habitats, and that these modifications influence selection pressures acting on one or more recipient populations (Laland et al. 2016). Coral reefs, soil building by lichens, and plants or earthworm activity in soil are conspicuous examples of niche construction, ecological succession being perhaps its most noticeable effect. More immediate examples include the dams built by beavers, hives of social bees, ant nests and spider webs. Although rooted in population genetics, the niche construction model has strongly appealed to researchers in anthropology, archaeology, psychology, social sciences and other human-related fields (see, for example, Gintis 2011; Rendell et al. 2011; O’Brien and Laland 2012; Flynn et al. 2013). Indeed, humans modify the environment more quickly and more extensively than any other species and transmit the changes to next generations; likewise, humans produce and transmit culture in a manner that leads to quantitative growth across generations, a process labelled as “cultural niche construction” that profoundly affects human evolution (Laland and O’Brien 2011). While the existence of niche construction is not disputed, its impact on evolutionary theory remains controversial (see below). Modern biological thinking owes much to Richard Dawkins, former Professor for Public Understanding of Science at the University of Oxford, UK. In the book “The selfish gene” (1976), Dawkins observes that the genes are transmitted across generations and persist much longer than individual organisms, which in contrast are ephemeral entities whose ultimate function is gene perpetuation. Natural selection operates on the organisms, namely on phenotypes, but the biological units actually selected are the single genes. Genes belonging to the same genotype cooperate with each other because and to the extent that this improves their individual chances of perpetuation. In spite of its apparent simplicity, the “selfish gene” notion has multifaceted implications (for example the fallacy of group-selection models) that have changed the way biologists think about evolution (Grafen and Ridley 2006).

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A second deep-impacting idea introduced by Dawkins is that genes use the organisms to modify the surrounding environment, which thus becomes itself an extended expression of the genotype, namely an “extended phenotype” (Dawkins 1999). The extended phenotype concept focuses on the selective feedback from the environmental modification to the responsible gene or genes, without considering evolutionary effects on other species. Proponents of the niche construction theory regard the extended phenotype as a particular variant of niche construction in which the organism that is adaptively affected by the environmental change is the same that causes the change (Laland et al. 2016). Extended phenotypes have a solid Darwinian explanation in the benefit that they impart to the genes responsible for their expression at the expense of alternative alleles (Dawkins 2004). In contrast, there is little evidence for Darwinian mechanisms underlying niche construction in which the activity of one species affects the evolution of another. Dawkins (2004) points to the need to distinguish environmental changes that are simple by-products of organismal activity from adaptive changes. For example, atmosphere oxygenation by cyanobacteria (Chap. 5), the reduction of atmospheric carbon dioxide to few hundreds parts per million by plants (Chap. 11), or simple ecological successions do not imply any backward selection pressure on the responsible organisms, yet they perfectly fit the niche construction evolutionary model. As already observed, advocates of the niche construction perspective and sceptics do not question whether niche construction exists (it clearly does) but disagree about its implications for evolutionary theory. Advocates see them as profound and reject Dawkins’ idea of organisms as simple gene vehicles, sceptics see no reason why niche construction poses any problems for standard evolutionary theory, much less any reason for a fundamental revision (Scott-Phillips et al. 2013; Laland et al. 2014; Wray et al. 2014). The role of symbiosis as a major driver of evolution has been recognized since the end of nineteenth century by several important forerunners, notably Konstantin Mereschkowski (Kutschera 2011). In the 1960s and 1970s, Lynn Margulis reelaborated and brought to the scientific mainstream Mereschkowski’s ideas on the origin of the eukaryote cell, starting with her famous paper “On the origin of mitosing cells” signed with her husband’s surname (Sagan 1967). The bacterial origin of mitochondria and chloroplasts is today well-consolidated knowledge (Archibald 2015), and symbiosis is recognized as a major mechanism underpinning the evolution of complexity (Douglas 2014; Rosenberg and Zilber-Rosenberg 2016). Multicellular eukaryotes host a diversity of commensal, symbiotic and pathogenic microorganisms including bacteria, archaea, protists, fungi and viruses, collectively referred to as the “microbiota” or “microbiome”. It is estimated, for example, that the digestive tract of humans hosts about 1014 microbes (Salvucci 2016; Rosenberg and Zilber-Rosenberg 2016). Lynn Margulis used the term “holobiont” in 1991 to describe a host and its symbionts (Margulis 1991). Expanding the notion of “holobiont” as an evolutionary unit, Zilber-Rosenberg and Rosenberg (2008) have added the term “hologenome” to describe the sum of the genetic information of the host and its symbiotic microorganisms. In the form currently proposed, the hologenome concept holds that the holobiont is a unitary biological entity defining a level of selection in biological evolution; this implies that

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selection on constituent genomes acts primarily to increase fitness at the level of the holobiont, thus suppressing conflicts between the evolutionary interests of host and symbionts (Rosenberg and Zilber-Rosenberg 2016). Evidence for rampant conflict between symbionts even in highly integrated holobiomes has raised criticism about the general applicability of the hologenome concept and even its usefulness (Moran and Sloan 2015). The introduction of techniques for rapid sequencing of DNA (or of its transcript RNA, notably ribosomal RNA) permits the collection and comparison of a growing mass of data on a great number of organisms. With the use of powerful software, the affinity and divergence in homologous sequences from different organisms can be evaluated quantitatively. This area of research, known as molecular phylogeny, is revolutionising traditional systematics. One the major achievements of molecular phylogeny is the discovery that the tree of life has not two but three major branches: the Bacteria, Archaea and Eukarya (Box 1.1). While splitting the living world into three deeply divergent groups, molecular analysis also revealed that these share a number of complex characters that could hardly have evolved independently. This indicates that life on Earth is monophyletic: all extant organisms, however diverse, arise from a common ancestor. The only possible exception are viruses, which anyway fall outside the definition of life given above. We now know with reasonable confidence that the common ancestor of all living beings, called LUCA from the Last Universal Common Ancestor, had a cellular organization, a genome of at least 500 genes and a genetic code based on 64 triplets (Koonin 2003; Koonin and Wolf 2009; Pace 2009; Cavalier-Smith 2010; Koumandou et al. 2013). The evolutionary phase that preceded LUCA has proved so far inaccessible to both palaeontology and molecular analysis, so much so that it has been labelled as the “dark age” in life history (Nitschke and Russell 2010). We will see that novel geochemical insight has changed the way we think about the origin of life, providing a more solid framework for experimental modelling of initial steps in biological evolution. The story that is being presented is a sequence of long periods of stasis and sudden, dramatic changes that transfigured the whole planet, each time adding higher levels of complexity. The first fundamental innovation considered is, quite obviously, the appearance of life. The transition from non-living to living is a complex problem still eluding full understanding; analysing possible scenarios will help us reflect on fundamental aspects of life. The second revolutionary innovation is photosynthesis, which enabled life to use light energy from the Sun, thus opening access to novel habitats. Following primitive forms of photosynthesis that used reduced compounds of geochemical origin, the real change was oxygenic photosynthesis, a pathway that utilizes water as a source of electrons for carbon dioxide reduction, and makes molecular oxygen as a byproduct. This fundamental shift severed ancestral dependence on geochemistry and enabled life virtually to colonize the whole surface of the planet. Oxygen appeared as a stable component of the atmosphere about 2.40 billion years ago. Because of its dramatic effects on life and global geochemistry, the

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oxygenation of the planet is treated here as the third major transition. A far-reaching consequence of oxygenation was the emergence of aerobic metabolic pathways; by using oxygen as an electron acceptor, aerobic metabolism not only increased energy production by orders of magnitude but also permitted the emergence of novel molecules with important biological properties. All the changes mentioned above occurred in the prokaryotic world. The fourth revolution was the birth of the eukaryotic cell, a type of cell with a novel structural and functional design. Innovative properties of the eukaryotic cell were pivotal to the fifth fundamental transition: sexual reproduction or meiotic sex, a novel mechanism of reproduction in which a new organism arises not from a single cell but from two cells that merge and combine their whole genomes. The sixth transition, multicellularity, appeared independently in several lineages under selection pressure for increased fitness from cellular specialization and supra cellular levels of organization. Multicellularity increased ecosystem complexity by introducing novel trophic levels in the food chain. The evolution of the chloroplast, namely the transfer of photosynthesis from the prokaryotic to eukaryotic world, is considered here as the seventh major transition. Photosynthetic eukaryotes spread throughout the world, substantially increasing global productivity and giving a major contribution to planet oxygenation. The emergence of animals, land plants and humans profoundly affected planet’s appearance, global ecology and geochemistry, the three events being thus treated as the eighth, ninth and tenth transition, respectively. The above innovations proceeded by steps, each of which were in turn a complex evolutionary change. The details are only partially known, numerous missing parts of the picture remaining the object of speculation and continuing research. With the dramatic force of a great novel, the story that follows begins with a cloud of dust in a corner of the Galaxy and, covering an immense lapse of time, terminates with an organism that ponders about the texture of the Universe.

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Chapter 2

The Origins

I believe a leaf of grass is no less than the journeywork of the stars. Walt Whitman (1900)

Abstract Primordial nucleosynthesis produced most of the hydrogen and helium present in our Universe. The other elements, including those essential for life on Earth, formed within the stars, spread in space and were incorporated into new solar systems. The Earth developed in the “habitable” zone of our system about 4.55 GYA, mostly by accretion of chondritic material. Gravitational separation of an iron/nickel core and a silicate-rich mantle generated the planetary engine that drives tectonic activity. Oxygen isotope analysis of ancient zircons suggests that tectonics started about 4.5 GYA; comparative analysis of zircons of different ages dates the start of modern plate tectonics to 2.9–2.5 GYA. Major outcomes of tectonic activity include (a) the formation of an atmosphere, an ocean and continental masses; (b) the activation of a planetary thermostat based on carbonate/silicate inter-conversion, essential to maintain the average temperature of the Earth surface in a range compatible with life; (c) the recycling of elements essential to life; (d) the maintenance of chemical disequilibria on the planetary surface, which created the conditions necessary for the development of life. The Earth was probably a habitable planet from about 4.4 GYA.

2.1

Introduction

Energy is the only universal currency: one of its many forms must be transformed to another in order for stars to shine, planets to rotate, plants to grow, and civilizations to evolve. (Smill Vaclav 2000)

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_2

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The hydrogen and helium existing in the Universe were almost entirely produced by primordial nucleosynthesis within the first few minutes after the Big Bang, the event that gave birth to our cosmos about 13.798 GYA (Fig. 2.1). The same process also produced lithium and traces of beryllium; all other elements, including those that are fundamental to life on Earth, formed later from hydrogen and helium by nuclear fusion within the stars (stellar nucleosynthesis) and by other processes during the final demise of stars (https://en.wikipedia.org/wiki/Big_Bang). The first stars appeared about 400 MY (million years) after the Big Bang (Fig. 2.1). Since then, billions and billions of stars have developed within huge aggregates of matter called galaxies (Spier 2010; Alles 2014). Stars form via gravitational collapse of clouds of gas and dust (Fig. 2.2) and their life cycle proceeds through a succession of phases that varies depending on initial mass. Massive stars, with eight times or more the mass of our Sun, end their life cycle with a spectacular supernova explosion, during which much of the initial matter is expelled to space. Less massive stars such as our Sun have much a longer and relatively quiet life cycle, yet they also go through a turbulent phase during which a part of their mass is expelled in space. During the so-called “main sequence” of stars’ life cycle, hydrogen is converted into helium; this phase may last from few million years in the case of the most massive stars to tens of billion years in the case of low-mass stars. When the hydrogen fusion pathway is exhausted, further fusion processes may be ignited, first turning helium into carbon and then proceeding to increasingly heavier elements in the case of high-mass stars. The fusion of lighter nuclei into heavier nuclei is a process that liberates energy until the formation of iron (average atomic mass ¼ 55.845 Da). The formation of nuclei of higher mass than iron is an endergonic process that requires temperature and density conditions almost exclusively produced in the supernova explosions that terminate the existence of massive stars. These extremely violent events have produced almost all the iron and heavier elements that are present today in the Universe and in part are used by life on Earth (https://en.wikipedia.org/wiki/Stellar evolution). Over the time, the conversion of hydrogen and helium into other elements by stellar nucleosynthesis has modified the hydrogen/helium ratio in the Universe, which initially was about 3/1 in terms of mass and 10/1 in terms of atoms’ number.

2.2

Birth of the Solar System

The Solar system started developing about 4.57 GYA. In the course of the preceding 9 billion years, a great number of stars had completed their life cycle and the novel chemical elements produced in their core had been disseminated in space. In mass terms, the Solar system today consists of about 70% hydrogen and 27.5% helium, the remaining 2.5% comprising nearly all the other known elements, notably oxygen (about 0.6%), carbon (0.3%), neon (0.15%), nitrogen (0.11%), iron (0.11%),

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Fig. 2.1 Schematic representation of the temporal evolution of the Universe. Starting from the Big Bang on the left, space has been expanding and the average temperature and density of the Universe, initially approaching infinity, have been steadily decreasing. Protons, neutrons and electrons (as well as a variety of other subatomic particles) formed within 1 s after the beginning. Whereas protons and electrons formed in equal numbers, protons outnumbered neutrons by a ratio of about 5 to 1. Within the first few minutes, protons and neutrons aggregated into nuclei of helium and tiny amounts of deuterium, tritium, lithium and beryllium; most protons remained free as hydrogen nuclei. A few minutes after the Big Bang, nucleosynthesis stopped because the temperature and density of the Universe had become too low. Had the early Universe expanded at a slower rate, almost all matter would have become iron, the most stable chemical element. About 380,000 years later, the temperature had decreased to about 3000  C, thus allowing atomic nuclei to capture electrons and form atoms, a process known as recombination. With the disappearance of free electrons, space became transparent to electromagnetic radiation (indicated as “light” in the figure). The radiation emitted by hot matter during this short phase of Universe’s evolution dispersed in the expanding space; this radiation is still present in the Universe today in a “diluted” form known as the cosmic microwave radiation, as the original wavelength has been stretched by nearly 14 billion years of cosmic expansion. The following phase is known as the dark age because there was no further emission of light: during this period, matter started aggregating gravitationally around slightly denser areas that were a relic of random fluctuations from the very early Universe. About 400 MY later, the first aggregates became hot and dense enough to start new nucleosynthesis processes: the first stars. Since then, billions and billions of stars have formed, giving rise to galaxies and clusters of galaxies. The rate of star formation reached its peak between 0.5 and 1 GYA and has been declining ever since. The figure shows the evolution of the Universe over time across a horizontal axis, but actually the processes described, first of all cosmic expansion, have occurred homogeneously in all directions. It is estimated that the Universe observable today from the Earth is a sphere with a diameter of approximately 93 billion light-years, placing the edge at about 46–47 billion light-years away from us. Hydrogen and helium are converted into heavier elements in the core of stars; the predominance of these elements in the ordinary matter of the observable Universe indicates that this is still at the very beginning of its evolution. (From: https:// commons.wikimedia.org/wiki/File%3ACMB_Timeline300_no_WMAP.jpg Credit: NASA/ WMAP)

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Fig. 2.2 This image shows a huge mass of gas and dust in our galaxy (enclosed in the oval) that is home to a large number of developing stars, some of which are in the T-Tauri phase. The area, named Lupus 3, lies in the Scorpio constellation, about 600 light-years from the Sun. The Sun and neighbouring stars most likely developed in a cloud like this, about 4.6 billion years ago. (Credit: ESO/F. Comeron)

silicon 0.06%). The elemental composition of the Earth is much different, with a large prevalence of iron (32.1%), oxygen (30.1%) and silicon (15.1%), indicating that the elements distributed unevenly during the development of the solar system (Rollinson 2007; Hazen 2012). When the ancestral cloud began to collapse, in a few million years most of the matter (over 99.8% of the total) gathered in the central area, giving rise to a T-Tauri protostar, an object as massive and hot as a star but not yet dense enough for nuclear fusion to occur. This stage characterizes the initial development of stars with a mass below two solar masses. The remaining matter formed a protoplanetary disk rotating around the centre, wherefrom the planets and the other bodies present in the solar system today took origin (Fig. 2.3).

2.2 Birth of the Solar System

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Fig. 2.3 Artistic representation of an early phase in the development of our Solar system. Most of the matter aggregated into a solar embryo at the centre, while the rest formed a protoplanetary disc from which the planets and the other bodies now orbiting around the Sun developed

Because of the high temperatures produced by gravitational collapse, the primordial Sun in the T-Tauri phase emitted an intense flow of radiation and charged particles that blew away the hydrogen, helium and most other volatile molecules (e.g. noble gases, methane and other hydrocarbons) present in the internal part of the system. Because of this, the planets that developed here (Mercury, Venus, Earth and Mars) are much smaller than the external ones and are made mainly of silicates and iron, whereby they get the name “rocky planets”. Rocky planets are of great interest not only because the Earth is one of them but also because these planets are the most likely celestial bodies conducive to life (Lineweaver and Chopra 2012). The Earth is the planet with the highest density (5.515 g/cm3) in the Solar system, followed at a short distance by Mercury (5.427 g/cm3). The great planets external to Mars probably also have an inner core of iron and silicates, but most of their mass consists of hydrogen, helium and other volatile molecules such as nitrogen, water and methane, thus they have much a lower overall density. The Earth retains a reminder of the original nebula in the form of traces of noble gases in its inside; probably these elements were immobilized within solid bodies before the nascent Sun was hot enough to expel them to more external regions of the system (https://en. wikipedia.org/wiki/Solar_System). About 4.55 GYA, the T-Tauri protostar at the centre of the early Solar system reached a temperature and density sufficient to ignite hydrogen nuclear fusion, thus

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giving birth to the Sun, a star that astrophysicists put in the class G2 of the Main Sequence (https://en.wikipedia.org/wiki/Main_sequence). The Sun initially had a luminosity equivalent to about 70% of the present one; over time, it has become hotter and more luminous, possibly due to the conversion of a part of the hydrogen into helium, which increased the overall density of the star and accelerated hydrogen fusion. Before entering the Main Sequence, the nascent Sun emitted an intense burst of high-energy radiation and charged particles that cleaned the internal region of the planetary system from residues of volatile molecules still present. It is estimated that today’s Sun “burns” about 600 million tons of hydrogen and produces 596 million tons of helium per second.

2.3

The Earth

The Earth and the other rocky planets developed from smaller bodies in the internal area of the protoplanetary disk via “accretion”, a process essentially driven by gravity that was virtually completed in about 50 MY. The current model of planetary accretion comprises three phases (Hazen 2012; Morbidelli et al. 2012). During phase I, the prevailing force was not gravity but chemical interactions, i.e. covalent bonds and electrostatic and Van der Waals interactions; under the effect of these forces, gases and dust condensed into bodies of sizes ranging from less than 1 mm to a few tens of cm. The process was controlled by temperature, initially risen to over 1500  C in the more internal part of the protoplanetary disc and slowly decreased thereafter. Scarcely volatile substances such as iron and silicates, and reactive ones such as water and ammonia condensed relatively quickly, whereas volatile substances still present in the region remained in gaseous form. Random collision of two small bodies with much different angular speeds usually results into fragmentation rather than aggregation, gravity being too weak to exert any effect. In order to overcome this theoretical difficulty, planetologists postulate that the small bodies produced by chemical interaction aggregated into swarms by effect of local turbulences in the rotating disc; when these swarms became dense enough, planetesimals started to form by gravity-driven accretion and grew up to 100 km in diameter. This process was relatively slow, thus phase I probably lasted several dozens million years whereas later phases were much faster. As soon as a substantial population of planetesimals had formed, planetary accretion entered the second phase, dominated by gravitational interactions between pairs of bodies. During phase II, larger bodies rapidly increased in size by capturing smaller bodies one by one, thus giving rise to planetary embryos with a diameter of the order of 1000 km. Phase II was probably the shortest of the three phases, lasting no more than 1 million years. At the end, the orbital area of each prospective planet contained a number of planetary embryos and a residual population of planetesimals, with only traces of volatile material dispersed in space. The removal of gas by solar wind had a destabilising effect on the orbits of rotating bodies, which increased the frequency of collisions.

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During phase III, giant collisions of planetary embryos led to the formation of the planets as we know them. Fragments of differentiated planetesimals or planetary embryos (see below) ejected into deep space during collisions produced the so-called rocky, rocky-metallic and metallic meteorites. A different class of meteorites, known as chondrites, are instead bodies or fragments of bodies that never differentiated: as the oldest leftovers of the protoplanetary disc, their composition provides essential clues on the primordial material that gave rise to the Earth and the other rocky planets (Rollinson 2007). As soon as they attained a sufficient size – around a hundred km in diameter – the bodies produced by accretion started warming up until their inside was hot enough to melt (Zahnle et al. 2007). The temperature rise was caused in part by the heat from impacts; it is estimated that the gravitational energy converted into heat during the formation of Earth was equivalent to the energy received from the Sun in 200 MY. However, most of the heat came from the radioactive decay of unstable elements present in the original material, notably aluminium-26, potassium-40, thorium-232, uranium-235 and 238, and plutonium-244 (Box 2.2). The larger the bodies, the slower was heat loss to space and the faster the rise of internal temperature. Melting caused gravitational separation of denser materials, which migrated towards the centre, from lighter materials that remained near the surface. The result was differentiation of a core, mainly made of metallic iron and nickel, and an external layer rich in silicates (Fig. 2.4). The core of the early Earth grew larger by incorporating the core of captured planetesimals and planetary embryos; at a point, its

Fig. 2.4 The Earth has a diameter of 12,756 km at the equator. The external part of the planet consists mainly of silicates and comprises a crust with a thickness of 5–100 km, depending on the location, and a mantle with a thickness of about 2900 km. The mantle in turn consists of an upper and lower mantle, separated by a transition zone. The planet’s core, with a radius of about 3400 Km, is mainly made of metallic iron and nickel and encompasses a solid inner core and a liquid outer core. (From: https://fr.wiktionary.org/wiki/lité#/media/File:Earth-cutaway-schematic-english.svg)

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Fig. 2.5 Interaction of the solid core with the outer liquid shell of iron/nickel in the inside of the Earth creates a powerful magnetic field that shields the planet from solar wind. (Credit: NASA [Public domain], via Wikimedia Commons)

interior solidified, thus producing further heat, whereas the outermost part remained liquid (Fig. 2.4), probably as an effect of the presence of impurities such as sulphur, oxygen and silicon. The liquid core of today’s Earth has an estimated temperature of about 4000  C, whereas the solid, inner core probably reaches 6000  C. The physical and chemical disequilibrium between the inside and outside of the planet created the conditions for the emergence of life about half a billion years later (Chap. 3). Interaction between the solid and liquid core generates a powerful magnetic (or geomagnetic) field that protects the Earth from solar wind, an intense flow of charged particles (mainly electrons, protons and helium nuclei) emitted from the surface of the Sun (Fig. 2.5). If the Earth did not have a geomagnetic field, there would probably be no life as we know it, at least not on the surface of the planet, as solar wind is extremely harmful to biological structures, especially DNA (Hancock et al. 2000; Meissner 2002; Rollinson 2007). Of utmost importance is also the fact that the Earth is located in the habitable zone of the Solar system, viz. the range of orbits within which a planetary surface can support liquid water under sufficient atmospheric pressure. The bounds of the habitable zone in a planetary system depend on the amount of radiant energy emitted by the star. Besides the Earth, Mars also lies in the habitable zone of our system; Venus touches the inner boundary of the habitable zone only when it is at the maximum distance from the Sun (the position known as the aphelion), whereas Mercury’s orbit entirely lies is in the non-habitable inner zone (Fig. 2.6). The giant planets beyond the orbit of Mars (Fig. 2.6) developed from rocky embryos that probably formed as described above for the internal planets but were much larger (over ten Earth masses for the rocky embryo of Jupiter). Stronger gravity enabled these bodies to capture hydrogen and helium present in abundance

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Fig. 2.6 Main bodies of the Solar System. The system encompasses four rocky planets (Mercury, Venus, Earth and Mars), two gaseous giants (Jupiter and Saturnus), and two ice giants (Uranus and Neptune). These planets developed from the same protoplanetary disk, thus they orbit the Sun on the same plane and in the same direction, albeit at different speeds as described by Kepler’s laws. For reasons to be determined, Pluto, now classified as a “dwarf planet”, orbits on a different plane. The dimensions of the bodies and their relative distances are not to scale. (Adapted from: https://en. wikipedia.org/wiki/Solar_System)

in the outer part of the protoplanetary disc, thus growing to giant sizes (about 318 Earth masses in the case of Jupiter). The giant planets formed before the rocky planets, and their gravity influenced the development of the latter. An adjustment in the orbits of giant planets, around 4.55 GYA, probably forced smaller bodies that currently populate the area named “asteroid belt” to move closer to the Sun from their earlier location, thus contributing to the final accretion of rocky planets. Bodies from the asteroid belt were probably a major source of water and carbon-rich molecules for the Earth. Had this event not occurred, the Earth would have much less water and carbon, and life might not have appeared (Lunine 2006). Because most of the iron present in the original material from which the Earth took shape segregated into the core of the planet, the mantle was strongly depleted of this element. Iron is a chemical reductant, viz. an element that reduces hydrogen ions under standard conditions), thus its loss turned the mantle material into a weaker reductant (or, in more precise terms, increased its average reducing potential Box 3.1). This had major implications for the nature of gases emitted by the mantle and the type of atmosphere that developed on the primordial Earth. A geochemically minor consequence, yet of importance for life and humans, is that siderophilic (iron-loving) metals such as cobalt, molybdenum and gold migrated to the core during planetary differentiation, thereby their abundance in the mantle was strongly reduced (Rollinson 2007). A fundamental event in the history of Earth was the birth of the Moon, its satellite. The Moon has a diameter of 3474 km, a mass of about 1/80 of terrestrial mass, and a

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density of 3340 kg/m3. The gravitational interaction of the two bodies induces the tides; the energy lost by the Earth/Moon system in consequence of tides has caused the elongation of the Earth’s rotation period from a few hours initially to today’s 24 h, and an increase in the distance of the two bodies of about 4 cm per year. According to the currently leading model, the Moon formed about 4.53 GYA, at the end of phase III of accretion, from a glancing collision between the young Earth and Theia, a hypothetical planetary embryo with a mass at least one tenth of our planet’s (Sleep et al. 2014; Hartmann 2014; Stevenson and Halliday 2014). The model holds that the impact caused the vaporisation and expulsion to space of superficial material from both bodies, whereas the two metallic cores merged into a single core in the Earth’s interior. The expelled material, almost entirely derived from the mantles of the two bodies, remained in orbit around the Earth and gradually aggregated by accretion to form the Moon. According to some scientists, the tilt of 23.7 of the Earth’s rotation axis relative to its orbital plane, which is responsible for the cycle of seasons on our planet, is a consequence of the tangential collision with Theia. This model explains why the Moon has a very small metallic core, lacks a geomagnetic field and has a lower density than Earth’s. An alternative model posits that the Earth and the Moon developed simultaneously from the impact of two planetary embryos, each with a mass of about 0.5 Earth masses, which merged and immediately separated into two bodies of different masses because of an excess in angular momentum. Whether either competing model is correct or things went differently bears little importance for our story; what matters is that, about 4.53 GYA, Earth had attained its current size and had a satellite, an iron/nickel core and a geomagnetic field. Most likely, however, it was not yet a habitable planet.

2.4

The Activation of Tectonics

Before becoming suitable to life, the Earth went through other major changes, the most crucial of which was the start of tectonic activity, or tectonics. The term “tectonic” comes from the Greek word tectonicòs that means “pertaining to building”. Earth tectonic activity builds continental masses and modifies them continuously, thus exerting a profound influence on the surface of the planet. To understand the importance of tectonics for life, let us examine the situation on today’s Earth (Hancock et al. 2000; Meissner 2002; Hazen 2012; http://www.ucmp.berkeley.edu/ geology/tectonics.html). The solid surface of our planet consists of an outer layer, named crust, and an underlying mantle. The two layers are sharply different in chemical composition, density and temperature. The crust and the upper part of the mantle are rigid and jointly form the so-called lithosphere. Below the lithosphere lies a mantle layer known as the asthenosphere, with a thickness of around 700 km and an average temperature above 1300  C; because of the presence of small amounts of water, the asthenosphere tends to behave as a fluid, albeit extremely viscous material. Current models hold that heat transferred from the liquid iron/nickel core produces huge

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Fig. 2.7 The internal engine of tectonics. Ocean ridges are submarine mountain ranges associated with a valley, or rift, running along their spine. It is widely maintained that ocean ridges are the external manifestations of gigantic convection cells generated in the asthenosphere by heat from the liquid core. The ridges are areas where astenosphere material rises to surface; due to lower pressure, this material undergoes a transition phase from nearly solid rock to fluid magma that builds new oceanic crust on either side (arrows). Slow-spreading ridges, such as the Mid-Atlantic Ridge, have relatively high reliefs (up to about 1000 m), whereas fast-spreading ridges such as the East-Pacific Rise are almost flat; the rift valley associated with ocean ridges ranges from few hundred meters to 20 km in width. Convection cells may also form under continental plates, where they produce “rifts” that may eventually break the continent into two or more plates. For example, the Red Sea formed about 25 MY ago, due to the separation of the Arabic plate from the African Plate. The Rift Valley is a 6000-km long rift in East Africa, whose evolution in the next 10 MY will cause the African Plate to split into a Somali Plate on the east, and a Nubian Plate on the west. (Figure adapted from https:// en.wikipedia.org/wiki/Tectonics)

convective currents in the asthenosphere, which reach the surface and return to the inner depths after cooling down (Fig. 2.7). The mantle upwelling areas active in today’s Earth usually lie at the ocean bottom and are named “oceanic ridges”. As mantle material rises towards the surface, it melts because of lower pressure and emerges to surface in the form of lava. The lava emitted builds new oceanic crust on either side of the ocean ridges, whereas the preexisting crust moves away. The spreading rate varies from 10 to 40 mm per year (comparable to the growth rate of human nails) to about 160 mm per year (comparable to the rate growth of human hair). While this might appear quite a slow motion, the process may actually cause continental-sized masses to move over distances up to 160 km in 1 million year, a short interval at the geological scale. The driving power of these titanic movements likely comes from friction exerted by the mantle flowing under the lithosphere and from the dragging action exerted by the slugs of oceanic lithosphere sinking into the mantle (see below).

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The crust produced at ocean ridges, or oceanic crust, is rich in magnesium and iron silicates; it is only a few km thick near the ridges, but over time it thickens up to 100 km by incorporating material from the underlying mantle, thus producing the oceanic lithosphere. As time goes by, this grows heavier and eventually sinks into the mantle at some distance from the ridge, in a process known as subduction. A different type of crust forms at subducting areas, the continental crust. This arises in part from the melting of the outermost mantle layer, in part from oceanic lithosphere and sediments that escape subduction and are incorporated in the magma or are converted into metamorphic rocks (see below). The ocean water has a central role in these processes because it permeates the mantle in subducting areas and lowers its solidus point, viz. the temperature below which a material is completely solid (Grove et al. 2012). The magma produced in subducting areas is in part emitted to the surface in the form of effusive volcanic rocks (rhyolites, trachytes, andesites) and in part remains in the underground, where it cools down and slowly crystallizes, forming a class of rocks collectively named granites. Like the oceanic crust, the continental crust coalesces with material from the mantle underneath, in the process forming the continental litosphere, a rigid layer with a thickness of about 200 km (Hawkesworth et al. 2010). Both types of crust ultimately derive from the mantle, yet they have sharply different compositions: the oceanic crust is rich in magnesium and iron silicates (mafic rocks), whereas the continental crust is enriched in aluminium, sodium and potassium (felsic rocks). This difference arises from the fact that when basalt (the main component of the oceanic crust) is subducted, it fractionates into a part that melts and contributes to form the continental crust, and another that remains solid and sinks into the mantle (Sect. 2.6). Fractionation processes in subducting areas are responsible for the genesis of the diversity of minerals present on the Earth’s surface (Hazen 2008). The emission of mafic rocks rich in chemically active minerals produces chemical disequilibria that might have provided the conditions necessary for the emergence of life (Chap. 3). Being relatively light, the continental crust tends to remain on the surface, thus escaping subduction. Because of this, the continental crust is much more long lasting than oceanic crust; about 7% of extant continental crust was generated before 2.5 GYA and still persist in areas known as cratons. In contrast, the oldest oceanic crust known to science dates back “only” to the Jurassic era, about 180 MYA. The main mechanism that destroys continental crust is erosion by rain and wind. Fragments produced by erosion are accumulated as sediments, converted into sedimentary rocks and eventually transported to subducting areas; here they are in part incorporated into the mantle, in part brought back to surface as metamorphic rocks after exposition to high temperature and pressure. The primordial Earth had no continental crust; before the formation of the oceans, the planet’s surface was probably covered with a layer of basalt formed from solidified superficial magma (Hazen 2012). The first felsic rocks might have appeared about 4.4 GYA. Continental crust accumulated with time, producing the continents we inhabit today; these cover about 43% of the planet’s surface but account for only

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0.35% of total mass. Planetologists believe that continental crust is a feature unique to the Earth of all the bodies in the Solar system. Following the appearance of life and its spread in the ocean, interaction with living organisms probably enhanced the hydration of the ocean crust, thus accelerating the formation of new continental crust. Because of the rise of convective currents in the deep mantle (Fig. 2.7), the solid surface of the Earth is fragmented into large pieces, or tectonic plates, usually comprised of both oceanic and continental lithosphere. Being averagely less dense than the mantle underneath, tectonic plates float and move around, thus continuously changing their relative positions. Today’s Earth has eight major plates (African, Antarctic, Eurasiatic, North American, South American, Pacific, Indian, Australian) and numerous minor plates, including the famous Nazca plate (Hancock et al. 2000; Meissner 2002). The boundaries between plates are areas of intense tectonic activity. Three types of inter-plate boundaries are distinguished (Fig. 2.8): Transform boundaries, where the plates slide past each other along transform faults, such as the San Andreas Fault in California. Divergent boundaries, where new oceanic crust is produced and the plates drift apart (e.g. the Mid-Atlantic Ridge or the East African Rift). Convergent boundaries, where the plates enter into collision; in the case of oceanic plate/oceanic plate convergent boundaries (e.g. the Japanese Islands), the older and denser oceanic plate slides under the younger one via subduction. In the case of oceanic plate/continental plate convergent boundaries, the oceanic plate is subducted, whereas the continental plate is deformed and uplifted to produce mountain ranges (e.g. the Andes in South America). High mountain ranges (e.g. the Himalayas and the Alps) also develop at continental plate/continental plate convergent boundaries, where both plates are compressed, folded and uplifted. All types of plate boundaries exhibit intense seismic activity; in addition, divergent boundaries and convergent boundaries associated with subduction are the seat of volcanic activity, although of different types in the two cases. No volcanic activity is associated with transform boundaries and continental collision areas. Today, the relative movements of the plates are monitored with great precision by remotesensing satellites, to predict possible earthquakes and mitigate damages to humans.

2.5

Tectonic Processes Are Essential to Life

Most likely life would have never appeared on Earth, or certainly would not have lasted for billions of years, if our planet lacked tectonic activity. Tectonics maintains conditions suitable to life through two major mechanisms.

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Fig. 2.8 (a) At transform boundaries, neighbouring tectonic plates grind past each other; the friction forces involved produce intense seismic activity. (b) At divergent boundaries, new oceanic crust forms and the plates move away from each other. (c) At the convergent boundary of an oceanic plate (left) and a continental plate (right), the former is subducted, whereas the latter is uplifted to form a mountain chain. (Figure credit: By domdomegg – Own work, CC BY 4.0. https://commons. wikimedia.org/w/index.php?curid¼50782243,¼50772217,¼45874902. Credit: TBC)

2.5.1

Tectonics Drives a Powerful Global Thermostat

With time, the continental crust emerged above the oceans and formed continents of increasing extension. Chemical interaction of silicates in surfaced rocks with rainwater and atmospheric carbon dioxide activated a self-regulating mechanism that controls the superficial temperature of the planet (Fig. 2.9). Without tectonics, the atmospheric carbon dioxide would be irreversibly sequestered in sediments as carbonate, and its greenhouse effect (Box 2.1) would reduce to the point that the

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Fig. 2.9 Tectonic activity is essential for planetary thermoregulation. At the continental crust/ atmosphere interface, rock silicates react with atmospheric carbon dioxide, producing silicic acid (H4SiO4) and calcium/magnesium bicarbonate. Transported to the ocean by rainwater, these form silica (SiO2) and carbonate, which accumulate at the ocean bottom. Living organisms producing silica- or carbonate-rich skeletal structures enhance the process. In subduction areas, silica and carbonates are reconverted to silicates and carbon dioxide, which return to surface through volcanic emanations and tectonic uplifting. Why does the same reaction proceed to opposite directions at the crust/atmosphere interface and in subduction areas? The reason is that, in either situation, the removal of a part of the reagents prevents the attainment of equilibrium. At the crust/atmosphere interface, the rain removes silicic acid and bicarbonate, thus atmospheric carbon dioxide continues reacting with rock silicates; in subduction areas, volcanic activity removes carbon dioxide and silicates in the form of gaseous emanations and magma effusions, respectively. Rock weathering is sensitive to temperature. If the surface temperature increases, silicate weathering accelerates and greater amounts of carbon dioxide leave the atmosphere. In contrast, if the surface temperature decreases, the reaction slows down and the atmospheric concentration of carbon dioxide rises because of unchanged input from volcanos. Because carbon dioxide is a greenhouse gas (Box 2.1), this mechanism functions as a global thermostat that keeps the surface temperature of the planet in a range compatible with life. The interruption of any part of the process would stop the thermostat and, in a few million years, the Earth would become as hot as hell or an immense glacier, depending on whether carbon dioxide removal or carbon dioxide emission is affected, respectively. The Earth of 4.4 GYA probably had tectonic activity and oceans, thus the planetary thermostat was already active. However, the continental crust took quite a long time to form in sufficient amount and the young Sun was weaker than today, so the thermostat of early Earth stabilized at much a higher carbon dioxide level than today (estimated to be around 15,000 ppm 2.8 GYA). Go to 2.5 for further details

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Box 2.1: The Greenhouse Effect Radiant energy from the Sun reaches the surface of the Earth mainly in the form of light (viz. visible radiation) and is in part reflected back to the outer space as such, in part absorbed and re-emitted as infrared (which we cannot see, but perceive as heat). It is estimated that, without the effect of the atmosphere, the surface average temperature of the Earth would set around 17  C instead of 14  C actually measured; even worse conditions would have set on primordial Earth because the young Sun was weaker than today. The Moon has no atmosphere and its surface temperature oscillates from about 130  C during the day to a minimum of about 110  C during the night. Acting as a screen, mainly due to the formation of clouds, the atmosphere attenuates diurnal warming up; at night, in contrast, the atmosphere absorbs infrared radiation from the solid surface of the Earth and the ocean and reduces its dispersion to the outer space, thus preventing excessive cooling down. This mechanism is known as the “greenhouse effect” because it is exploited to moderate greenhouse cooling down during the night. The greenhouse effect is due to the presence in the atmosphere of molecules made of three or more atoms; this configuration enables the molecules to absorb infrared radiation and emit at a slightly longer wavelength. The result is that a large part of the heat emitted by the solid surface and oceans remains in the atmosphere, which behaves as a blanket covering the planet and keeping it warm during the night. The atmospheric gases endowed with this remarkable property are known as “greenhouse gases”. Dominant greenhouse gases in today’s atmosphere are, in order of decreasing importance, water vapour (H2O), carbon dioxide (CO2), methane (CH4) and ozone (O3). Differences in the effectiveness of these gases depend on their concentration and persistence time in the atmosphere, besides their intrinsic capacity to disperse infrared radiation (Lenton and Watson 2011). There is strong concern that the rise of carbon dioxide concentration due to the use of fossil fuels by humans may cause a significant increase in the average global temperature, with potential catastrophic consequences on economy, geopolitics and living conditions. The global thermostat will react to the temperature rise and will eventually reduce atmospheric carbon dioxide to the pre-industrial level. The core of the problem is that the global thermostat works at time scales of a hundred thousand years or longer, whereas the changes predicted will occur within the next 100 years (Solomon et al. 2007; Breecker et al. 2010).

average global temperature would fall below water freezing point and the Earth would be covered with ice from pole to pole (Kasting 2008). The average temperature of the Earth depends on a number of variables including the brilliance of the Sun, volcanic activity, atmospheric composition, biological activity and the distribution of continental masses. The planetary thermostat

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responds to temperature variations by acting on the atmospheric concentration of carbon dioxide. This mechanism managed to maintain the average temperature of the planet in a range compatible with life for most of the past 4 billion years of Earth history (with some interruptions known as “snowball glaciations”, 5.4). The temperature at which the planetary thermostat is set depends on spontaneous equilibriums between chemical and geological processes, therefore in the following discussion we will refer to it as the equilibrium temperature. The equilibrium temperature may oscillate within a range depending on the numerous variables involved, thus permitting the alternation of relatively cool and warm phases. We will see that, for reason yet to be completely understood, the Earth went through episodes of extreme cold known as global glaciations, during which the global thermostat temporarily stopped working. Life managed to survive these extreme events, probably finding refuge in the depths of the ocean or in relatively warm “oases” in volcanic areas or near the equator (Chap. 5).

2.5.2

Tectonics Recycles Bioelements

Let’s consider the case of phosphorous, an essential bioelement used for making nucleic acids and cell membranes, and for a multitude of other functions. Phosphorous is present as phosphate in living systems and, unlike carbon, nitrogen or sulphur, it does not form gaseous composts, so it cannot pass from the rocks or the ocean to the atmosphere. Phosphate incorporated in biological structures is set free with decomposition and is thus reused multiple times in the food chain (Filippelli 2002). Over time, however, a part of the phosphate stock unavoidably gets lost with sediments in the depth of the oceans, thus escaping biological cycling. Let us imagine that tectonic activity suddenly stopped, leaving all the rest unchanged. Being trapped in sediments and no longer recycled, the phosphorous stock available to life on the surface of the planet would be exhausted in about 29,000 years. The sulphur inventory would not take much longer to end up in sediments as pyrite or sulphate. Even worse, the atmospheric inventory of carbon dioxide would disappear in sediments as carbonate, plunging the planet in a catastrophic global glaciation in less than 1 million years. The same applies to several other bioelements such as calcium, magnesium, iron, nickel, cobalt, manganese and copper (Lenton and Watson 2011). Not only does tectonics recycle the bioelements trapped in sediments, but also replenishes their stocks by replacing the fractions lost in sediments. Two mechanisms are involved. The first is volcanic activity associated with oceanic ridges and subduction areas, which produces new rock rich in mineral nutrients; the second is the uplifting of continental masses, which brings the rocks (including sedimentary ones) to surface and exposes them to weathering. We can conclude that, without tectonics, the life on Earth would disappear in a geologically very short time span. A striking example of the importance of the processes mentioned above is the Australian continent. Because volcanic activity was completely exhausted at least

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60 MYA, this continent has extremely poor soils. Over a total of 762 million hectares of potentially cultivable land, less than one tenth is actually utilized, mainly for cereals and pasture, and only with addition of fertilizers. Only for a few bioelements, biological availability would not change dramatically if tectonics stopped. Nitrogen would probably remain available thanks to the chemical inertia of its elementary form (N2), the strong solubility of its main inorganic composts (nitrate, nitrite, ammonia) and biological mechanisms of inter-conversion, namely nitrogen fixation, ammonification and anaerobic ammonia oxidation, nitrification and denitrification (Galloway et al. 2004). Being highly soluble, potassium ions would remain abundant in the oceans but would be rapidly lost from soil.

2.6

Birth of the Atmosphere-Ocean-Continental Crust System

The atmosphere, ocean, and continental crust form a closely integrated system (AOC system) that is essential for planet habitability. Notably, an ocean is essential for tectonic activity and for the genesis of continental crust, whilst interaction of the atmosphere with the continental crust and ocean maintains the temperature on the planet surface within a defined range. How much water is there on Earth? The water on our planet forms a superficial domain comprising oceans, glaciers, rivers and lakes, as well as the water in soil, superficial rocks and atmosphere, and an internal domain localized in the mantle. The superficial water domain amounts to about 1.41021 kg, which is only 0.02% of total planet mass. More uncertain is the size of the internal domain; current estimates give a value between 0.16% and 0.26% of the mantle mass. This may seem very little, but the mantle is several thousand km thick; thus, if the estimate is correct, the mantle contains about four to eight times the amount of superficial water (Rollinson 2007). The two domains are not separate: substantial amounts of water pass continuously from the ocean to the mantle in subduction areas, in either a free form or chemically bound to the oceanic crust; simultaneously, equivalent amounts of water move from the mantle to the surface through volcanic emanations. Overall, the water content of the Earth is not much more than 0.1% of the planet total mass. This is much less than the average water content of chondrites, which are considered remnants of the primordial material wherefrom the Earth and the other rocky planets took origin (Sect. 2.3). Ordinary chondrites, the most abundant type of chondrites in our system, have a water content of about 0.3%, and carbonaceous chondrites may have as much as 10%. This suggests that much of the water present in the chondritic material that built the Earth was lost. A part of the water present in the original material was probably lost as vapour in the course of the accretion process, because of the rise of the internal temperature of developing bodies (Sect. 2.3). Other substantial amounts were probably lost in consequence of giant impacts in the final phase of accretion. The hypothetic impact

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that generated the Moon might have been so violent as to cause the ocean (if present) to be completely vaporized. According to this model, a part of the ocean water was lost to outer space immediately after the impact, whereas a part was retained by Earth’s gravity, absorbed by the superficial magma and released again as vapour when the magma cooled down, in a time span of about 30 MY (Zahnle et al. 2007). The isotopic composition of ancient zircons (Box 2.2) suggests that liquid water was already present on the surface of the Earth around 4.4 GYA. Tectonic activity at

Box 2.2: The Isotopes: A Present of Nature to Scientists The isotopes (from Greek ìsos, same, and tòpos, place) are variants of the same chemical element, which have equal atomic number (the number of protons) but different atomic masses (the sum of the mass of protons and neutrons). The isotopes of each element are placed in the same position in the periodic table (whence the name), the value indicated for atomic mass being the average of the different masses corrected for the relative abundances of the isotopes in nature. Isotopes are usually specified by the name of the element followed by a hyphen and the atomic mass, or by the element symbol with a superscript reporting the atomic mass at the upper left, e.g. carbon-12 (12C), carbon-13 (13C) and carbon-14 (14C). As an exception, the isotopes of hydrogen have been assigned their own names and symbols: protium (H, hydrogen-1), deuterium (D, hydrogen-2) and tritium (T, hydrogen-3). The relative abundances of the different isotopes of the same element as well as the abundances of the different elements in the Universe depend on the proton-to-neutron ratio in the atomic nuclei, which affects their stability and the probability of formation by nucleosynthesis. Because the chemical properties of elements essentially depend on the atomic number, the isotopes of the same element are nearly, yet not completely, indistinguishable from the chemical perspective. We could drink a glass of D20 with no relevant consequences (though I gathered that a particularly inquisitive researcher tried this and noticed a “dazing” effect). Because they have different masses, however, the isotopes differ in massdepending properties, notably the speed by which they, or the molecules containing them, move by diffusion. At invariant temperature, the diffusion rate is inversely proportional to the mass, namely lighter isotopes are more mobile than heavier ones. The more pronounced is mass difference relative to total mass, the larger is the difference in diffusion rate; for example, deuterium and tritium have masses two and three times larger than protium’s, respectively, thus differences in mass-depending properties of these three isotopes are quite pronounced. Heavier isotopes, for example the iron isotopes 54Fe, 56 Fe, 57Fe, 58Fe, have much slighter relative differences. However small or large, these differences make the isotopes a precious tool in many fields of scientific research (Budzikiewicz and Grigsby 2006; https://en.wikipedia.org/ wiki/Isotope). (continued)

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Box 2.2 (continued) Many isotopes tend to decay over time, producing other elements and emitting ionizing radiations (usually electrons, positrons and α-particles). The decay rate is expressed in terms of decay constant, or half-life, which is the time span for half of the isotope amount initially present to decay. The halflife depends only on intrinsic properties of the atomic nucleus and may be extremely long, even billions of years, or so short as to make it problematic the identification of certain isotopes. The spontaneous decay of some isotopes provides a natural clock that permits an estimation of the age of natural materials such as rocks or fossils, or man-made artefacts, a technique known as radiometric dating or radiometry. By measuring the amounts of a natural isotope and of its decay product(s) in a material, radiometry estimates the time past from the formation of the material to present with a calculation based on the half-life of the isotope. For the estimate to be reliable, it is essential that the concentration of decay product(s) in the sample at time zero was negligible, and that the sample behaved as a closed system, i.e. the original isotope and its decay product(s) were immobilized and there was no contamination from the outside. Radiometry permits the determination of the absolute age of rocks and fossils, thus being a fundamental tool in geological and paleontological research; radiometry has also found applications in numerous other fields, not the least the verification of the authenticity of archeologic finds and pieces of art. Isotopes with half-lives of different lengths are employed according to time lengths involved. For example, for dating rocks with ages of several hundred MY, radiometry utilizes uranium-235 (half-life ¼ 704 MY) or uranium238 (half-life ¼ 4.47 BY), whereas for historical artefacts, the isotope of choice is usually carbon-14 (half-life ¼ 5.730 KY). There are 81 stable elements on the Earth; of these, 26 have only one isotope, the others two or more. Tin (Sn) has as many as 10 natural isotopes, whereas sulphur (S) has 25, with atomic numbers between 26 and 49, only four of which are stable: 32S (95.02%), 33S (0.75%), 34S (4.21%), 36S (0.02%). So far, 339 natural isotopes have been identified on the Earth. Of these, 288 are primordial isotopes (i.e. nuclides already present in the nebula that formed the Solar System), the others have been produced by decay of instable primordial isotopes or by interaction of pre-existing isotopes with cosmic rays. Many processes, both biological and non-biological, discriminate the isotopes, a phenomenon called isotopic fractionation. Barring particular cases of mass-independent fractionation, isotopic fractionation depends of mass differences. At the same temperature, heavier isotopes (and molecules containing them) averagely have a lower vibrational energy and, if free to move, lower diffusion rates relative to lighter isotopes, thus they require higher activation energy to get involved in chemical reactions. In addition, because heavier (continued)

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Box 2.2 (continued) isotopes form slightly stronger bonds, the molecules containing them have a lower tendency to react. An example is the way isotopic fractionation affects the behaviour of water molecules. These are a mix of “light” molecules containing the isotopes H and 16O, and “heavy” molecules containing D, T, 17 O and 18O in various combinations. When exposed to air, heavier water molecules are less prone to evaporate, i.e. to abandon the liquid phase and pass to the gas phase; on the contrary, heavier molecules have greater tendency to leave the gas phase and condense in liquid form. The same principle applies for the transition from vapour to solid (deposition) and solid to vapour (sublimation) for any chemical species. Because of this, the water present on the surface of the planet in liquid or solid form is enriched in heavy isotopes, whereas the water in the atmosphere is depleted. Likewise, the plant body is enriched in heavier isotopes of hydrogen and oxygen, because plants continuously emit water as vapour by transpiration and the heavier isotopes left back are in part immobilized in organic form, for example cellulose in cell walls. Quite common in living systems also is isotopic fractionation associated with metabolic activity. A classic example is photosynthesis, the process that converts carbon dioxide into carbohydrates (Chap. 3). RubisCO, the key enzyme of photosynthetic carbon assimilation, tends to prefer molecules of carbon dioxide containing 12C vs 13C. Consequently, the organisms that use RubisCO for carbon dioxide fixation (as well as organisms directly or indirectly feeding on these) are 13C-depleted relative to the abundance of this isotope in nature. Some plants utilize phosphoenolpyruvate carboxylase instead of RubisCO for the primary fixation of carbon dioxide; this enzyme weakly discriminates the two carbon isotopes, thus the plants that use it and the animals feeding on these plants show much lower levels of 13C-depletion (Box 12.1). The amounts involved in natural fractionation processes are extremely small, but they are usually measurable with great precision with an instrument named mass spectrometer. In this type of analysis, the fractionation of an element X is expressed as the δX deviation relative to a standard reference material, according to the relation:
δX ¼ Rsample =Rstandard  1  1000 where Rsample and Rstandard are the ratio between the amounts of the isotope X and of another isotope of the same element (for example, 13C/12C) in the sample and the standard, respectively. The relation above assigns a δX ¼ 0 to the standard. If the sample examined is depleted of the isotope X relative to the standard, its δX will be negative; if it is enriched, the δX will be positive. The two stable natural isotopes of carbon, 13C and 12C, occur in nature in the ratio of about 1/99 (0.0101010). The reference standard for carbon is the (continued)

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Box 2.2 (continued) Pee Dee Belemnite (PDB), a limestone rock found in the Pee Dee Formation in South Carolina (USA) and derived from fossilized shells of Belemnitella americana, an extinct cephalopod. The PDB has an unusually high 13C/12C ratio (0.0112372) relative to the average ratio in nature, so its use as a standard assigns a negative δX to most materials. For example, the carbon emitted by volcanoes (mainly as carbon dioxide and methane) has a δ13C of about 5.5‰. The elevated 13C/12C ratio of PDB is a consequence of carbon isotope discrimination by photosynthetic organisms living at the time of its formation. Photosynthetic activity preferentially removed carbon dioxide containing 12C, whereas the heavier form was left in the ocean and used up by Belemnitella for building its shell (shell formation in molluscs involves non-enzymatic carbonate precipitation with virtually no isotope discrimination). Generally, a high 13 12 C/ C ratio in carbonate sediments reflects a parallel sequestration of organic matter with a low 13C/12C ratio, thus providing useful cues to estimate global productivity in past eras. The three natural isotopes of oxygen are, in the order of abundance, 160 (99.76%), 180 (0.2%), 170 (0.04%). The standard for oxygen is the standard mean ocean water (SMOW). The isotopic ratio usually considered is the δ180, but in some cases the δ170 is used as well. Certain isotopic ratios are kind of a signature for specific biological or nonbiological processes; isotopic analysis, therefore, has become a technique of fundamental importance in numerous research areas (Budzikiewicz and Grigsby 2006). An example of the striking informative power of isotopic analysis comes from zircons. Made of zirconium silicate, zircons are among the most durable minerals known. Once formed, zircon crystals survive the dissolution of their parent rock, are transported by water to sediment basins and may be incorporated into novel rocks of sedimentary origin, being able to experience many of these cycles without alteration. It is not surprising, therefore, that relatively young rocks may contain much older zircons. How did we learn that? Uranium-238 decades to lead-208 with a half-life of 4.47 billion years, and uranium-235 decades to lead-207 with a half-life of 704 MY. Satisfying the conditions required for radiometric dating, uranium atoms present in the parent rock are readily incorporated into the crystal lattice during zircon crystal growth, taking the place of zirconium atoms, whereas lead atoms in the parent rock are not allowed to contaminate the crystal. Most conveniently, uranium atoms incorporated in zircons and lead atoms produced by uranium decay do not escape the crystal lattice. Thanks to these remarkable properties, zircon crystals behave as atomic clocks that keep working for billions of years from time zero. (continued)

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Box 2.2 (continued) Zircons extracted from sedimentary rocks at Jack Hills, Australia, have revealed an age of about 4.4 GY, which means they are only 130 MY older than the Solar system (Wilde et al. 2001). Determining the antiquity of these zircons was only the starting point. As long as they last, zircons retain the oxygen isotopic signature of their parent rock; a high δ180 and the presence of inclusions of silicon dioxide in Jack Hills zircons suggest that these crystals formed in granite rock that had interacted with liquid water. This suggests that the Earth of 4.4 GYA already had continental crust and an ocean (Sect. 2.5), thus it was tectonically active. Likewise, the discovery of carbonaceous inclusions with a δ13C as low as 24  5‰ in zircons aged 4.1 GY suggests that life was already present on Earth at that time (Bell et al. 2015).

a global scale possibly started at the same time. Water confers the deep mantle the fluidity necessary for the development of convection currents, whilst interaction with ocean water at ocean ridges and subduction areas induces the melting of the mantle, thus driving the formation of volcanoes, oceanic crust and continents (Grove et al. 2012). During accretion, the primordial Earth possibly acquired significant amounts of gas from the ancestral nebula, so it may have had a primary atmosphere rich in hydrogen, helium, methane and noble gases. If ever present, such an atmosphere quickly vanished to space due to combined effects of solar wind and impact with large bodies. The same forces also depleted the Earth of other volatile substances present in the primordial material. For example, this is how the Earth probably lost about 50% of the original nitrogen stock (Zahnle et al. 2010). It is estimated that, in the immediate aftermath of the impact with Theia, the Earth surface was covered by a magma ocean at a temperature around 6000  C, remaining as bright and hot as a small star for about a thousand years (Fig. 2.10). The Earth cooled down by convection from deeper parts to the surface, and radiation from the surface. In a few million years, the superficial magma formed a layer of solid rock, and magma degasification produced an atmosphere of several hundred bars of water and 100–200 bars of carbon dioxide plus molecular nitrogen and traces of methane, sulphur dioxide, hydrogen, ammonia and noble gases (Sleep 2010; Zahnle et al. 2010; Sleep et al. 2014). When the average surface temperature was sufficiently low, liquid water precipitated: a huge flood probably lasting several thousand years gave rise to the ocean, thus paving the way to the development of life. Experiments with special devices that produce extremely high temperatures and pressures have provided novel insight into the mineralogical evolution of the early Earth (Hazen 2008, 2012). Olivine, a magnesium silicate, is the first mineral that separates in the solid state from a “synthetic” magma with the chemical composition of the mantle when the temperature drops to 1500  C. Being denser than the magma, olivine crystals sank to the depth and formed the stunning green rock named dunite,

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Fig. 2.10 In the immediate aftermath of the hypothetic impact with a planet embryo named Theia, the Earth was probably entirely covered with a “magma ocean”. A large mass of fragments from the mantle of the two bodies remained orbiting around the Earth and generated the Moon by accretion. The Earth’s primordial atmosphere and a part of the ocean, if present, were lost to space in consequence of the impact; the superficial magma absorbed the rest of the water. In the following 10 MY, intense volcanic activity produced an ocean and a CO2-rich atmosphere. (Image Credit: Alan Brandon/Nature)

which occasionally appears on the surface in consequence of tectonic uplifting and erosion. Because of the separation of olivine, the superficial magma was impoverished of magnesium and enriched in calcium and aluminium. Further cooling down induced the separation of other minerals, mostly pyroxenes, a class of aluminosilicates of calcium, magnesium and minor amounts of other metals. Mixed with olivine crystals, the pyroxenes formed the peridotite, a greenish-black dense rock that like olivine sank down, leaving magma on the surface. The process continued until all the superficial magma solidified into a peridotite layer with a thickness of several tens km. The formation of a peridotite crust was only a short, juvenile phase in Earth evolution. Being solid, peridotite could not transfer to surface the heath from the radioactive decay of instable nuclides trapped in it. The peridotite layer, therefore, warmed up until melting, facilitated in this by chemical interaction with water. Once again, melting affected only a part of the original rock and produced mafic magma enriched in calcium and aluminium and, to a lower extent, in iron and silicon.

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According to the reconstruction by Hazen (2012), peridotite melting on the early Earth generated a mighty burst of volcanic activity that in a few tens MY covered the planet surface with a novel type of rock, the basalt. When the basalt layer attained a sufficient thickness, the process described for peridotite reiterated; at a temperature of about 1200  C, basalt melting produced felsic magma enriched in silicon, sodium and potassium, and depleted of iron, magnesium and calcium. Due to the pressure exerted by dissolved volatile substances, the felsic magma rose to the surface through cracks in the overlying layer of solid basalt and produced a novel rock, less dense than basalt (2.7 vs 3 g/cm3), known as granite. Made of quartz (silicon dioxide) crystals, feldspate (sodium, potassium and calcium aluminosilicates) and varying amounts of iron minerals (amphibole, pyroxene, mica), granite accumulated on the planet surface and formed the embryos of prospective continents (Campbell and Taylor 1983; Hazen 2012). The processes described above are still active today and are responsible for the emission of mafic magma along volcanic ridges and through volcanoes at hot spots (a sort of chimneys in the inside of tectonic plates), and of felsic magma through volcanoes at subduction areas. The ancient atmosphere was much richer in carbon dioxide than today. Dissolved in the ocean, carbon dioxide brought the pH to about 5.5 (today’s seawater is lightly alkaline, with a pH in the range 7.5–8.4). The atmosphere also contained more methane and other small hydrocarbons than today, which probably gave the Earth a yellowish colour very unlike its present bluish aspect. A shallow ocean covered the early Earth almost entirely, with a thick layer of basalt at the ocean bottom scarred by numerous volcanoes produced by uprising plumes of magma. Only occasionally did volcanic edifices manage to emerge from the ocean but these did not last long, being rapidly eroded by the huge tides induced by the Moon, then much closer to the Earth than today. One might think that the primordial ocean was less salty than today, assuming that marine salt (mainly sodium chloride, NaCl) took a long time to get to present concentrations. Most likely, instead, salt accumulated very early and the ancient ocean was more salty than today. The reason is that almost half of the salt present on the Earth’s surface today is in halite ores formed by desiccation of ancient seas, a huge amount of salt that was at least in part dissolved in the ocean of the early Earth, as initially there were no continents (Knauth 2005). Because of the paucity of superficial rocks, the silicate/carbonate cycle was scarcely active and consequently carbon dioxide concentration remained very high until the start of tectonics, thus contributing to compensate for the weak Sun (see below). It is unclear how long a warm CO2 greenhouse persisted. Volcanic activity initially depended on hot-spot-like magmatic plumes. The occurrence of quartz inclusions (a mineral common in granite but rare in other rocks) in Jack Hills zircons suggests that granite was already present on Earth around 4.4 GYA. Isotopic analysis of detrital zircons from Jack Hills suggests that tectonics and continental crust formation started as early as 4.4 GYA (Harrison et al. 2005). In contrast, a worldwide compilation of U/Pb, Hf-isotope and trace-element data on ancient zircons from 4.5 to 2.4 GYA

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suggests that Earth’s crust was essentially stagnant and dominantly mafic in composition (i.e. directly derived from the mantle) until about 3.4 GYA, and that modern plate tectonics started as late as 2.9–2.5 GYA (Griffin et al. 2014). Cawood and Hawkesworth (2019) argue that continental area and thickness varied independently and increased at different rates and over different periods, in response to different tectonic processes. In their scenario, modern plate tectonics driven by convective cells (Fig. 2.7) started around 3 GYA. From about 4 GYA, however, pre-plate tectonics driven by mantle plumes had produced relatively thin and mafic continental crust, attaining a dynamic equilibrium at about 40% of Earth’s surface. With the start of plate tectonics, the continental crust became more felsic and increased in thickness, but its overall surface area remained virtually unchanged in the last 3 GY. The emergence of continental crust from the ocean accelerated the silicateweathering cycle, thus reducing the atmospheric concentration of carbon dioxide and the average global temperature. Data from ancient soils (paleosols) suggest that 2.8 GYA carbon dioxide was around 1.5% (15,000 ppm) (Sheldon 2006). The spread of the first microorganisms possibly gave a substantial contribution to the geochemical evolution of the Earth. Notably, subsurface rock-microbe interactions may have enhanced the hydration of early oceanic crust, which in turn promoted bulk melting and the formation of the first evolved fragments of continental (felsic) crust at subduction areas. Thus, by facilitating hydrothermal alteration of oceanic crust, life probably promoted mineral diversification and accelerated the development of surface environments and microcontinents on the young Earth (Grosch and Hazen 2015). Around 4.5 GYA, the Sun luminosity was ~70% of the present value and 2.8 GYA had risen to ~80%. An atmospheric carbon dioxide level of 15,000 ppm is extremely high compared to today’s level (~400 ppm), but still too low for producing the greenhouse effect expected to maintain the Earth’s average temperature above the freezing point with a faint Sun (Zahnle et al. 2007). To do so without the participation of other forces, carbon dioxide should have had a concentration of at least 30% (300,000 ppm), a level 20 times higher than estimates for the Earth of 2.8 GYA. In the absence of other balancing mechanisms, the Earth should have plunged into a global glaciation, with the ocean frozen to a depth of several Km and the continents covered by ice from pole to pole. Large glaciations leave persistent signs on continents; indeed the study of fragments of ancient continental crust has demonstrated that the Earth did experience global or almost global glaciation episodes 2.4 and 0.7 GYA. Nevertheless, remains of ancient continental crust show no sign of glaciation before 2.4 GYA. The contrast between geologic evidence for the presence of liquid water on the Earth’s surface in the deep past (Mojzsis et al. 2001) and lower irradiance from a faint early Sun has been cast as “the faint young Sun paradox” (Haqq-Misra et al. 2008).

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Several mechanisms have been postulated for explaining the “missing glaciation” of Hadean Earth (Rosing et al. 2010). The inside of the young Earth was probably significantly hotter than today and heat transferred to the surface by intense volcanic activity might have helped prevent ocean freezing (Zahnle et al. 2007). A second possible factor was a lower albedo (Glossary). Much of the albedo of today’s Earth is due to clouds, whose formation is stimulated by transpiration from land plants on continental masses (11.9), and by emission of dimethylsulfoniopropionate by eukaryotic algae in the ocean, which enhances the nucleation of water droplets in the atmosphere (Feulner et al. 2015). Eukaryotic algae probably appeared only about 0.8 GYA and land plants around 0.5 GYA; therefore the Hadean Earth probably had a less dense cloud cover than in the more recent past (Rosing et al. 2010). A third possible mechanism is a stronger greenhouse effect due to methane (Kasting and Ono 2006; Haqq-Misra et al. 2008). Methane is present in today’s atmosphere at much a lower concentration (~1.8 ppm) that carbon dioxide (~400 ppm), and its contribution to greenhouse warming is relatively low (~7% vs. ~20% of carbon dioxide). In today’s atmosphere, methane has a relatively short lifetime (~12 years vs 30–95 years for carbon dioxide) because of light-mediated oxidation by oxygen. Before 2.45 GYA, however, oxygen was virtually absent in Earth’s atmosphere and methane lifetime was about 1000 times longer, thus increasing the greenhouse impact of this gas. On today’s Earth, the main source of methane is methanogenic archaea, either free or living in symbiosis with animals; a second important source is the reaction of carbon dioxide with iron silicates in mafic rock, a geochemical process known as “serpentinization” (Chap. 3). It has been suggested that, in the absence of free oxygen, methane emissions of the same magnitude as observed today might have provided the additional greenhouse warming necessary for preventing global freezing of the early Earth (Haqq-Misra et al. 2008). This model implies that the Archaea, the only life form known to make methane, have been in existence before 2.8 GYA, an assumption not unanimously accepted in current evolutionary models (Chaps. 3 and 6). Whichever the mechanism(s) involved, the early Earth avoided being trapped in a succession of global glaciations that might have confined life to the ocean bottoms forever or even prevented its very appearance. Before starting our adventurous journey, let us have a look at the geologic time scale. Covering about 4.6 billion years from the formation of Earth to present, this features four Eons (Hadean, Archean, Proterozoic, Phanerozoic) and a number of shorter intervals, the transition from one to the other marked by geological, geochemical or biological events (Fig. 2.11).

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Fig. 2.11 Geologic time scale. The Hadean (4.6–4.0 billion years ago, GYA) is the eon preceding the formation of the first known rock. The Archean spans from 4 to 2.5 GYA. The Proterozoic, the eon of microscopic life, began 2.5 GYA and terminated 541 MYA. The Phanerozoic is the eon of macroscopic life. Phanerozoic is the most recent and best-known eon, as reflected by extensive subdivision into lower-rank intervals

References Alles DL (2014 The evolution of the Universe. http://fire.biol.wwu.edu/trent/alles/Cosmic_Evolu tion.pdf Bell EA et al (2015) Potentially biogenic carbon preserved in a 4.1 billion-year-old zircon. Proc Natl Acad Sci U S A 112:14518–14521 Breecker DO, Sharp ZD, McFadde LD (2010) Atmospheric CO2 concentrations during ancient greenhouse climates were similar to those predicted for A.D. 2100. Proc Natl Acad Sci U S A 107:576–580 Budzikiewicz H, Grigsby RD (2006) Mass spectrometry and isotopes: a century of research and discussion. Mass Spectrom Rev 25:146–157 Campbell IH, Taylor SR (1983) No water, no granites – no granites, no continents. Geophys Res Lett 10:1061–1064 Cawood PA, Hawkesworth CJ (2019) Continental crustal volume, thickness and area, and their geodynamic implications. Gondwana Res 66:116–125 Feulner G, Hallmann C, Kienert H (2015) Snowball cooling after algal rise. Nat Geosci 8:659–662 Filippelli GM (2002) The global phosphorus cycle. Rev Mineral Geochem 48:391–425 Galloway JN et al (2004) Nitrogen cycles: past, present, and future generations. Biogeochemistry 70:153–226 Griffin WL et al (2014) The world turns over: Hadean-Archean crust-mantle evolution. Lithos 189:2–15

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Grosch EG, Hazen RM (2015) Microbes, mineral evolution, and the rise of microcontinents – origin and coevolution of life with early Earth. Astrobiology 15:922–939. https://doi.org/10.1089/ast. 2015.1302 Grove TL, Till CB, Krawczynski MJ (2012) The role of H2O in subduction zone magmatism. Annu Rev Earth Planet Sci 40:413–439 Hancock PL, Skinner BJ, Dineley DL (2000) The Oxford companion to the Earth. Oxford University Press, Oxford Haqq-Misra JD, Domagal-Goldman SD, Kasting PJ, Kasting JF (2008) A revised, hazy methane greenhouse for the Archean Earth. Astrobiology 8(6):1127–1137 Harrison TM et al (2005) Heterogeneous Hadean hafnium: evidence of continental crust. Science 310:1947–1950 Hartmann WK (2014) The giant impact hypothesis: past, present (and future?). Phil Trans R Soc A 372:20130249 Hawkesworth CJ et al (2010) The generation and evolution of the continental crust. J Geol Soc 167:229–248 Hazen RM (2008) The emergence of chemical complexity: an introduction. In: Zaikowski L, Friedrich JM (eds) Chemical evolution across space & time, ACS symposium series, vol 981. American Chemical Society, Washington, DC, pp 2–13. https://doi.org/10.1021/bk-2009-1025. ch001 Hazen RM (2012) The story of the Hearth. The first 4.5 billion years from start dust to living planet. Viking, New York Kasting JF (2008) The primitive Earth. In: Wong JTF, Lazcano A (eds) Prebiotic evolution and astrobiology. Landes Bioscience, Austin. http://www3.geosc.psu.edu/~jfk4/PersonalPage/ PDFs.htm Kasting JF, Ono S (2006) Palaeoclimates: the first two billion years. Philos Trans R Soc B 361:917– 929 Knauth LP (2005) Temperature and salinity history of the Precambrian ocean: implications for the course of microbial evolution. Palaeogeogr Palaeoclimatol Palaeoecol 219:53–69 Lenton T, Watson A (2011) Revolutions that made the earth. Oxford University Press, Oxford Lineweaver CH, Chopra A (2012) The habitability of our Earth and other Earths: astrophysical, geochemical, geophysical, and biological limits on planet habitability. Annu Rev Earth Planet Sci 40:597–623 Lunine JI (2006) Physical conditions on the early Earth. Philos Trans R Soc B 361:1721–1731 Meissner R (2002) The little book of planet Earth. Copernicus Books, New York Mojzsis SJ, Harrison TM, Pidgeon RT (2001) Oxygen-isotope evidence from ancient zircons for liquid water at the Earth’s surface 4,300 Myr ago. Science 409:178–181 Morbidelli A et al (2012) Building terrestrial planets. Annu Rev Earth Planet Sci 40:251–275 Rollinson H (2007) Early Earth systems. A geochemical approach. Blackwell Publishing, Oxford Rosing MT et al (2010) No climate paradox under the faint early Sun. Nature 464:744–747 Sheldon ND (2006) Precambrian paleosols and atmospheric CO2 levels. Precambrian Res 147:148– 155 Sleep NH (2010) The Hadean-Archaean environment. Cold Spring Harb Perspect Biol 2:a002527. https://doi.org/10.1101/cshperspect.a002527 Sleep N, Zahnle KJ, Lupu RE (2014) Terrestrial aftermath of the Moon-forming impact. Phil Trans R Soc A 372:20130172 Solomon S et al (2007) Climate change 2007: the physical science basis. Cambridge University Press Cambridge., https://www.ipcc.ch/pdf/assessment-report/ar4/wg1/ar4_wg1_full_report. pdf. Accessed 22 June 2018 Spier F (2010) Big history and the future of humanity. Wiley-Blackwell, Chichester Stevenson DJ, Halliday AN (2014) The origin of the Moon. Phil Trans R Soc A 372:20140289 Vaclav S (2000) Energies: an illustrated guide to the biosphere and civilization. Mitt Press, Cambridge, MA. The MIT Press Website on Tectonics.: http://www.ucmp.berkeley.edu/geol ogy/tectonics.html. Accessed 20 Mar 2018

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Whitman W (1900) Leaves of grass. David McKay, Philadelphia Wilde SA et al (2001) Evidence from detrital zircons for the existence of continental crust and oceans on the Earth 4.4 Gyr ago. Nature 409:175–178 Zahnle K et al (2007) Emergence of a habitable planet. Space Sci Rev 129:35–78 Zahnle K, Schaefer L, Fegley B (2010) Earth’s earliest atmospheres. Cold Spring Harb Perspect Biol 2:a004895

Chapter 3

The Birth of Life

Events in molecular evolution that are unlikely at limited laboratory scales of space and time may, nevertheless, be inevitable on an Earth-like planet at time scales of a billion years. Robert Hazen (2017)

Abstract Life was most likely present on Earth as early as 3.5 GYA and probably made its first appearance around 4 GYA. Alkaline hydrothermal vents discovered in 2000 are presently considered a likely setting for the origin of life because they could provide organic matter, chemical disequilibria and compartmentation. Simulation experiments show that the synthesis of simple organic molecules from CO2 and H2 and of peptides from free amino acids is thermodynamically favoured under hydrothermal vent conditions. Abiotic synthesis of nucleotides and RNA is more problematic due to intrinsic instability of RNA and ribose under alkaline conditions. Association with abiotic peptides might have stabilized abiotic RNA, leading to the emergence of self-replicating ribonucleoprotein complexes (RNPs). It is suggested that a crucial step towards life was the appearance of “protoribosomes”, viz. RNPs capable of making peptides with a sequence determined by cognate RNAs under the rules of a primordial genetic code, and “protoviruses”, viz. RNPs that replicated RNA templates from free nucleotides under the rules of base complementarity. Although the two classes of RNPs most likely evolved independently, they established stable associations by moving with water currents and binding to peptide-mineral protomembranes in hydrothermal vents. Protoribosomes and protoviruses were ancestral to ribosomes and chromosomes, respectively. Incorporation of polar lipids into protomembranes produced biological membranes. DNA replaced RNA as a more stable repository of genetic information at a very early stage of evolution. Chemiosmosis, the universal energy-harnessing mechanism of life, probably appeared in a rudimentary form at a prebiotic stage, and was certainly operative in LUCA. Early life was probably autotrophic, obtaining organic carbon and energy from carbon dioxide reduction with hydrogen of geochemical origin.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_3

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3.1

The Birth of Life

Introduction

Any local rise in complexity must inevitably have been accompanied by a larger rise of disorder elsewhere. Given this situation, how could complexity have emerged all by itself? Fred Spier (2010)

The origin of life is the most complex and recalcitrant issue of modern biology. Living systems exist in a physical state that is extremely far from thermodynamic equilibrium or, equivalently, has extremely low entropy and thus low probability. To maintain this condition, living systems need coupling with an external disequilibrium, namely a supply of “negentropy” (Branscomb and Russell 2018a). The loss of thermodynamic coupling is what we call “death”, dead organisms rapidly and irreversibly degrading to the equilibrium. Explaining how this fundamental property emerged spontaneously during the transition from “inanimate matter” to living systems is the major difficulty encountered in efforts to reconstruct the origin of life. Said that, let us begin this story with insight from paleogeochemistry. The earliest signs of life on Earth are particles of graphite (a carbon mineral) with a δ13C value compatible with isotopic fractionation by living organisms (Box 2.2). The oldest report is from graphite-containing zircons aged 4.1 GY (Bell et al. 2015). 13Cdepleted graphite also occurs in rocks from the Itsaq Gneiss Complex and Isua Greenstone Belt (Greenland), both aged about 3.7 GY (Rosing 1999; Ohtomo et al. 2013). Apatite with graphite coatings within iron formations (BIF) from the 3.830 GY-old Akilia supracrustal belt in southwest Greenland are interpreted as the metamorphosed product of biogenic matter (Mojzsis et al. 1996). Direct signs of the existence of life come from fossils, viz. remnants, impressions or traces of ancient organisms. The first organisms appeared on Earth most certainly were unicellular forms of microscopic size. The identification of billion-year-old microfossils is an extremely difficult task because of the highly metamorphic nature of old sedimentary rocks. Structures interpreted as microfossils occur in sedimentary rocks from the Strelley Pool Formation (Pilbara Craton, Australia) and the Buck Reef Chert (South Africa), aged about 3.4–3.5 GY (Brasier et al. 2006; Schopf 2006; Wacey et al. 2011; Sugitani et al. 2015; Schopf et al. 2017). Putative microfossils associated with 13 C-depleted carbonaceous material have been reported in the Nuvvuagittuq belt in Canada, dated to at least 3.77, possibly 4.29 GYA (Dodd et al. 2017). This discovery is of special interest not only for the exceedingly old age of putative fossils, but also for their association with coeval submarine hydrothermal vents, currently considered as the most likely environment for the early evolution of life. Much easier to identify as biological products are stromatolites, layered biochemical accretionary structures formed in shallow water by the trapping, binding and cementation of sedimentary grains by biofilms secreted by microbial communities. When the sediment becomes so thick as to prevent light or nutrient interception by underlying cells, a new layer of microorganisms develops on the free surface, thus reiterating the process. Over time, this may generate structures of considerable sizes (even meters), which may be incorporated into sedimentary rocks and fossilize, although only occasionally do they retain recognizable cellular remnants (Schopf

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2006). The occurrence of fossil stromatolites in the Strelley Pool Chert is a likely indication of the existence of photosynthetic life about 3.4 GYA (Wacey 2010). Overall, the available evidence suggests that life was present on Earth around 4.0 GYA and was widely distributed on the planet surface by 3.4 GYA, in the form of microscopic organisms similar to extant bacteria. Since the definitive demonstration by Pasteur in 1864 that life (including microscopic life) does not develop spontaneously from organic matter, the origin of life has been at the focus of attention in scientific debate, yet it remains a most elusive issue. We shall not consider religious models, these being not liable to scientific discussion, or extra-terrestrial scenarios such as the panspermia hypothesis that life appeared somewhere in the universe (presumably a more permissive place than the Earth) and spread to the Earth and other “habitable” planets by travelling through space in the form of resting spores (Chandra 2011). That life may have appeared somewhere else in the Universe besides the Earth is a very likely possibility, considering the immense spatial and temporal scale involved and the increasing number of “hospitable” planets being discovered even in the immediate surroundings of the Solar System (Lineweaver and Chopra 2012; Petigura et al. 2013). There is no evidence, however, that supports the panspermia hypothesis. Assuming that life appeared on Earth autonomously, the following discussion will consider where and how this extraordinary event could take place. Basic principles of the “terrestrial scenario” presented in this chapter are probably of general application to other “Earth-like” planets. The first scientific approach to the issue was the famous experiment performed in 1952 by Stanley Miller, a student working for his doctorate at the Chicago University. Miller introduced a mixture of hydrogen, ammonia, methane and water in a sealed system, and the mixture was exposed to sparks from an electrical device. The system operated under sterile conditions (i.e. care was taken to ensure that no microorganism contaminated the inside of the device) and permitted the operator to take small aliquots at intervals for chemical analysis. After few days, Miller detected small organic molecules such as formaldehyde. In the following months a number of more complex molecules appeared, including sugars and amino acids. Miller’s report of this experiment appeared in 1953 on the scientific journal Science (Miller 1953), raising enormous interest in the scientific community as well as in the media and public. Ensuing experiments showed that the addition of hydrocyanic acid to the original mixture induced the formation of adenine, one of the four organic bases in DNA. Later it was found that a gas mixture more similar in composition to the likely atmosphere of the Earth around 4.4 GYA (Sect. 2.6), viz. with a prevalence of nitrogen and carbon dioxide and minor amounts of hydrogen and methane, was able to generate a mixture of simple organic molecules, including key components of living systems. Miller’s experiments revealed that simple components of living systems such as amino acids, sugars and organic bases are promptly produced in abiotic systems in the presence of a source of energy. In line with this observation is the discovery of

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organic compounds in interstellar gas, probably produced from inorganic precursors under the effect of high-energy radiation from stars (Chan et al. 2018). The core of the issue is that living systems are much more complex than their elementary components, namely they have emergent properties that are not explained from the properties of lower-level parts. In William F. Martin’s words (Martin 2011), “life is a chemical reaction perpetually far from the equilibrium, not stirred organic soup”. For a start, most of the molecules utilized by life can exist in two or more forms, or “stereoisomers”, which differ only in the spatial arrangement of functional groups. Abiotic reactions as those involved in Miller’s experiments produce mixtures of stereoisomers, whereas life invariably uses only one of the possible forms of each compound. A kind of stereoisomers of special importance in biology are chiral isomers, or enantiomers, molecules that exist in two forms that are non-superposable mirror images. For example, the α-amino acids (the molecular units of proteins) have two enantiomeric forms, named L and D, that differ in the relative spatial arrangement of the four groups linked to a central carbon, or α-carbon. The α-amino acids produced in Miller’s experiments are a mixture of the L and D isomers, whereas biological proteins are made of the L-isomers only. In fact, the living world is entirely homochiral: none of the thousands molecules used by living organisms occurs as a random mixture of stereoisomers; quite the opposite: whenever a molecule can express chirality, life has chosen to use only one of the two possible enantiomers. From bacteria to animals and plants, proteins are built from the L isoforms of twenty α-amino acids, including L-alanine and L-glutamate. D-alanine and D-glutamate are instead specifically employed by bacteria in the production of peptidoglycan, a major component of the cell wall. There are 16 possible enantiomers of the aldohexoses, a class of simple sugars with six carbon atoms, but only three of these exist in nature, namely D-glucose, D-mannose and D-galactose. D-glucose is the main substrate of respiration and the precursor for numerous essential metabolites. Our intestine would not absorb L-glucose, nor would the intestinal microflora metabolize it, although this molecule differs from D-glucose only for the orientation of a –OH group linked to carbon 5. A more radical problem comes, as already observed, from thermodynamics. Life is an emerging property of systems of linear polymers (nucleic acids and proteins) with specific monomer sequences. The spontaneous polymerization of monomers in solution proceeds up to an equilibrium characterized by small amounts of polymers with random sequences and a majority of free monomers. The polymerization of monomeric units is generally a thermodynamically disfavoured reaction because it involves an increase of free energy (ΔG > 0); for the equilibrium to favour polymer formation, polymerization must be coupled with a reaction with a negative ΔG. Even more complex and thermodynamically unlikely is the polymerization of monomers in non-random sequences. In living systems the polymerization of proteins and nucleic acids uses “activated” monomers (aminoacyl~tRNAs and nucleoside-triphosphates) that satisfy this necessity (Deamer and Weber 2010).

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A third difficulty comes from the fact that cells, the structural units of life, are not simple pots of chemicals. The electro-chemical mechanism almost universally used to drive carbon and energy metabolism depends on sophisticated ion-tight phospholipid membranes and protein nanomachines with moving parts (Box 3.1). Membranes, protein nanomachines and vectorial biochemistry must have arisen by selection in an environment in which vectorial ion flux occurred spontaneously. Life has six fundamental properties: carbon capture, energy transduction, heredity, metabolism, compartmentation and excretion (Lane 2015). Essential conditions for the spontaneous emergence of these properties are: • Physical compartmentation, e.g. barriers separating environments with different levels of entropy. • A source of energy preventing the attainment of thermodynamic equilibrium and driving spontaneous vectorial ion fluxes across barriers. • Inorganic catalysers promoting abiotic synthesis of small organic molecules including amino acids and nucleotides. • Abiotic polymerization of monomeric units. • Accumulation of organic molecules including polymers to concentrations high enough to establish pre-biotic metabolic networks. • Continuous disposal of waste by-products, necessary for pre-biotic systems to get rid of entropy and to remain in a state far from equilibrium.

3.2

Seafloor Hydrothermal Vents as Settings for the Emergence of Life

The tension between CO2 dissolved at relatively high atmospheric pressure in the Hadean ocean, and H2 generated as ocean water oxidized ferrous iron during convection in the oceanic crust, was resolved by the onset of life. Russell and Hall (2006)

Where could so special conditions exist on early Earth? Certainly not in a simple “primordial soup” as imagined by Darwin and more recently by Oparin and Haldane, whose thought inspired Miller’s experiments. After Miller, the scientific literature has seen a multitude of variously elaborate “organic soup” models. For example, Skoblikow and Zimin (2018) postulate massive production of organic compounds on early Earth by explosive volcanic activity, followed by spontaneous “polycondensation” and polymerization. Higgs (2016) proposes wetting and drying cycles as a mechanism favouring polymerization in aqueous solution. None of these models, which Branscomb and Russell (2018a) dub “frankenstein models”, provides a thermodynamically plausible scenario for the emergence of life, i.e. a long-lasting, stable source of disequilibrium. An alternative scenario emerged in the late 1970s, with the discovery of an extraordinary geochemical phenomenon associated with mid-ocean ridges (Hannington et al. 2005; https://en.wikipedia.org/wiki/Hydrothermal_vent). Superheated water, mostly produced by magma degasification (Chap. 2), emerges from

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Fig. 3.1 Black smokers host a rich community of animals ultimately depending on sulphideoxidizing chemosynthetic bacteria. The picture on the left shows a colony of Riftia pachyptila living in the proximity of a vent. This worm lives in the inside of a chitinous tube, exposing only the branchial apparatus, of a vivid red colour due to hemoglobin dissolved in blood. Remarkably, Riftia hemoglobin binds both oxygen and sulphide ions and transports these molecules to the trophosome, a modified intestine that hosts chemosynthetic bacterial symbionts. The worm lacks a mouth and anus and lives on organic matter produced autotrophically by its bacterial symbionts. The symbionts are acquired horizontally during the larval stage. Riftia pachyptila may grow to a length of 1.5 m in 2 years, the highest growth rate recorded for invertebrates. (Credit: Danny Preiss, Deep sea creatures)

fractures in the ocean crust close to the ridges. At emission points, the water has a temperature of 300–400
C and is strongly enriched in dissolved minerals (notably iron sulphide) and gases due to interaction with mafic rock (Sect. 2.4). When this hot solution gets in contact with the cold ocean water (about 2
C), a part of the minerals dissolved precipitate producing black emissions, whence the denomination “black smokers” given to these vents (Fig. 3.1). Despite total absence of light, black smokers host living communities comparable to coral reefs in the richness of forms and abundance of biomass. At the base of the food chain, there are chemosynthetic bacteria that aerobically oxidize the sulphides using oxygen dissolved in seawater: H2 S þ 2O2 ! SO4 2 þ 2Hþ þ energy S2 þ 2O2 ! SO4 2 þ energy Bacterial mats growing all around the vents are “grazed” by small invertebrates (mainly amphipod and copepod crustaceans and gastropod molluscs), which in turn are predated by other animals including cephalopods and fishes. Particularly striking is

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the adaptation of Siboglinida, annelid worms that obtain food directly from chemosynthetic bacteria growing within their modified gut (Hilàrio et al. 2011; Fig. 3.1). The discovery of black smokers led German chemist Günther Wächtershäuser (1988) to suggest that similar environments were the incubators of life in the Hadean Earth. According to his model, the major source of organic compounds and energy required for starting life was the reaction of carbon monoxide (CO) with iron sulphide (FeS), two compounds present in the emanations of black smokers. The reaction can proceed spontaneously and produces a small amount of free energy (ΔG < 0) that in principle might promote reactions of organic synthesis. A second type of hydrothermal vents, currently known as “white smokers” or alkaline vents, was discovered in 2000 (Kelley et al. 2001). Characterized by white emissions and alkaline pH, these vents arise from the interaction of seawater with newly formed oceanic crust, and are much less hot than black smokers. Water penetrates through breaks in the basalt rock and chemically interacts with some of the minerals present. Known as serpentinization (Russell et al. 2010) this process produces hydrogen and methane by oxidizing ferrous iron. The reactions below show the balance of serpentinization of fosterite and fayalite, two common components of basalt, by water (Eq. 3.1) or water plus carbon dioxide (Eq. 3.2). forsteriteþfayaliteþwater ! serpentineþmagnetiteþhydrogen 9Mg2 SiO4 þ3Fe2 SiO4 þ14H2 O ! 6Mg3 Si2 O5 ðOHÞ4 þ2Fe3 O4 þ2H2

ð3:1Þ

forsteriteþfayaliteþwaterþcarbon dioxide ! serpentineþmagnetiteþmethane 18Mg2 SiO4 þ6Fe2 SiO4 þ26H2 OþCO2 ! 12Mg3 Si2 O5 ðOHÞ4 þ4Fe3 O4 þCH4

ð3:2Þ Being strongly exothermic, serpentinization raises the temperature of circulating water up to 260
C; in addition, the water is enriched in calcium/magnesium hydroxides and carbonates, thus becoming alkaline. In the process, substantial amounts of water bind to the rock, whose density changes from about 3.0 to 2.7 g/ cm3 with a concurrent volume increase of about 30–40%. Because of the pressure generated by the heat, a part of the water is forced to return to the surface, with a temperature ranging 40–90
C; in contact with ocean water, dissolved minerals precipitate, forming solid mounds of a mixture of silicates, clays, carbonates and sulphides with a finely compartmentalized structure (Fig. 3.2). More than chimneys, these formations resemble gothic cathedrals towering over 50 meters above the sea floor (Colín-García et al. 2016)., The field discovered in the mid-Atlantic Ocean in 2000, named Lost City, contained about 30 alkaline vents. Despite the name, the site hosts a living community nearly as rich and diverse as the ecosystems associated with black-smoker fields. The primary producers here are autotrophic archaea that synthesize methane from hydrogen and carbon dioxide; methanotrophic bacteria use methane as a source of organic carbon and support a food chain including numerous invertebrates (Kelley et al. 2005). After Lost City, other white-smoker fields have been discovered elsewhere in the planet.

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Fig. 3.2 Alkaline vents at Lost City. (A) active and (B) inactive vent. (From: National Oceanic & Atmospheric Administration (NOAA) Central Library. https://www.photolib.noaa.gov/htmls/ expl1170.htm)

Alkaline vents depend on newly formed oceanic crust for activity; once the reactive minerals are exhausted, the vents die out, but others develop on new oceanic crust. Because of this, active alkaline vents develop in the proximity to oceanic ridges but, unlike black smokers, are not directly associated with magma emission. Alkaline vents have an average duration of about 104 years, whereas black smokers last only a few hundred years. Both are geochemical systems far from thermodynamic equilibrium and exhibit other properties considered conducive to the evolution of life (Sleep et al. 2011): • A continuous supply of reactive compounds at a low reduction potential relative to the ocean water, mostly sulphides in black smokers, hydrogen, methane and minor amounts of hydrogen sulphide, ammonia, thiomethane (CH3SH), formate (HCOO), cianide (CN), and nitrogen oxide (NO) in alkaline vents; these conditions favours the formation of organic compounds from carbon dioxide (Amend et al. 2013). • An abundance of minerals containing elements with catalytic properties such as iron, nickel, molybdenum, tungsten, vanadium, cobalt; these elements are universally used in extant organisms for the catalysis of redox reactions (SchoeppCothenet et al. 2012; Nitschke et al. 2013). • Temperatures high enough to accelerate chemical reactions but compatible with the stability of organic molecules. • The possibility to discharge waste in the ocean, thus preventing unfavourable equilibria. Both types of vent produce precipitation structures where the issuing fluids encounter oceanic water. Black smokers, however, are largely characterized by high-temperature flow focussed in channels, whereas alkaline vents exhibit a more distributed flow of fluids at moderately high temperatures. The force driving the growth of black-smoker chimneys is thermal diffusion, whereas the growth of alkaline vents depends on much slower chemical diffusion. Additionally, water at

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high temperature contains higher concentrations of dissolved chemicals, which rapidly precipitate in contact with cold water at the ocean bottom. Because of this, black smokers form solid mounds with a scarcely porous inner structure, in which water flow is mainly channelled through large openings; in contrast, alkaline vents produce mounds with a porous microstructure and water flows slowly across myriads of apertures, with more prolonged interaction between the issuing fluid and ocean water (Cardoso and Cartwright 2017). The alkaline vents have additional properties theoretically conducive to the emergence of life, which are lacking in black smokers: • Mineral precipitation on pore walls forms membrane-like barriers with catalytic properties and selective permeability. Experiments simulating conditions likely existing in the early Earth (notably absence of oxygen and high levels of Fe and Ni dissolved in ocean water) have demonstrated the formation of active “membranes” of mackinawite (iron and nickel sulphide), greigite (Fe3S4), silica (SiO2) and fougerite (a complex of ferrous-ferric iron, hydroxide ions and carbonate also present in soil) (Russell and Hall 2006; Russell et al. 2010, 2013; Sojo et al. 2016). • There is a pH and redox potential gradient between the alkaline inside of the chimneys, rich in hydrogen and other reducing compounds, and the ocean water. This gradient was probably more pronounced in the Hadean Earth than today (at least four pH units), due to higher concentration of carbon dioxide. A pH gradient is not only a potential source of energy, but also influences the equilibrium of redox reactions involving protons (Box 3.1), for example the reduction of carbon dioxide to organic carbon by molecular hydrogen (Sect. 3.6). A gradient in pH and redox potential is also present in black smokers (whose emissions are strongly acid), but these have little or no compartmentation, thus preventing gradient coupling with endergonic reactions. Under thermal and/or ionic gradients simulating alkaline vent conditions, iron sulfide/ silicate films give appreciable yields of energy-rich pyrophosphate, an inorganic analogue of ATP (Barge et al. 2014). Because of their distinctive features, alkaline vents have been proposed as a setting for the birth of life, with inorganic micro-compartments behaving as “prebiotic cells”, and mineral catalysts promoting abiotic formation of a diversity of organic compounds (Martin and Russell 2003, 2007; Koonin and Martin 2005; Branciamore et al. 2009). In this scenario, the starting materials were hydrogen, carbon dioxide, ammonia and phosphate dissolved in the circulating water, and the necessary energy input was provided by gradients of pH and reduction potential across mineral interfaces in contact with ocean water (Lane et al. 2010; Russell et al. 2013; Sojo et al. 2016). Isotopic analysis has produced evidence of abiotic synthesis of simple organic compounds in Lost City hydrothermal fields (Proskurowski et al. 2008; Lang et al. 2010). In-vitro experiments simulating alkaline vent conditions demonstrated spontaneous formation of numerous organic compounds including amino acids and small peptides (reviewed by Colín-García et al. 2016). Particularly informative are experiments on simulated vent microcompartments containing nickel-doped mackinawite,

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a catalytic Fe(Ni)S mineral. Under prebiotic ocean conditions, these structures were able to catalyse the formation of simple organic molecules including formaldehyde from CO2 and H2, using energy from a proton gradient. In addition, they were able to catalyse abiotic synthesis of a range of sugars including ribose and deoxyribose (the sugar moieties of RNA and DNA) from added formaldehyde, and to concentrate the organic pigment fluorescein up to ~5000-fold (Herschy et al. 2014). Most remarkably, the formation of simple organic molecules from CO2 and H2, and the polymerization of free amino acids into peptides turned out to be exergonic, thermodynamically favoured reactions under hydrothermal vent conditions (Lemke et al. 2009; Amend et al. 2013). A theoretical approach has shown that the synthesis of cell mass (essentially amino acids, nucleotides, fatty acids and sugars) from H2, CO2, and NH3 in hydrothermal vent conditions is exergonic in the absence of oxygen (as most likely was the case on Hadean Earth), but extremely endergonic in the presence of even small traces of oxygen (Schönheit et al. 2016). The porous structure of the mounds of alkaline vents is conducive to accumulation of nucleic acids and other macromolecules under the action of a thermal gradient (Baaske et al. 2007). Computerized simulations showed that a temperature gradient across a dead-end channel may drive two entangled processes: (i) the solution moves up and down the channel by laminar thermal convection and (ii) the heavier molecules in solution remain behind and accumulate at one end of the channel, a phenomenon known as “thermophoresis”. Accumulation grows exponentially with the size of the molecules and the length of the channel. Under a thermal gradient of 30
C, a dead-end channel with a cross section of 145 μm and a length of 5 mm was estimated to accumulate 22-base-long RNA up to six-fold the original concentration, and 100-base-long double-stranded DNA up to 20,000-fold. The estimated accumulation for a 1000-base-long DNA was 1015-fold, a level comparable to DNA concentration in prokaryotic cells. These accumulations might further increase in a concatenated system of channels such as those present in the mounds of Lost City vents. Remarkably, the application of temperature gradients across a glass microcapillary containing the necessary chemical set was found to drive both replication and accumulation of short DNA molecules in a process called “convective polymerase chain reaction” (Mast et al. 2013).

3.3

A Primordial Role for RNA?

In living organisms, proteins, RNA and DNA mutually interact in a closed circuit in which each type of molecule depends on the others. Proteins catalyse the synthesis of RNA and DNA from nucleotides; RNA provides the information required for amino acid polymerization in cognate sequences; DNA provides the information for the synthesis of preordained sequences of RNA and for its own replication. It is extremely unlikely that these three polymers and their complex interrelationships emerged together from scrap. Unlike DNA, RNA is a flexible polymer that can produce a diversity of molecular architectures with alternating single- and

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double-filament regions and interconnecting loops (Hiller and Strobel 2011). The discovery that RNA not only carries genetic information (for example in RNA viruses) but is also able to function as a catalyser in certain reactions, led to the hypothesis that RNA preceded proteins and DNA in a pre-biotic evolutionary phase dubbed the “RNA World” (Robertson and Joyce 2012). Research has revealed several instances of RNA sequences with non-canonical functions that might be heritages of the putative “RNA World”. Among these are riboswitches, non-coding traits of mRNA that bind specific metabolites and may affect gene expression at the transcription or translation level (Breaker 2010). Even more interesting in the present context are ribozymes, RNA sequences that perform enzymatic functions. The best-known example of a ribozyme is the peptidyl-transferase activity responsible for the formation of the peptide bond between the amino acid and elongating peptide during protein synthesis. This catalytic activity lies in the large ribosome subunit and depends on a region of rRNA known as the peptidyl transferase complex (PTC), with no protein directly participating in the process. With a turnover rate of 10–20 peptide bonds per second and an error frequency of 104, the PTC has a catalytic efficiency comparable to that of many protein enzymes (Fox 2010; Moore and Steitz 2010). A second example of ribozymes is from “self-splicing” sequences that catalyse their own excision from longer RNA filaments. These include type-I and II introns present in mRNA, tRNA and rRNA precursors in all three domains, as well as hairpin and hammerhead ribozymes common in viruses but also found in plants and animals (Woodson 2005; Ferré D’Amaré and Scott 2010; Lambowitz and Zimmerly 2011). A third type of ribozyme is RNA in spliceosomes, which directly participates in the excision of spliceosomal introns in precursors of messenger RNAs, a type of introns unique to eukaryotes (Will and Lührmann 2011). It is worth noting that mono or di-nucleotides such as ATP, GTP, coenzyme-A, NAD and FAD, which perform essential functions in all living organisms, also are possible remnants of a “RNA world”. Although RNA may have enzymatic properties, no RNA is known that can replicate autonomously, namely without the assistance of a protein enzyme (e.g. a RNA-dependent RNA polymerase). Several research groups have tried to produce self-replicating RNA molecules by natural selection. Random RNA sequences were copied through numerous replication cycles mediated by a RNA-dependent RNA polymerase; with time, many variant copies were produced due to copying errors. The products were tested at intervals and molecules with catalytic properties were selected for further cycles of replication and selection. This process eventually led to the identification of RNA sequences that were able to function as a RNA ligase, i.e. to bind together short RNA sequences by phosphodiester bonds. A self-replicating system was assembled using two of these ribozymes, each able to produce copies of the other by linking together four shorter sequences in the right order (Yoice 2009). This remarkable result highlights the evolutionary potential of RNA, yet no ribozyme has been found so far with the catalytic properties of a protein RNA replicase, viz. the ability of copying RNA templates using single nucleotides as precursors.

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Besides phosphoryl transfer (the cleavage and/or ligation of the RNA phosphodiester backbone) and peptide bond formation, the catalytic repertoire of ribozymes includes RNA aminoacylation, a reaction analogous to amino acid activation by aminoacyl~tRNA synthetases (Chumachenko et al. 2009; Yarus 2011). Proteins are by far more versatile catalysers than RNA because they have a much greater diversity of functional groups, including alkyl, hydroxyl, carboxyl and amino groups. In addition, RNA monomeric units (the nucleotides) are relatively complex molecules made of three components (a base, a sugar and phosphoric acid) and their synthesis in experimental abiotic systems occurs with very low yields. Geochemically plausible pathways have been described for the abiotic synthesis of pyrimidine (cytosine and uracil) but not purine nucleotides (Sutherland 2010). Nam et al. (2018) have reported spontaneous synthesis of purine and pyrimidine ribonucleosides within aqueous microdroplets containing phosphoric acid, ribose, nucleobases and magnesium ions as a catalyser. The authors suggested that alignment at the air–water interface of microdroplet surfaces permitted the reactants to overcome the thermodynamic barrier for these condensation reactions. Formamide generated from ammonia and formate can behave as a precursor for abiotic synthesis of nucleic acid bases and purine acyclonucleosides (i.e with the sugar in non-cyclic form) in conditions simulating alkaline vents. In the same set of experiments, pre-formed nucleosides were spontaneously phosphorylated by phosphate minerals to cyclic nucleotides, and guanine- and adenine 30 , 50 cyclic nucleotide monophosphates spontaneously polymerized into chains with a length of up to 30 nucleotides (Saladino et al. 2012a, b). Despite these encouraging results, a major theoretical problem comes from the tendency of RNA to hydrolyse spontaneously into free nucleotides in alkaline solution, which would prevent accumulation of abiotic RNA in alkaline vents (Bernhardt 2012). In fact, experiments simulating conditions in alkaline hydrothermal vents using circular ribonucleotides, imidazole-activated ribonucleotides with montmorillonite catalyst, or ribonucleotides in the presence of lipids only managed to produce RNA oligomers up to four units in length (Burcar et al. 2015). In contrast, Da Silva et al. (2015) report mononucleoside polymerization into RNA chains with a length of 20–100 units, upon exposition to multiple cycles of hydration-dehydration at elevated temperatures and in presence of monovalent salts, once again suggesting the involvement of molecular alignment at water/air interfaces. These results, as well as similar results by Nam et al. (2018), appear to be more consistent with “organic soup” models than the hydrothermal vent hypothesis, and might inspire research for mechanisms of molecular alignment at interfaces in hydrothermal vents. A second concern in modelling early steps of life evolution raises from intrinsic chemical instability of ribose in solution. It has been proposed that ribose in prebiotic systems was stabilized by adsorption on borate-containing minerals. The occurrence of borates such as colemanite, ulexite and kernite at pre-biotic times (over 4 GYA) depends on whether a granitic continental crust had already differentiated (Sect. 2.5), because in its absence there would be too little borate in the ocean (Grew et al. 2012).

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Why RNA (and consequently DNA Sect. 3.7) only uses four bases? As observed by Maynard Smith and Szathmáry (1995), assuming that the active site in ancestral ribozymes was a string of four monomers (as typical of extant ribozymes and protein enzymes as well), the number of different active sites that a ribozyme of four different monomers could make was 44 ¼ 256. By comparison, the number of active sites in proteins is 420 ¼ 160,000. In both cases, these numbers can significantly increase by post-polymerization modification of monomers, but the principle is clear: the efficiency and versatility of ribozymes could increase dramatically if more than four bases were used. Using more bases, however, would increase the mutation rate and decrease replication fidelity. We may thus conclude that the fourbase genetic alphabet was the cost/benefit optimum choice adopted by natural selection.

3.4

The Genetic Code

There could be no cell, however rudimentary, without a genetic code, so this must have evolved at a pre-biotic stage. The origin of the genetic code is one of most perplexing problems in evolutionary biology, so much so that little novel insight has emerged in the 20 years after Maynard Smith and Szathmáry (1995) reviewed the topic, despite spectacular progress in molecular biology. In living organisms, the amino acid sequence of proteins assembled by ribosomes mirrors the nucleotide sequence of messenger RNAs (mRNAs) under the rules of the 64-triplet code. The link is established by “transfer” RNAs (tRNAs) that function as adaptors. The most critical step in the process is the binding of free amino acids at the CCA end of cognate tRNAs, namely tRNAs bearing a nucleotide triplet (anticodon) complementary to a triplet (codon) for the same amino acid. Aminoacylation of tRNA is catalysed by aminoacyl~tRNA synthetases, a family of enzymes with medium specificity (the rate of mis-aminoacylation is one in 104 cases, implying a frequency of error of 3% for a protein long 300 amino acids). The code is virtually universal among extant life forms and is therefore known as the Standard Genetic Code (SGC); although many deviations from the SGC exist, particularly in organelles and prokaryotes with small genomes, these are limited in scope and obviously secondary in origin (Maynard Smith and Szathmáry 1995; Koonin and Novozhilov 2017). The design of the translation system in even the simplest modern cells is extremely complex. At the heart of the system is the ribosome, a large complex of at least three RNA molecules and 60–80 proteins arranged in a precise spatial architecture and interacting with other components of the translation system in a most finely choreographed fashion. Other essential components include the complete set of tRNAs for the 20 amino acids (only about 40 tRNA species, due to the general occurrence of isoacceptor tRNAs), a set of aminoacyl~tRNA synthetases, and a complement of at least 7–8 translation factors (Smith et al. 2008; Fox 2010; Moore and Steitz 2010).

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More simple, albeit less efficient solutions must have preceded such a complex biosynthetic system. The observation that aminoacyl~CCA complexes (CCA is the three-nucleotide tail shared by all tRNAs) can participate in peptide bond formation in ribosomes suggests that protein synthesis began as a non-coded process and that the tRNA adaptors were a late addition introduced when the system was able to produce coupling enzymes (aminoacyl~tRNA synthetases) with the necessary specificity (Fox 2010). Maynard Smith and Szathmáry (1995) suggested that the ancestral aminoacyl~tRNA synthetases were ribozymes, later replaced by protein enzymes; this hypothesis has recently received support from the observation that in-vitro evolved short RNA molecules are able to catalyse RNA aminoacylation (Chumachenko et al. 2009; Yarus 2011). The structure of the code is non-random and ensures high robustness to mutational and translational errors. For example, for most codons, the third base may be one of the two purines (adenine and guanine) or one of the two pyrimidines (uracil and cytosine) without changing the meaning. This suggests that the primordial genetic code was based on couples of nucleotides, which allowed for 16 possible codons (the number of combinations with repetition of four elements in groups of two is 42). Thus, the amino acids initially used in peptide synthesis might have been less than 20, the others being co-opted later, when a more complex metabolism had developed and the translation system had become more efficient. Ten amino acids are consistently produced in prebiotic chemistry experiments, in the following order of relative abundance: glycine, alanine, aspartic acid, glutamic acid, valine, serine, isoleucine, leucine, proline, tyrosine. This order reflects the free energy of their synthesis, the first being those thermodynamically more stable. The same amino acids, with the same relative abundances, occur in meteorites. Several lines of evidence suggest that these ten amino acids are older than the others, in the sense that they were probably present in the first proteins whereas the others were not (Koonin and Novozhilov 2017). With the addition of a third nucleotide, stereospecificity and the accuracy of synthesis increased, but the possible combinations became 64 (43). As the number of amino acids deployed in protein synthesis was ancestrally set to 20, the genetic code underwent “degeneracy”, with most amino acids being assigned two or more codons. Code degeneracy had the positive effect of alleviating the consequences of point mutations (mutations that substitute a single nucleotide for another). A further increase in the number of encoded amino acids would enhance the diversity and structural versatility of peptides chains but would probably also lessen the accuracy of translation, so setting the number to 20 was probably the optimal solution between the two opposite effects (Maynard Smith and Szathmáry 1995). Codes based on codons of four or more nucleotides would imply a greater level of degeneracy, therefore enhanced resilience to point mutations, and would increase stereospecificity, but arguably they would also be slower and more expensive than shorter codification systems. The final solution selected by evolution, a code based on 64 triplets, succeeded as the best possible compromise, thus becoming universal.

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The debate on the origin and evolution of the genetic code currently revolves around four competing perspectives. (i) The stereochemical hypothesis suggests that direct affinity between single amino acids and codons (or anticodons) played a pivotal role in primordial translation before being replaced by the extant indirect mechanism. (ii) The coevolution model proposes that the code structure coevolved with amino acid biosynthesis pathways. (iii) The error minimization scenario assumes that the code emerged from selection to minimize adverse effects of point mutations and translation errors. (iv) The frozen accident idea holds that the standard code has no special properties but was fixed simply because all extant life forms share a common ancestor, with subsequent re-assignment of codons generally precluded by deleterious effects on protein structure. These four perspectives are not mutually exclusive, thus adding further complexity to the problem. For example, mathematical analysis has shown that the SGC is more robust than approximately every million randomly chosen codes. Nevertheless, the SGC is far from being the best possible: given the astronomical number of codes that are theoretically possible with three-base codons and four bases (>1084), there are billions of variants more robust than the actual universal code. We can infer that, once attained a sufficient level of reliability, the SGC was irreversibly fixed in evolution, becoming in part a “frozen accident” (Koonin and Novozhilov 2009, 2017; Facchiano and Di Giulio 2018). Modern rRNAs and tRNAs are chiral molecules containing D ribose, and during translation they work together to make chiral proteins exclusively with L-amino acids. This is highly advantageous to modern organisms because mixed chirality would interfere with self-organization of structural motifs such as α-helices and β-sheets, which are fundamental in modern proteins. Structural analysis of the ribosomal peptidyl transferase complex indicates that the chirality of the sugar ring in RNA is well paired with the choice of L-amino acids, possibly being another instance of “frozen accident” (Fox 2010). As already observed, homochirality is not only a property of proteins but virtually applies to any biological compound that may exist in isomeric forms. In modern living systems, biological molecules are the products of enzyme-catalysed chemical reactions, and homochirality is maintained by stereospecific interaction with the active site of enzymes. There is no reason to exclude that enzymes working with different stereoisomers of the same compound may appear by mutation. Indeed, mutations affecting enzyme stereospecifity do certainly occur. Mutant enzymes of this sort, however, are quickly eliminated by selection because their products cannot interact with other enzymes in the metabolic network. Was a similar constriction present in pre-biotic systems? The answer is “probably yes”, if these were to evolve a well-integrated metabolic network. Initially, the choice between enzyme/substrate variants was probably random; yet, selection had to choose one working isoform and eliminate the other(s) to avoid competition and futile cycles, thus making homochirality a general property of the living world. Once again, it is important to remember that this did not involve a sort of hindsight: alternative competing solutions disappeared because of lower efficiency or simply by chance.

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For a more detailed analysis of the different scenarios mentioned above and supporting evidence, the reader is invited to consult the excellent reviews by Wolf and Koonin (2007) and Koonin and Novozhilov (2009, 2017).

3.5

A RNA-Protein World

In contrast to nucleotides, spontaneous amino acid synthesis is relatively easy in the conditions existing in alkaline vents and, due to the thermal gradient between the inside and outside, amino acids may theoretically accumulate in micro-cavities of hydrothermal edifices to concentrations conducive to polymerization. Numerous mechanisms have been proposed for prebiotic amino acid polymerization, but experimental evidence is still poor. The data, however, supports the hypothesis that Hadean alkaline vents accumulated a diversity of peptides (short proteins) of abiotic origin, with random lengths and sequences. Amino acid polymerization initially proceeded spontaneously with low yields, but it probably accelerated over the time, due to the appearance of peptides with catalytic properties (Lemke et al. 2009; Colín-García et al. 2016; Kitadai and Maruyama 2017). Because nucleic acid synthesis is more complex than peptide synthesis and nucleic acids alone are instable, the pre-biotic phase probably encompassed a period with peptides but no nucleic acids, followed by a period with both polymers but still without a genetic code (Milner-White and Russell 2011). Pre-code peptides were probably small, simple and heterochiral in nature. Although theoretically not limited to the 20 amino acids in current proteins, amino acid occurrence was governed by ease of abiotic synthesis, thus most likely with a preponderance of glycine and a few other amino acids. Because of heterochiral composition, pre-code peptides probably lacked regular architectures such as the α-helices and β-sheets present in modern proteins, and were more variable and motile in their three-dimensional structure. In addition, because of the lack or sporadic occurrence of amino acids with prominent side chains, peptide reactivity depended on the CONH backbone rather than amino acid side chains. Interestingly, metal-, metalsulphide- and phosphate-binding structural motifs of many modern proteins only employ main chain atoms, suggesting that these motifs first appeared in pre-code peptides. Milner-White and Russell (2011) propose that precode peptides chelated inorganic clusters and took over the roles of minerals as compartment walls and catalysers. For example, abiotic peptides might have replaced mineral catalysers in the synthesis of pyrophosphate, trimetaphosphate or acetylphosphate, all analogues of ATP. Allegedly, these peptides deployed the pH gradient between the internal alkaline fluid and acidic ocean water as a source of energy, and might have been ancestral to pyrophosphatases, proton pumps universally present in extant organisms (Box 3.1) (Koonin and Martin 2005; Russell and Hall 2006; Mulkidjanian et al. 2009; Lane and Martin 2012; Russell et al. 2013). Spontaneous formation of ATP analogues might have favoured the synthesis of nucleotides and nucleic acids from free precursors under catalysis by pre-biotic peptides. For the sake of simplicity, we assume that the first nucleic acid in prebiotic

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systems was RNA; it is quite possible, however, that primordial nucleic acids were similar but not identical to RNA (“XNA”), and that RNA took over at a later stage (Schrum et al. 2010; Robertson and Joyce 2012; Yu et al. 2012). Thanks to its negative charge, RNA formed complexes with peptides rich in basic amino acids, the association probably providing mutual protection from chemical degradation and disassembly (Kovacs et al. 2017). Today, proteins and RNA are the major components of living systems, accounting for about 50% and 25% of their dry weight, respectively (excluding cellular envelopes and storage materials). Proteins and RNA form the ribosomes, universal components of cells, and other complexes displaying remarkable properties such as the spliceosomes and RNA viruses. Comparing three-dimensional structures of ribosomes across the tree of life, Petrov et al. (2015) reconstructed a stepwise increase in rRNA folding. An analogous analysis of ribosomal proteins points to a parallel increase in protein folding complexity (Kovacs et al. 2017). Based on the assumption that the frequency of random protein sequences that are competent to fold to domains is very nearly zero, these studies suggests that protein-RNA co-evolution favoured the emergence of structured protein domains. In an attempt to convert the observations above into an operative model, we propose that a primary step on the way to life was the appearance of ribonucleoprotein complexes (RNPs) with the ability to catalyse peptide synthesis from free amino acids, and RNA synthesis from free nucleotides or shorter RNA fragments. Working cooperatively, these catalytic systems could produce peptides and RNA that spontaneously assembled into new complexes, thus falling under the action of Darwinian selection for more efficient self-replication (Koonin and Martin 2005). Because of this, RNP evolution probably favoured certain peptide and RNA sequences from the very beginning, although no mechanism was initially in place linking peptide to RNA sequences. A second critical step was possibly the appearance of RNA replicases, enzymes capable of catalysing nucleotide polymerization into RNA sequences mirroring the sequence of a pre-existing RNA template. No RNA with this enzymatic property is known, nor attempts at producing such a type of RNA by in vitro evolution (Sect. 3.2) have met with success, thus it is reasonable to assume that RNA replicases have been proteins from the beginning. Why did a genetic code evolve? Arguably, natural selection favoured abiotic systems routinely producing peptides that were able to bind RNA and build new RNPs similar to pre-existing ones. Any improvement in the accuracy of peptide synthesis that enhanced the replication of the complexes was therefore subject to strong positive selection. Once a number of functional sequences were established, the genetic code became unmodifiable: any change in the meaning of a triplet would cause major variations in the sequence of all the proteins codified, therefore the likely extinction of all RNPs. With time, RNA templates increased in number and sizes by co-opting new sequences through primitive recombination processes, thus producing novel, more complex peptides.

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The scenario drafted above postulates the emergence, at a pre-cellular stage of evolution, of RNP complexes with the following properties: • Type-1 RNPs that polymerized amino acids into non-coded peptides; • Type-2 RNPs that polymerized nucleotides into RNA whose sequence was loosely determined by pre-existing RNA. Under the action of natural selection for increased efficiency in cooperative replication (Koonin and Martin 2005), type-1 and type-2 RNPs respectively evolved into: • Type-3 RNPs that produced peptides with a sequence determined by cognate RNA, under the rules of a genetic code; the core component of these complexes was RNA with peptidyl transferase activity. • Type-4 RNPs that replicated RNA templates from free nucleotides, under the rules of base complementarity; the core component of these complexes probably included a peptide with RNA replicase activity and the RNA sequence encoding this enzyme. Assuming a low level of fidelity in RNA replication, the mutation rate was initially very high; in addition, the RNP complexes were probably able to break up into fragments and re-aggregate, thus producing myriads of new variants by recombination. Arguably this entailed extremely high evolution rates, yet it is most unlikely that the four types of complexes described above evolved in the same vent, because alkaline vents remain active only for some thousand years, which is presumably too short a time span to cover such a complex evolutionary transition. More likely, type-1 and type-2 complexes evolved several times independently and were able to “colonize” new vents by moving with ocean currents, possibly bound to organic-mineral pellicles or to mound fragments. Migration enabled the complexes to persist longer than the “life time” of a single vent. When type-1 and type-2 complexes found themselves in the same system, they co-operated with mutual benefit and with time evolved into type 3 and type 4 RNPs. Type-3 RNPs were able to make peptides from free amino acids under the control of RNA templates, therefore we call them protoribosomes; the peptides so produced self-assembled with RNA (including the peptidyl transferase complex) to build new protoribosomes. Type-4 RNPs replicated RNA molecules that self-assembled with peptides including a RNA replicase, thus forming new copies of type-4 complexes. In a way, type-4 RNPs were similar to RNA viruses, so we call them protoviruses. Pervasive horizontal gene transfer in pre-biotic systems was an essential part of early evolution, without which the transition to the cellular level of complexity would have probably never occurred. At the same time, HGT was a powerful factor selecting for a universal code, the persistence of different codes being incompatible with integration of genetic information from different sources (Sengupta et al. 2014).

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With the appearance of protein-synthesizing protoribosomes and RNA-replicating protoviruses linked to each other by a genetic code, for the first time a change in RNA templates could be translated into a novel protein structure. Depending on each other, these complexes could survive only by establishing stable associations, possibly binding to organic-mineral pellicles coating the micro-compartments of host vents; besides stabilizing the complexes, this facilitated simultaneous migration of genetically interlinked complexes to new vents. Thus, prior to the emergence of life, protocell-like structures were more like ecological communities than organisms, with gene-like elements shifting regularly between quasi-cells (Sterelny 2011). The postulated mobility of RNPs was an essential factor in pre-biotic evolution. As observed by Koonin and Martin (2005), the development of new compartments during the growth of vent chimneys and geological formation of new vents can be viewed as the abiogenic predecessors of cell division; likewise, colonization of new vents by mutually replicating RNPs was analogous to a viral infection. With the appearance of the first cells, protoribosomes that had adopted the Standard Genetic Code evolved into ribosomes, whilst protoviruses evolved into gene-storing complexes ancestral to chromosomes. The protoviruses possibly also generated the first RNA viruses, although viruses most likely evolved multiple times independently (Forterre 2006). The nucleotide sequence of rRNA includes a common core that is the structural and functional foundation of rRNAs of all extant life (Bernier et al. 2018), possibly a legacy of a pre-biotic phase of evolution. According to Kovacs et al. (2017), the primary driving force for early protein evolution was association with RNA in (proto)ribosomes, free catalytic proteins (enzymes) being a late by-product of this process. We suggest that the first enzymes were RNPs, possibly evolved from protoribosomes or protoviruses, whose possible descendants are the plethora of modern enzymes that use nucleotides (e.g. NAD and FAD) as co-factors. Two fundamental components were still necessary for making a cell: a bounding membrane and an autonomous metabolism.

3.6

Biological Membranes

Far from being a simple bag for living matter, the cell membrane is a highly selective barrier that controls the inflow and outflow of substances. Most importantly, the cell membrane is able to build electrochemical gradients and use them for making ATP or for transporting solutes against their electrochemical gradient (Box 3.1). Because of this, any attempt at reconstructing the evolution of life should address not only the emergence of a genetic system and a metabolism, but also the origin of cell membranes. Besides an enveloping membrane, many cells have internal membranes sharing the same basic organization. In the following discussion, therefore, we will make use of the general term “biological membranes”, or simply “membranes”.

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Cells do not make new membrane by merely putting together single molecules from naught, as a bricklayer does when making a brick wall. The cells can only expand preexisting membranes by adding new molecules one by one into their framework, and then pinch off them into separate compartments. This is the way membranes are transmitted to daughter cells during cell division. Thus, much like chromosomes, biological membranes are hereditary structures (Cavalier-Smith 2001, 2004). The basic components of membranes are amphipathic lipids, molecules consisting of a hydrophilic (polar) and hydrophobic (non-polar) part. Interacting with water, these lipids spontaneously arrange into bilayers that isolate the hydrophobic parts in the inside, exposing the hydrophilic parts superficially on either side (Chen and Walde 2010; Fig. 3.3). Besides lipids, biological membranes contain amphipathic proteins that insert in the lipid bilayer according to the same principle: the hydrophobic parts in the inside and hydrophilic ones exposed on one or both surfaces (Fig. 3.4). An important property of membranes is that they always form closed compartments. Mechanically fragmented membranes extracted from living cells and suspended in water seal spontaneously, forming vesicles of varying size with an inside isolated from the outside. If amphipathic lipids are capable of spontaneously organizing into bilayers in an aqueous environment, why do living cells produce new membrane only from preexisting membranes? There are several reasons. The first is a contingent reason: since cells need to have a (continuous) bounding membrane, the best way to ensure that daughter cells receive a cell membrane is to expand the pre-existing one before cell division. The same applies to intracellular membranous compartments. Besides, biological membranes have intrinsic structural traits, including bilayer asymmetry, which are essential for their functions and are not reproducible without a pre-existing template. A mechanistic reason is that the enzymatic system responsible for lipid synthesis is an integral component of membranes that directly inserts the lipids into the membrane framework. The insertion of integral proteins is more complex as these must be synthesised on ribosomes, which lie in the cytosol. Proteins are inserted in the membranes by special translocons named SecYEG in bacteria and archaea (du Plessis et al. 2011), and Sec61 in eukaryotes (Nyathi et al. 2013). The Sec systems in prokaryotes and eukaryotes display structural homologies, indicating an origin from an ancestral system present in the common progenitor (LUCA). Sec translocons recognize the proteins destined to be inserted in or translocated across a membrane from a signal sequence that is the first part of the chain emerging from the ribosome during synthesis. The signal sequence mediates adhesion of the ribosome to a target membrane and “instructs” the Sec traslocon to insert the elongating protein within the membrane, or to drive it across the membrane. The membrane that separates the internal cellular environment from the outside is generally knowns as the cell membrane or plasmalemma. As already said, the cells often have other membranes besides the cell membrane. For example, Gram-negative bacteria (the largest and most diverse group of bacteria) have a second membrane external to the cell membrane. In addition, the cyanobacteria have a

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Fig. 3.3 Polar lipids can spontaneously form micelles and bilayered vesicles in water suspensions. This behaviour depends on water expelling the lipid hydrophobic tails from its molecular network. The vesicles are able to grow by merging with other vesicles or incorporating lipid micelles. They may divide by mechanical deformation (due for example to energetic mixing of the suspension) or by thermodynamic instability when they grow above a threshold size

system of inner membranes named “thylakoids”, which host the molecular complexes responsible for photochemical conversion (Chap. 4). Particularly complex is the endomembrane system in eukaryotic cells, which encompasses the endoplasmic reticulum (ER), the Golgi apparatus and numerous other membrane-bound compartments (Schlacht et al. 2014). These membranes share the same basic structure, but differ in composition and function. Cavalier-Smith (2001, 2004) makes a distinction between “genetic” (or “hereditary”) membranes and “nongenetic” membranes. The former are membranes that necessarily arise from membranes of the same type, the latter secondarily derive from other types of membranes. The most obvious example of a genetic membrane is the cell membrane, which is transmitted to daughter cells during cell division; other examples are the outer membrane in Gram-negative bacteria, thylakoids in cyanobacteria, the ER and perhaps the Golgi system in eukaryotes. Examples of non-genetic membranes are the

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Fig. 3.4 Diagrammatic representation of a biological membrane. Phospholipids are orderly arranged in a bilayer, exposing their hydrophilic heads on the surface, whereas the hydrophobic tails are segregated in the oily inside. Integral proteins are inserted in the lipid bilayer according to the same principle. This type of structure is called a fluid crystal, because the molecules are orderly arranged yet at the same time are relatively free to move along the horizontal plane; in contrast, vertical movement (from one layer to the other) is strongly constrained. Peripheral proteins are linked to the surface of membranes without entering the hydrophobic inside of the lipid bilayer. Consisting of two molecular layers, biological membranes have a thickness of only 5–8 nanometres, depending on the length of lipid hydrophobic chains, yet they are extremely efficient barriers to molecular diffusion

nuclear envelope and the bounding membranes of vacuoles, lysosomes, endosomes and other types of vesicles, all arising from ER or Golgi. If by hypothesis a genetic membrane is lost, for example the ER in a eukaryotic cell or the thylakoids in a cyanobacterium, the cell would no longer be able to produce it. The ER is essential in eukaryotes and its loss would be lethal, but a cyanobacterial lineage is known (Gloeobacter) that lacks thylakoids and uses the cell membrane for photosynthesis. If membranes are essential to life and cells can make them only from pre-existing membranes, how did membranes evolve? Describing alkaline vents, we have mentioned minerals with catalytic properties that tend to precipitate and form thin pellicles coating the walls of micro-compartments within the chimneys. Theoretical models suggest that at an early stage of precellular evolution, peptides of abiotic origin bound to mineral pellicles and modified their properties. Indeed, the addition of peptides and RNA to the solution circulating in a simulated hydrothermal vent was found to modify the elemental stoichiometry of the chimneys, perhaps epitomizing the putative takeover of abiotic metabolism by organic polymers (McGlynn et al. 2012). Under the repulsive force of water, peptide-mineral pellicles might have incorporated amphipathic lipids, isolating their hydrophobic tails from water circulating on either side. It is not necessary to postulate the existence of lipids as complex as those forming modern membranes, simple long-chain organic acids or monoglycerides being more likely precursors in a prebiotic world (Mulkidjanian et al. 2009; Mansy 2010; Schrum et al. 2010; West et al. 2017).

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Current models postulate that protocells used organic-mineral membranes to harness chemiosmotic energy (Box 3.1) of geochemical origin (Russell and Hall 2006; Lane and Martin 2012; Russell et al. 2013; Sousa et al. 2013). For these systems to become independent free-living cells, their bounding membrane needed two major additions: (i) incorporation of a ion-pumping mechanism for autonomously producing electrochemical gradients, and (ii) incorporation of the machinery for membrane growth, viz. lipid-synthesising enzymes and the protein insertion machinery. These changes converted the protomembrane into an “encoded membrane” (Lane and Martin 2012), a hereditary structure vertically transmitted from cell to cell. Most likely, the Last Universal Common Ancestor had a bounding membrane containing all fundamental components of modern membranes including an ATP synthase (Box 3.1), and the Sec protein insertion machine (Jekely 2006). Reiterating Virchow’s classic aphorism “omnis cellula e cellula” (every cell from a cell), cell biologist Günter Blobel (1980) wrote “omnis membrana e membrana”, every membrane from a membrane. Membranes are so fundamental to life that in some models membrane-bound protocells and a rudimentary form of membrane heredity are assumed to predate RNA and genetic heredity (West et al. 2017). Because life is almost certainly monophyletic, this implies that all membranes existing today, however modified and specialized, arise from one ancestral membrane. Yet, because of major divergences in membrane architecture between the archaea and the other organisms, some researchers have proposed a dual origin. We will return on this issue in Sect. 3.8. Putative self-replicating RNPs probably bound to membranes and, as suggested for organic-mineral pellicles, this might have facilitated vent-to-vent migration of these complexes. A legacy of this ancestral association is still evident in extant cells. In prokaryotes, the chromosome remains linked to the cell membrane throughout the cell cycle, the site of attachment controlling the separation of sister chromosomes during cell division (Toro and Shapiro 2010). In eukaryotes, chromosome association with the nuclear envelope controls the spatial arrangement of chromatin during interphase; in addition, in many eukaryotes the nuclear envelope persists during mitosis and the chromosomes remain bound to it throughout the cell cycle (Box 6.3).

Box 3.1: Chemiosmosis Biological membranes are responsible for a fundamental property of life: the creation of ion gradients and their use as a source of energy. British biochemist Peter Mitchell described the underlying principle in 1961 and called it chemiosmosis. Confirmed by subsequent investigation, the chemiosmotic model earned Mitchell the Nobel prize for chemistry in 1974. Chemiosmosis consists in the creation of a gradient of hydrogen (H+) or, in some cases, sodium (Na+) ions across the membrane and its use as a source of energy to make ATP from ADP and phosphate (Deamer and Weber 2010; Schoepp-Cothenet et al. 2013). Before the discovery of chemiosmosis, (continued)

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Box 3.1 (continued) biochemists thought that the only way for cells to make ATP was substratelevel phosphorylation (SLP), a process that produces ATP or GTP by direct transfer of a phosphoryl (PO32) group to ADP or GDP from another phosphorylated compound. SLP is convenient because it is a very fast process, yet it is little efficient because it does not permit cells to utilize all the energy potentially available in substrates. Like the genetic code and membranes, chemiosmosis is a universal property of life, possibly emerged even before the evolution of true cells. The use of protons is a convenient choice as these ions are produced by water dissociation, thus being present wherever water is; other small ions such as sodium ions may work as well as protons and in fact are used for chemiosmosis instead of protons in some prokaryotes. The proton (or sodium) electrochemical gradient needed to support chemiosmosis is built using energy released from the transfer of electrons from a chemical species to another. The energetic variation associated with these reactions is: ΔG0 ¼  nFΔE0 where: ΔG0 is the free standard energy of the reaction, in joules per mole (J/mol); the minus sign means that the reaction produces energy (i.e. ΔG0 < 0) when the electrons move from a molecule with a lower reduction potential to one with a higher reduction potential; n is the number of moles of electrons transferred per mole of molecules oxidized; F is the Faraday constant, about 96,500 coulomb/mole; ΔE0 is the difference between the standard reduction potential of the oxidant (the electron acceptor) and reductant (the electron donor). As an example, let us consider the reaction: NADH þ Hþ þ ½O2 ! H2 O þ NADþ where: n ¼ 2 (1 mole of NADH donates 2 moles of electrons). the standard reduction potential (at pH 7) of the couple oxygen/H2O is +0.81 V, the standard reduction potential (at pH 7) of the couple NAD+/NADH is 0.32 V. (continued)

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Box 3.1 (continued) Therefore: ΔG0 ¼ 2  96,500ð0:81 þ 0:32Þ ¼ 2  96,500  1:13 ¼ 218:000 J=mole ¼ 218 kJ=mole: Standard redox potentials are of course only first approximations of the free energy available in a given electrochemical disequilibrium between the electron donor and electron acceptor. Mass-action ratios, pH-values, number of transferred electrons all play a role in determining the true electrochemical ΔG exploitable by an organism at a given time and in a specific growth environment (Schoepp-Cothenet et al. 2013). The reduction potential of chemical species participating in reactions involving the transfer of protons besides electrons decreases by about 0.06 V for each pH unit above zero. For example, the half-reaction O2 + 4H+ + 4 e/2H2O has a standard reduction potential of +1.23 V at pH 0, but only +0.81 V at pH 7, whereas the reference half-reaction 2H+ + 2e/H2 has a standard reduction potential of 0.41 V at pH 7 (all values being relative to a standard hydrogen electrode at pH 0, which is conventionally assigned a potential of 0.0 V). Because the inside of cells has a pH close to 7 (with the exception of some specialized compartments), biochemists generally use standard potentials at pH 7, usually indicated with the symbol E00 . It is useful to remember that the oxidation potential is equal to the reduction potential changed of sign, thus the equations above are valid also for oxidation potentials but it is necessary to invert the position of standard potentials. As for pH, the reduction potential is an important physiological parameter subject to strict homeostatic control. The reduction potential is also a major environmental parameter that influences the distribution and metabolic activity of living organisms, from bacteria to plants and animals. Enzymatic complexes located in membranes couple exergonic redox reactions to proton translocation across the membranes; because protons are electrically charged, this accumulates electrochemical energy in the form of a difference of electrical potential (Vm, expressed in volts) and a difference of proton concentration (in pH units) between the two sides of the membranes. The overall energy stored by this mechanism is referred to as the proton motive force (PMF) and is expressed in volts (V) according to the equation: PMF ¼ 0,0591ΔpH þ Vm where ΔpH is the pH difference and Vm the electrical potential difference between the two sides of the membrane. (continued)

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Box 3.1 (continued) Thus, electron flow from organic substrates to oxidants such as oxygen in aerobic respiration, sulphate, nitrate or nitrite in anaerobic forms of respiration, drives protons across membranes against electrochemical gradient. The cell membrane of a bacterium or a mitochondrion, with a thickness below 10 nm, can build a PMF of about 0.18 V, equivalent to an electric field of about 180 million V/m, comparable in strength to lightning-generating fields that develop during storms. Other PMF-building processes include the oxidation of inorganic molecules such as hydrogen, sulphide ions or Fe+2 ions in lytoautotrophic bacteria, and electron flow in photosynthesis (Chap. 4). In some cases (e.g. acetogenic bacteria and methanogenic or halophilic archaea) Na+ ions are used instead of protons to build an electrochemical gradient, but the general principles underlying the process are the same (Sousa et al. 2013; Ducluzeau et al. 2014). The membranes usually have very low permeability to protons; the only way protons can flow downhill along their electrochemical gradient is through integral protein complexes known as proton pumps. There are several families of proton pumps. Two of these, the A-ATP synthases and F-ATP synthases, produce ATP when crossed by protons under physiological conditions, although in appropriate experimental conditions they may work the other way round. The A-ATP synthases occur in the archaea and in some cases use sodium ions instead of protons; the F-ATP synthases occur in bacteria and eukaryotes. A third family of proton pumps known as V-ATPases occurs in eukaryotes; under physiological conditions these consume ATP to generate PMF (Müller and Grüber 2003; Jefferies et al. 2008; Junge and Nelson 2015). All these proton pumps are multi-protein complexes consisting of a Fo domain embedded in a membrane and a F1 domain protruding superficially. They operate with a mechanism of “rotational catalysis” described by Paul Boyer, Nobel laureate for chemistry in 1997, and subsequently defined in detail (Nakanishi-Matsui and Futai 2008; Junge and Nelson 2015). When crossed by protons, the Fo domain rotates, thus forcing a conformational change in the F1 domain. This reacts by binding ADP and phosphate into ATP (A- and F-ATP synthases, Fig. 3.5) or hydrolysing ATP into ADP and phosphate (V-ATPases). Looking at the F1 domain from above, the rotation is anticlockwise in F-ATP synthases, clockwise in V-ATPases, but becomes clockwise in F-ATP synthases forced to work as ATPases by inverting the pH gradient across the host membrane. The direction of rotation in A-ATP synthases has yet to be determined. A- and F-ATP synthases require the translocation of 3 or 4 protons for each ATP molecule produced, but different stoichiometric ratios have been reported in some cases. The data currently available suggest that the V-ATPases translocate two protons for each ATP molecule hydrolysed; this stoichiometry is thermodynamically favourable to (continued)

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Box 3.1 (continued)

Fig. 3.5 Diagram showing the functioning of an F-ATP synthase. This enzymatic complex uses the PMF built by redox reactions (in blue) to produce ATP

ATP hydrolysis rather than ATP synthesis, and enables the V-ATPases to function in the acidification of intracellular compartments or of the extracellular medium. Besides a similar molecular architecture, the A, F and V pumps shows sequence homologies in some of their protein components, suggesting a shared origin from an ancestral complex present in the Last Universal Common Ancestor. Archaeal A pumps and eukaryotic V pumps are more similar to each other than each is to bacterial F-pumps, not only in protein sequence but also for the shared presence of a “C subunit” lacking in F-pumps. Because of these similarities, sometimes archaeal ATP synthases are reported as VATPases. The occurrence of V-ATPase DNA sequences in the genome of some bacteria is ascribed to horizontal gene transfer. Eukaryotic F-ATP synthases are localized in the mitochondrial cristae (Kühlbrandt 2015) and chloroplast thylakoid membranes (Junge and Nelson 2015), in both cases having a bacterial ancestry because these organelles arise from endosymbiotic bacteria (Chaps. 6 and 9). Measures of ATP/2e ratios and H+/2e ratios suggest that three protons are translocated for each ATP molecule produced in mitochondria, and four in chloroplasts. Emerging structural data, however, call into question the assumption that the stoichiometry is the same in all organisms and even that the number of protons utilized per each ATP molecule is an integer. (continued)

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Box 3.1 (continued) V pumps only occur in eukaryotes (apart from the likely instances of horizontal gene transfer mentioned above), being usually localized in the bounding membrane of intracellular compartments such as the lysosomes and vacuoles, but in some cases also in the cell membrane, e.g. in mammal kidney cells and osteoclasts (Beyenbach and Wieczorek 2006; Jefferies et al. 2008). Besides A, F and V pumps, two other families of pumps are known: the P-ATPases or P pumps (Bublitz et al. 2011; Palmgren and Nissen 2011), and proton-pumping pyrophosphatases (H+-PPases) (Baltscheffsky et al. 1999). The P-ATPases are a large family of single-protein pumps present in all three domains, which transport protons and other ions against electrochemical gradient using energy from ATP hydrolysis. The suffix “P” indicates that while functioning these pumps are temporarily linked to a phosphate group; by irreversibly binding to P-pumps in the place of phosphate, the vanadate ion behaves as a specific inhibitor of P-ATPases. P-ATPases are localized in the cell membrane in prokaryotes, both in the cell membrane and inner membranes in eukaryotes. Eukaryotic P-ATPases encompass at least five subfamilies whose members specifically transport ions (H+, Na+, K+, Mg2+, Ca2+, Ag+ e Ag2+, Zn2+, Co2+, Pb2+, Ni2+, Cd2+, Cu+ e Cu2+). A sixth subfamily of P-ATPases, known as “flippases”, transport phospholipids such as phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine from one layer of the membrane to the other. The main function of P-ATPases in prokaryotes seems to be the expulsion of potentially noxious heavy metals such as copper, lead or nickel. P-ATPases in eukaryotes have a central role in building the membrane potential (Vm) needed to support active secondary transport and excitability (e.g. in neurons and muscle cells). In plants and fungi the Vm is mainly built by proton-pumping P-ATPases, whereas in the animals the Vm mainly depends on the work of P-ATPases that transport sodium and potassium ions in opposite directions (the so-called sodium-potassium pumps). Of utmost importance in eukaryotes also are calcium pumps, a heterogeneous group of P-ATPases that maintain cytosolic calcium ions at very low concentration, thus permitting the cells to use these ions for signal amplification. Some eukaryotes use P pumps for the uptake of metals needed for metabolism or, as in prokaryotes, for heavy-metal detoxification. Proton-pumping pyrophosphatases (H+-PPases) are single-polypeptide integral proteins that transport protons across membranes against an electrochemical gradient using energy from the hydrolysis of pyrophosphate ions. These are dimers of phosphoric acid generated as by-products in numerous metabolic reactions; the use of pyrophosphate for building PMF permits the recovery of energy still present in the anhydride bond between the two phosphate groups, which otherwise would be lost. Genes for H+-PPases have been found in the (continued)

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Box 3.1 (continued) genome of bacteria, archaea and eukaryotes. The seemingly universal distribution and relatively simple structural design of H+-PPases suggest that these are the most ancient type of proton pumps (Seufferheld et al. 2011). Chemiosmosis permits the cells to store energy from redox reactions in the form of an electrochemical gradient that can be used to make ATP. Protein phylogenomics strongly favours chemiosmosis as life’s primordial way to harvest free energy (Ducluzeau et al. 2014). As already mentioned, the cells also use substrate-level phosphorylation (SLP) to couple exergonic redox reactions to ATP synthesis. For example, in the metabolic pathway known as glycolysis, the oxidation of 3-phosphoglyceraldehyde to 3-phosphoglycerate and subsequent conversion of the latter to pyruvate are coupled to the synthesis of two ATP molecules. ATP is an easily expendable energy currency but has a serious problem: there is only one note available, equivalent to about 40 kJ (10 kcal) per mole. To understand what this implies, let us imagine that all currency in circulation suddenly disappears except € 10 notes: we would be forced to pay € 10 also for less valuable goods, thus losing the difference (the same would happen if we paid by credit card if there were no currency units smaller than € 10). In biochemical terms, if cells only used SLP for making ATP, a lot of energy would be wasted. For example if a reaction produces 75 kJ/mol, it might be coupled to the synthesis of only one mole of ATP, storing about 40 kJ and losing the rest as heat. Likewise, a reaction producing 39 kJ could not be coupled to the synthesis of ATP. Chemiosmosis is more efficient than SLP because it uses energy units much smaller than ATP, i.e. single ions. Indeed chemiosmosis is the only way to make a live from reactions with very low energy yields, such as methane synthesis from hydrogen and carbon dioxide, a reaction with a ΔG0 as low as 10 kJ/mol or even less. The PMF produced by chemiosmosis is a source of energy not only for ATP production but also for transporting solutes against their electrochemical gradient (active secondary transport). The PMF can even support electron flow against a reduction potential, permitting the production of stronger reductants from weaker ones; this process, known as reverse electron transport is vital to several microorganisms, for example autotrophic methanogenic archaea (Sect. 5.2) and photosynthetic bacteria with a type-2 photosystem (Sect. 4.1) (Nelson and Cox 2017). Electron bifurcation is a recently recognized mechanism of energy conservation that adds up to chemiosmosis and substrate-level phosphorylation. Electron bifurcation simultaneously couples endergonic and exergonic oxidation-reduction reactions to circumvent thermodynamic barriers whilst minimizing free-energy loss (Buckel and Thauer 2013). As an example, electrons derived from hydrogen are simultaneously used to reduce low-potential ferredoxin and a still unknown higher potential acceptor in methanogenesis and acetogenesis, reduced ferredoxin being necessary for carbon dioxide assimilation (Lane and Martin 2012).

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From Geochemistry to Biochemistry: The Emergence of an Autonomous Metabolism

Metabolism (from Greek metabolè, change) is the set of life-sustaining chemical transformations that enable the organisms to produce building materials and to obtain energy. As for the informational system, it is necessary to postulate that a primitive form of metabolism predated life. Active oceanic vents present today on the Earth are rich with life, thus it is extremely difficult to produce experimental evidence for abiotic production of organic compounds. As already mentioned, radiometric investigation in nature, data from artificial systems and theoretical models suggest that, in the conditions presumably existing in alkaline vents in the Hadean Earth, the pH gradient across mineral pellicles supported the synthesis of energy-rich molecules such as pyrophosphate (HP2O73) and trimetaphosfate (H3P3O103). In addition, experimental evidence suggests that reductants of geochemical origin such as hydrogen, hydrogen sulphide and ferrous iron reacted with carbon dioxide and produced a diversity of organic molecules that accumulated in pre-biotic microcompartments by thermophoresis (Sect. 3.1) and/or by binding to mineral surfaces (Martin and Russell 2003, 2007; Proskurowski et al. 2008; Lang et al. 2010; Russell et al. 2013; Sojo et al. 2016; Branscomb and Russell 2018a). Proponents of the alkaline vent hypothesis (Branscomb and Russell 2018b) have vigorously rebutted criticism based on thermodynamic arguments (Jackson 2017; Ross 2018). Arguably, the appearance of peptides with catalytic properties vastly increased the diversity, abundance and complexity of organic molecules of abiotic origin. Bound to mineral surfaces, these polymers might have worked co-operatively with inorganic catalysers, generating metabolic networks of increasing complexity. The suite of metal-enzymes present in extant organisms, such as ferredoxin (iron-sulphur), cytochromes (iron), plastocyanin (copper), carbonic anhydrase (zinc), nitrogenase (molybdenum), urease and hydrogenase (nickel) may be modern descendants of peptides associated with metal catalysers at a prebiotic stage of evolution (Nitschke et al. 2013). Two further steps towards the emergence of an autonomous metabolism were the appearance of integral membrane peptides capable of enhancing the production of high-energy phosphates (pyrophosphate, metaphosphate, acetyl-phosphate, or ATP) and of peptides capable to build PMF (Box 3.1) using energy from chemical reactions. The former were probably ancestral to ATP synthases, the latter to modern PMF-building redox systems (Mulkidjanian et al. 2009; Lane et al. 2010; Lane and Martin 2012; Herschy et al. 2014; Lane 2017). The notion that energy harvesting in early life was chemiosmotic is virtually uncontested, yet it is debated whether the underlying ion gradient was ancestrally based on protons or Na+ ions (Mulkidjanian et al. 2008; Ducluzeau et al. 2014). As long as early life was associated with hydrothermal vents, chemiosmotic energy was freely available in the form of natural geochemical gradients. Current scenarios suggest that protocells harnessed energy from the disequilibrium between the acidic

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Fig. 3.6 According to the alkaline-vent model of the origin of life, proton-driven nanomachines made ATP or ATP analogues by harnessing the natural proton gradient between circulating alkaline solution and acidic ocean water. (Adapted from Lane 2017, license number 4458200716683)

ocean water and the alkaline solution circulating in vents (Fig. 3.6), implying that an ancestral ATP synthase predated active PMF-building mechanisms (Lane and Martin 2012; Sousa et al. 2013; Martin and Sousa 2016). Early membranes were probably leaky to protons. As long as a natural geochemical gradient was available, protocells could retain leaky membranes, which prevented the establishment of a Donnan equilibrium, viz. the rise of an electrical potential that would stop proton flow (Sousa et al. 2013). However, not all areas in vents provided strong enough gradients, and consequently selection pressure for adaptation to “poorer” areas (or aging vents) favoured the evolution of autonomous PMF-building mechanisms; this required a radical reduction in membrane permeability to protons, which was conducive to the establishment of a Donnan equilibrium. According to an elegant model by Sousa et al. (2013), the solution to the physiological conundrum faced by early life in alkaline vents was the evolution of a H+/Na+ antiport channel that coupled an endergonic outward flow of Na+ ions with exergonic inward flow of protons in a 1/1 ratio. By flowing backward from the outside to inside, Na+ ions fed an ancestral Na+-dependent ATP synthase. Only protocells that adopted this solution were able to make the transition to the freeliving style and leave the hydrothermal environment. The hypothesis explains why intracellular Na+ concentration is extremely low in all extant organisms, and is consistent with the fact that acetogenesis and methanogenesis (considered primordial

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metabolic pathways, see below) use a Na+-dependent instead of proton-dependent ATP synthase to convert chemiosmotic power into ATP. In the “primordial soup” scenario almost universally accepted until the end of the past century, early life was generally assumed to be heterotrophic (reviewed in Martin and Sousa 2016). With remarkable hindsight, in 1995 Maynard Smith and Szathmáry argued that life must have been ancestrally autotrophic. At high H2 activities as observed in seafloor vents and under strictly anaerobic conditions, the synthesis of cell mass from CO2 is thermodynamically favoured. By analogy with metabolic pathways in extant acetogenic bacteria (Eq. 3.3) and methanogenic archaea (Eq. 3.4), both strictly anaerobic, the current mainstream scenario holds that early life used hydrogen of geochemical origin to reduce carbon dioxide to acetate or methane through the acetyl-CoA pathway (Lane and Martin 2012; Sousa et al. 2013; Schönheit et al. 2016). 4H2 þ2HCO3 2 !CH3 COOþ2H2 O

ð3:3Þ

4H2 þCO2 !CH4 þ2H2 O

ð3:4Þ

Interestingly, this pathway not only permits cells to convert carbon dioxide into organic compounds (intermediates of the pathway in methanogens), but also to store energy in the form of a chemiosmotic gradient (Poehlein et al. 2012). A deep ancestry of the acetyl-CoA pathway is supported by phylogenetic analysis of the enzymes involved, such as the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-CoA synthase reducing CO2 to CO, the molybdo/tungstopterin proteins reducing CO2 to a formyl moiety, and [NiFe] hydrogenases extracting electrons from hydrogen (Sousa et al. 2013; Schoepp-Cothenet et al. 2013). Methanotrophy has been proposed as an ancestral carbon-fixing metabolism alternative to the acetyl-CoA pathway (Russell and Nitschke 2017). Present-living methanotrophic bacteria (α- and γ-proteobacteria) sequentially oxidise methane into methanol, formaldehyde, formate, CO2, utilizing oxygen as an electron acceptor (Eq. 3.5). CH4 þ2O2 !CO2 þ2H2 O

ð3:5Þ

These bacteria are autotrophic because they are able to use intermediate compounds of the pathway as carbon sources. The hypothesis is captivating, as serpentinization (Sect. 3.1) produces substantial amounts of methane that methanotrophs could use as a substrate. Hadean Earth, however, had only negligible amounts of free oxygen, and the atmosphere/ocean/crust system had much a lower average reduction potential than today (Chap. 5). A major problem for early life, therefore, was not in finding a suitable electron source (hydrothermal vents are rich

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sources of sulphides and other potential reductants besides hydrogen and methane), but strong enough oxidants. Aerobic methanotrophy could evolve only after the Great Oxygenation Event, about 2.4 GYA (Chap. 5). Anaerobic methane-oxidizing archaea living in syntrophic consortia with sulphate-dependent methanotrophic bacteria have been discovered in anoxic marine sediments (Knittel and Boetius 2009); more recently, a bacterium and an archaeon have been identified that couple anaerobic methane oxidation to nitrite or nitrate reduction, respectively, without the need for a syntrophic partner (Ettwig et al. 2010; Haroon et al. 2013). Until the appearance of molecular oxygen there was very little sulphate or nitrate/nitrite in the ocean, but paleogeochemical inference suggests that substantial amounts of nitrogen oxides could arise from reaction of atmospheric molecular nitrogen with carbon dioxide under the effect of lightning or meteorite impacts. Dissolved in the Hadean ocean, these compounds could be used as electron acceptors by early life, reforming molecular nitrogen (Ducluzeau et al. 2009; Wong et al. 2017). An alternative potential oxidant was ferric iron (Fe+3) generated by photo-oxidation of ferrous iron (Fe+2) by solar ultraviolet in surface ocean, with concomitant production of hydrogen, lost to space; being scarcely soluble, ferric ions precipitated to the ocean bottom, thus becoming available to early life for redox processes (Nitschke and Russell 2013; Russell et al. 2013). The spread of ancestral autotrophic life likely permitted ecological differentiation of heterotrophic life at some stage in evolution; ancestral heterotrophs could use waste organic compounds or dead cells as a substrate, most likely occupying marginal niches as long as life remained dependent on hydrothermal vents. Amino acid fermentation could not evolve under high partial pressure of hydrogen because of thermodynamic constraints (Schönheit et al. 2016). Non-fermentative heterotrophic pathways using nitrite or ferric iron (Fe+3) of abiotic origin as oxidants were possible alternatives. Several pathways are known that convert inorganic carbon into organic molecules besides the acetyl-CoA pathway, the most important one being the Calvin-BensonBasham cycle (Chap. 4). Unlike the acetyl-CoA pathway, which generates PMF, all alternative carbon-fixing pathways do consume PMF (at least in the form of ATP). In addition, for building PMF, these pathways employ a class of proton transporters named quinones (e.g. ubiquinone and plastoquinone), which do not participate in the acetyl-CoA pathway. Recent phylogenomic work depicts LUCA as an anaerobic, CO2-fixing, N2-fixing, H2-dependent and thermophilic organism (Weiss et al. 2016). Despite similar catalytic properties and partial homology of some enzymes, notably CO dehydrogenase, ferredoxin and soluble hydrogenases, the acetyl-CoA pathways in acetogenic bacteria and methanogenic archaea display major biochemical divergences. This is considered as evidence of early divergence of bacteria and archea (Martin and Russell 2007; Lane and Martin 2012; Sousa et al. 2013; Sojo et al. 2014).

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DNA Replaced RNA as the Repository of Biological Information

A network of entangled metabolic pathways and the genetic system evolved together. Evolutionary pressure for novel and more efficient protein catalysts promoted the diversification of RNA templates, or “replicators”. The persistence of numerous independent replicators enhanced recombination, as occurs still today in some viruses, but also increased the risk that cooperative sequences were separated and potential synergies dispersed (Maynard Smith and Szathmáry 1995). Aggregation of smaller replicators into larger molecules was positively selected because it favoured (a) the synthesis of novel, larger and more versatile peptides, and (b) coordinate expression, replication and transmission of functionally related sequences (Koonin and Martin 2005). At a point, the replicators attained sizes that raised problems of instability: being the RNA filament highly flexible, it tended to spontaneously break and/or to fold into structures that interfered with translation or replication. Thanks to its double-helix structure and the absence of the oxygen atom in the position 2 of the sugar (deoxyribose, instead of ribose), DNA is much more stable that RNA. This permits the use of DNA traces for crime investigation, or genomic analysis of fossils (Chap. 12). A further advantage of the double helix structure is that the two strands carry the same information, yet only one is actually decoded, the other being used for correcting errors during replication, thus affording the genetic system enhanced stability. As for other major innovations, the transition from RNA to DNA occurred gradually, for example first utilizing double-filament RNAs for information storage and replication, and single filament RNAs for expression, and then separating the two functions completely with the use of different nucleotide precursors: deoxyribonucleotides for double-filament replicators and ribonucleotides for single filament “messengers”. The separation also involved replacement of uracil with thymine in the replicator, thus reducing the risk of mutation due to the tendency of cytosine to convert into uracil by deamination; a misplaced uracil cannot be recognized in RNA, whereas it can be pinpointed as an alien base in DNA and removed by repair systems (Forterre et al. 2004). The adoption of DNA increased the efficiency of storage, replication and transmission of genetic information and activated a strong evolutionary pressure towards functional aggregation of genes. Less “co-operative” genes were unavoidably suppressed, favouring the emergence of gene communities, or genomes, with increasing efficiency in achieving genes’ only goal, perpetuation. The scenario presented for the emergence of life may now be resumed in the following sequence of events: 1. simple amino acids from abiotic synthesis ! peptides (accumulated by thermophoresis and binding to mineral “membranes”) 2. peptides + mineral “membranes” ! high-energy molecules (e.g. pyrophosphate) and nucleotides

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nucleotides ! RNA (random polymerization catalysed by peptides) RNA + peptides ! ribonucleopeptides (RNPs) Random RNPs ! mutually replicating RNPs (protoribosomes and protoviruses) Protoribosomes + protoviruses ! catalytic peptides (enzymes) ! increased metabolic complexity 7. Peptides + polar lipids + mineral “membranes”) ! liquid-crystal biological membranes 8. Biological membranes + protoribosomes + protoviruses + free enzymes ! protocells 9. RNA replicators ! DNA replicators ! first cells (Last Universal Common Ancestor). 3. 4. 5. 6.

3.9

The Bacterial-Archaeal Divide: Ancestral or Derived?

The transition from RNA to DNA was probably pivotal to the structural and functional compaction that permitted the evolution of metabolically autonomous, self-replicating cells no longer linked to the inorganic scaffold of vent chimneys. Based on membrane chemical composition, present-living organisms split into two assemblages: bacteria and eukaryotes on one side, archaea on the other. In both, the main membrane lipids are glycerophospholipids, amphipathic molecules made of glycerol bound to two long hydrophobic “tails” and to a phosphate group. Apart from this commonality, glycerophospholipids in the two assemblages could not be more different. In bacteria and eukaryotes, glycerol is in the L form, the hydrophobic tails are linear aliphatic chains bound to glycerol at the sn-1 and sn-2 positions (!Glossary) by ester bonds, and phosphate is bound at the sn-3 position. In the archaea, glycerol is in the D form, the hydrophobic molecules are isoprenoid chains bound at the sn-2 and sn-3 positions by ether bonds, phosphate is bound at the sn-1 position (Shimada and Yamagishi 2011; Fig. 3.7). Two independent non-homologous metabolic pathways of isoprenoid synthesis are known: the mevalonate (MVA) pathway in eukaryotes, a modified MVA pathway in archaea (Boucher et al. 2004), and the methylerythritol phosphate (MEP) pathway in bacteria. Photosynthetic eukaryotes obtained the MEP pathway through the chloroplast. The occurrence of the MVA pathway in some bacteria has been traditionally interpreted as the result of horizontal gene transfer (Box 1.1) from eukaryotic or archaeal donors. In contradiction with this claim, phylogenomic evidence indicates that the MVA pathway ancestrally occurs in all three domains and was probably present in the last common ancestor of extant life (Lombard and Moreira 2011). The real difference between the archaea and the other two domains is in the synthesis of glycerol from dihydroxyacetone phosphate. Bacteria and eukaryotes use a glycerol-3-P (G3P) dehydrogenase that produces glycerol-3-phosphate; in contrast, the archaea use a glycerol-1-phosphate (G1P) dehydrogenase that produces glycerol1-phosphate. The two enzymes have no sequence homology apart from weak similarities in the NADH-binding domain (Peretò et al. 2004; Lombard et al. 2012).

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Fig. 3.7 (1–5) Glycerolipid structure in the archaea: (1) isoprenoid chains, (2) ether bonds, (3) Dglycerol, (4) phosphate group. The isoprenoid chains are bound to glycerol at the sn-2 and sn-3 positions by ether bonds, phosphate is bound at the sn-1 position. (5–8) Glycerolipid structure in bacteria and eukaryotes. (5) Linear fatty acid chains, (6) ester bonds, (7) L-glycerol, (8) phosphate. The fatty acid molecules are bound to glycerol at the sn-1 and 2 positions by ester bonds, phosphate is bound at the sn-3 position. (9) Bilayered membrane organization as typical of bacteria and eukaryotes. (10) Unilayered membrane of tetraether lipids in hyperthermophilic archaea. Non-hyperthermophylic archaea have bilayered membranes as in bacteria and eukaryotes, but their glycerolipids have archaeal stereochemistry. From: https:// commons.wikimedia.org/wiki/File:Archaea_membrane.svg

The evolutionary significance of the membrane divide between G3P lipid stereochemistry in bacteria and eukaryotes and G1P in Archaea is uncertain. The current mainstream debate is focused on two competing scenarios. The first holds that the bacteria and archaea diverged from a common ancestor that either lacked true membranes (Martin and Russell 2003, 2007) or possessed heterochiral membranes made of both G3P and G1P lipids (Wächtershäuser 2003; Peretò et al. 2004; Lombard et al. 2012; Koga 2014). After splitting, the bacteria evolved/retained a G3P membrane stereochemistry, the archaea evolved/retained a G1P stereochemistry, the choice being driven by adaption to different environmental conditions. Under this scenario,

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the eukaryotes are viewed as a lineage derived by “fusion” of an archaeon and a bacterium, the latter being converted into the mitochondrion (Chap. 6). Cellular integration of the two symbionts ostensibly involved the loss of archaeal G1P lipid biosynthetic machinery and its replacement with the symbiont’s G3P machinery, thus explaining why eukaryotes have bacterial-type G3P membranes. The hypothesis of a heterochiral common ancestor for archaea and bacteria is consistent with the demonstration that heterochyral membranes not only are stable, but are even more resistant to high temperature (in terms of proton permeability) than homochiral G3P membranes (Shimada and Yamagishi 2011). Importantly, the Sec protein translocon is universal in the three domains, indicating that the last common ancestor did have a true membrane (Jékely 2006). The alternative scenario, mainly sponsored by Cavalier-Smith (2014), maintains that the three life domains derive from a common ancestor with G3P stereochemistry. In this perspective, the bacteria and eukaryotes retained G3P membranes, whereas the archaea secondarily evolved G1P membranes by adapting to hyperthermal acidic habitats. Like the competing hypothesis, this scenario implies an intermediate phase in which G3P and G1P lipids must have co-existed, but provides a strong selection pressure to explain the transition from one to the other. The archaeal domain typically encompasses hyperthermophilic and acidophilic forms with optimum temperature above 80
C and optimum pH below 3, yet mesophilic (optimum temperature in the 20–45
C range) and even psychrophilic archaea (20 to +10
C) are also known. The paradox is only apparent, as archaeal membranes are stable at high temperature but remain fluid and functional also at very low temperatures. A unique trait of hyperthermophilic archaea is that their membranes consist of only one layer of bipolar lipids with a tetraether structure (Fig. 3.6). These special membranes are more stable and less permeable to protons that doublelayered membranes in conditions of high temperature and low pH. Molecular evidence points to hyperthermophyly and underpinning tetraether G1P membranes as the ancestral condition in archaea (Gribaldo and Brochier-Armanet 2006). In this perspective, archaeal lineages with bilayered membranes are viewed as derived lineages that lost tetraether lipids by adapting to less extreme habitats, but retained isoprenoid G1P stereochemistry (Cavalier-Smith 2014). This scenario has received support from molecular work by Williams et al. (2017). Hyperthermophyly is also present among bacteria, and at least two bacterial lineages (the Thermotogales and Aquificales) possess tetraether lipids; these lipids, however have G3P stereochemistry and are made with non-isoprenoid fatty chains, thus they most likely evolved independently of archaeal tetraether lipids (Schouten et al. 2007; Glansdorff et al. 2008). Hyperthermophyly was suggested to be an ancestral trait of the Bacteria domain, possibly inherited from LUCA (Di Giulio 2003). Phylogenetic analysis by Lake et al. (2009) does not support a hyperthermophilic root of the tree of life. A phylogenetic analysis based on ribosomal proteins resolves the Thermotogales and Aquificales as sister groups and places them in a derived position in the bacterial tree (Yutin et al. 2012). In contrast, a more recent analysis based on a large dataset from metagenomic sequencing places these

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lineages in a basal position, thus supporting the view that hyperthermophyly is an ancient trait in the Bacteria (Schulz et al. 2017). Some archaea possess a G3P dehydrogenase besides a G1P dehydrogenase, whereas some bacteria have a G1P besides G3P dehydrogenase, both occurrences being interpreted as due to horizontal gene transfer (Peretò et al. 2004). The Firmicutes, a gram-positive group of bacteria, possess a geranylgeranylglyceryl phosphatase, the enzyme responsible for the formation of the ether bond in archaeal lipids, probably acquired from an archaeon by horizontal gene transfer (Valas and Bourne 2011). Spontaneous racemization (viz. the conversion of the L isoform into the D isoform) of aspartic acid residues in proteins increases exponentially with temperature, thus obliging microorganisms living at high temperatures to continuously replace their proteins to maintain enzymatic activity (Onstott et al. 2014). This is a major constraint limiting life at high temperature, its recognition providing robust evidence against hyperthermophyly as an ancestral trait of life.

3.10

Concluding Remarks

Life as we see it today is a highly refined product of billions of years of evolution, yet it retains traits that probably appeared in a very early phase of its history. Transition metals, ribozymes and proton gradients are likely legacies of a pre-cellular stage intermediate between the living and non-living world. A particularly remarkable example is carbon monoxide dehydrogenase/acetyl CoA synthase, the key enzyme of the acetyl CoA pathway for carbon fixation (Sect. 3.6); this is is an unusual hydrogenase with three Fe4S4 clusters, two of which bridge via sulphide to nickel. The appearance of the first cells – self-reproducing systems with a bounding membrane, a genetic code and a metabolism – was a crucial, irreversible step that put an end to pre-biotic evolution, as cells were immensely more efficient than any precellular system in exploiting environmental resources. After that, the chance for independent emergence of novel forms of life on Earth was cancelled forever, as preexisting life would immediately out-compete them. The same probably applies for hypothetical extra-terrestrial life reaching the Earth in the form of resting cells. The model presented here assumes that self-replicating molecules and a rudimentary metabolism appeared independently, and that life emerged when a genetic code linked them together, thus giving rise to the genotype/phenotype interdependence that is the very essence of life. The notion of living organisms as mortal vehicles for immortal genes as popularized by Richard Dawkins (1976) is a useful metaphor that helps catching the way natural selection works, yet biological inheritance implies not only the transmission of genes but also of the biochemical machinery necessary for their functioning. In order to replicate, genes must fit in a cell, a structured and predictable environment that depends on genes for maintenance and reproduction but cannot be built by genes from scratch. The long phase of prebiotic evolution was necessary not only to produce self-replicating molecules but also to accommodate

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Concluding Remarks

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them in a reproducible house: the cell. The transgenerational transmission of a cellular organization is thus the most ancient expression of ecological inheritance, or niche construction, the ability of life to modify the environment and transmit the changes to next generation (Laland et al. 2016). The following chapters deal with other remarkable instances of niche construction, from the oxygenation of the planet to colonization of land by plants and animals, culminating with the emergence of humans. A second point worth of attention is the relatively low chemical diversity of living systems. Estimates of the total number of carbon-based compounds with molecular masses in the same range as those of living systems, i.e. below 500 daltons, give numbers in excess of 1060. Life actually uses only a tiny fraction of this potential “chemical space” (Dobson 2004). The first explanation that comes to mind is that life chemistry is still focused on ancestral molecular diversity, viz. the few types of compounds of abiotic origin wherefrom life started and their close derivatives. This is unlikely. Under positive selection, billions of years of evolution would have certainly produced much greater chemical diversity. The most likely explanation stems from the fact that, for thermodynamic reasons, cells are crowded systems containing high concentrations of molecules, either free or in the form of polymers. This raises the problem of spontaneous, uncontrolled reactions that may interfere with metabolic pathways and/or damage key cellular components. Thus, life evolved under strong chemical constraints, being obliged to exclude a large number of potentially noxious molecular architectures. Even keeping chemical diversity under tight control, life had to accept compromises, a most prominent example being that of glucose. This is a six-carbon sugar with an aldehyde functional group, universally employed as a substrate in respiration. The highly reactive aldehyde group can easily be blocked by glucose polymerization (e.g. by conversion into glycogen), but the free form tends to react with amino groups in proteins, causing structural damage. Presumably, glucose emerged as the most abundant and widespread natural monosaccharide because it is less reactive with proteins than other monosaccharides. Damage to proteins exposed on cellular surfaces from high glucose concentration in the blood is at the origin of diabetes in humans. Is life a rare phenomenon, possibly a unique trait of Earth as Jacques Monod (1971) advocated, or a likely, almost necessary occurrence (Russell et al. 2013; Hazen 2017) whenever and wherever the right conditions happen to develop? Answering this fundamental question requires a better knowledge of other planets, first several candidates in our own system. Exploring Mars for traces of extinct or extant life is an important objective at hand in the near future. By now, let us continue our journey on Earth. According to the reconstruction presented, life was originally dependent on geochemical energy accessible at special spots associated with volcanic activity. For leaving the original birthplace and conquering the habitable zone of Earth completely, life had to find a more reliable and widespread source of energy. Next chapter deals with this fundamental transition.

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Chapter 4

Moving to the Light: The Evolution of Photosynthesis

Last night, after a day in the garden, I asked Robin to explain (again) photosynthesis to me. I can’t take in this business of eating light and turning it into stem and thorn and flower. . . (Mary Rose O’Reilley, The barn at the end of the world: the apprenticeship of a quaker, buddhist shepherd)

Abstract Photosynthesis enabled early life to severe its ancestral dependence on geochemistry. The paleogeochemical record suggests that photosynthetic life colonized the planet photic zone as early as 3.4 GYA. Photosynthesis evolved in the Bacteria domain, and initially utilized compounds of geochemical origin such as ferrous iron or hydrogen as sources of electrons, without producing oxygen. Several variants of anoxygenic photosynthesis are present in extant bacteria. The cyanobacteria evolved oxygenic photosynthesis, a pathway that deploys two types of photosystem working in series to sum the energy of two photons for each electron transported from water to carbon dioxide. Multiple sources of evidence suggest that the cyanobacteria and oxygenic photosynthesis appeared at least 2.7 GYA, viz. 300 MY before the stable oxygenation of the planet. Endosymbiosis horizontally transferred oxygenic photosynthesis to the eukaryotes. Major similarities in the molecular architecture of photosystems in extant bacterial lineages point to a monophyletic origin of the core photosynthetic machine, followed by horizontal transfer among distantly related taxa, duplication and neo-functionalization. The Archaea lack photosynthesis but independently evolved a phototrophic pathway based on rhodopsins. Among a diversity of metabolic pathways for inorganic carbon fixation, the RubisCO-based Calvin, Benson and Bassham cycle is by far predominant.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_4

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4 Moving to the Light: The Evolution of Photosynthesis

Introduction

Although lacunose and mostly hypothetical, the evolutionary scenario presented in the previous chapter shows how theoretical analysis combined with experimental insight may help to set the transition from inanimate matter to the first cells into a solid framework. According to the model currently favoured, early life was associated with submarine vents and was dependent on row-matter and energy of geochemical origin. This did not prevent a degree of freedom, which probably permitted these ancient organisms to move with ocean currents and colonize new vents, thus escaping extinction. Nevertheless, life might never have left submarine depths and spread in the habitable zone of the planet without becoming independent of geochemical power. Whilst life inhabiting ancient vents grew more efficient and competitive, selection pressure for novel energy sources must have become inescapable. The solution found was obvious and, together, extremely innovative: the conversion of light energy into chemical energy, a process known under the general term “phototrophy”, from Greek fotos, light, and fagein, to eat, literally “light eating” (Bryant and Frigaard 2006; Butterfield 2015). Electromagnetic energy from the Sun is the most abundant and widespread form of energy accessible on the surface of Earth. About half of the total is associated with wavelengths between 400 and 700 nm; the radiations within this narrow range are named “light” as they are visually perceived by the animals, including humans (Fig. 4.1); the radiations deployed by life as a source of energy are much in the same range. The transition from a metabolism based on geochemical energy to a metabolism based on light energy is probably second in importance only to the evolution of the genetic code. Phototrophy is by far the most important form of energy harnessing used by life on Earth, the alternative way being chemotrophy, which deploys energy produced by oxidation of organic or inorganic molecules from the environment. The simplest known form of phototrophy uses light energy for making ATP by a chemiosmotic mechanism. The process is mediated by rhodopsins, proteins localized in the cell membrane and covalently attached to a carotenoid named retinal. This form of phototrophy is typical of archaea living in hypersaline environments (Bryant and Frigaard 2006). Rhodopsins structurally and functionally akin to archaeal rhodopsin also occur in some bacteria, including marine proteobacteria (Béjà et al. 2000, 2001). Rhodopsin-like proteins are employed in animals for light perception and, in combination with protein G, for light signal transduction. The most important and widespread form of phototrophy is photosynthesis, a process that uses light energy to convert inorganic carbon (either carbon dioxide or its hydrated form, the bicarbonate ion HCO3
) into organic carbon. In chemical terms this is a reduction, so the process needs a source of electrons besides energy.

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Fig. 4.1 Spectra of solar direct radiation at the top of Earth’s atmosphere (yellow band) and at sea level (red band). The sun produces electromagnetic radiation with a distribution similar to that expected from a blackbody at 5525 K (5250  C, black curve), which is approximately the sun surface temperature. As radiation passes through the atmosphere, some is absorbed by gases with specific absorption bands (producing the gaps visible in the red band). A part of radiation is redistributed by Rayleigh scattering, which is responsible for the atmosphere’s blue colour. At sea level most energy is associated with radiations in the visible range (400–700 nm); a lower amount of energy is carried by ultraviolet (100–400 nm) and infrared (700 nm–1 mm). (Figure by Robert A. Rohde as part of the Global Warming Art project (http://www.globalwarmingart.com/ wiki/Global_Warming_Art:About))

The general equation of photosynthesis is: CO2 þ2H2 D!CH2 OþH2 Oþ2D

ð4:1Þ

where H2D is an electron donor, or reductant. The photosynthesis is based on chlorophylls, a class of molecules containing a tetrapyrrole ring similar to the heme group of hemoglobin, but linked to a magnesium ion instead of iron. An important distinction is to be made between anoxygenic and oxygenic photosynthesis. The first utilizes compounds other than water as reductants and does not yield molecular oxygen (dioxygen); the molecules more commonly employed as reductants in anoxygenic photosynthesis are hydrogen H2, hydrogen sulphide and sulphide ions H2S and S
2, ferrous ions Fe++, thiosulphate S2O3
2 (Eqs. 4.2 and 4.3). Oxygenic photosynthesis, instead, uses water molecules as electron donors and liberates dioxygen as a by-product (Eq. 4.4).

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Examples of anoxygenic photosynthesis: CO2 þ2H2 !CH2 OþH2 O

ð4:2Þ

CO2 þ2H2 S!CH2 OþH2 Oþ2S

ð4:3Þ

Oxygenic photosynthesis: CO2 þ2H2 O !CH2 OþH2 OþO2

ð4:4Þ

*Molecular oxygen comes from water, not carbon dioxide There are numerous types of chlorophyll, differing from each other essentially in the lateral groups bound to the tetrapyrrole ring. Chlorophylls absorb light mainly in the blue (400–500 nm) and red (600–700 nm) region of the spectrum, whilst they reflect or transmit in the green region (500–600 nm); these spectroscopic properties are responsible for the green colour of chlorophylls. For enhancing light interception, photosynthetic organisms evolved phycobilins and carotenoids, accessory pigments that integrate the spectroscopic properties of chlorophylls. All known photosynthetic pathways evolved in bacteria (Butterfield 2015). Oxygenic photosynthesis evolved in cyanobacteria and was subsequently transferred to eukaryotes with the chloroplast (Chap. 9). Let us now return to our initial problem. Sunlight cannot beam down much past 200 m (660 ft) in the water column before being completely absorbed; thus, solar radiation does not reach the ocean depths where vents are generally located. If the earliest forms of life were metabolically bound to submarine vents, how did photosynthesis evolve? At temperatures of 300–450  C, black smokes emit radiations in the far-red and infrared region of the spectrum in amounts that could theoretically support photosynthetic reactions (Perez et al. 2013). Indeed, an obligate photosynthetic green sulphur bacterium has been isolated from the effluent plume within 50 cm above the orifice of the 2391 m-deep TY black smoker in the East Pacific Rise. This bacterium had a radiation adsorption spectrum with a major peak in the far-red region, suggesting that it could live photoautotrophically utilizing radiation from the vent (Beatty et al. 2005). Thus, evolution of photosynthetic life in the ocean depth is not impossible. An alternative scenario, in which early life evolved infrared-sensitive pigments to detect new vents suitable for colonization and later deployed this system for photosynthesis, is unlikely as it implies unicellular organisms to swim across substantial distances. A third possibility is that early life still dependent on reduced compounds of geochemical origin (i.e. non-photosynthetic) managed to colonize shallow hydrothermal systems at subduction areas, arriving there with ocean currents or following the slow movement of oceanic plates (Russell and Hall 2006). From the geochemical point of view, hydrothermal systems associated with subduction areas are deeply different from alkaline submarine vents. Firstly, their emissions are acid and

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relatively poor in hydrogen, methane and other reducing compounds, and there is no reason to doubt that the same applied in the Archean eon. Secondly, these systems do not build the elaborate compartmentation that distinguishes alkaline vent edifices, thus the chemicals emitted immediately disperse in the ocean. This might have limited the growth of chemotrophic forms but possibly also stimulated the emergence of forms capable of using light energy to integrate geochemical resources. Alternatively, light-absorbing pigments may have evolved first as a protection from ultraviolet and then deployed for primitive forms of photosynthesis. The paleogeochemical record suggests that photosynthetic life colonized the planet photic zone very early in the Archaean. The most ancient possible sign of photosynthetic activity has been found in the Isua Greenstone Belt in Greenland (aged about 3.7 GY), in the form of graphite deposits with a δ13C between
25% and
19% (Box 2.2), compatible with a biological origin (Rosing 1999; Ohtomo et al. 2013). Better substantiated evidence comes from sedimentary rocks in the Buck Reef Chert (South-Africa) and the Strelley Pool Formation (Pilbara Craton, Australia), both aged about 3.4 GY and containing 13C-depleted carbonaceous material as well as structures recalling present-living filamentous photosynthetic bacteria (Brasier et al. 2006; Sugitani et al. 2015; Schopf et al. 2017). Again in South-Africa, the Fig Tree and the Upper Onverwacht Group formations, with an age of about 3.2 GY, contain barite deposits (barium sulphate, BaSO4) possibly produced by photosynthetic bacteria living in shallow basins exposed to sulphide volcanic emanations (Muller et al. 2017). The earliest fossil stromatolites so far detected, structures at least in part built by photosynthetic bacteria (Chap. 3), are aged about 3.4 Gy (Schopf 2006). Almost certainly, the earliest photosynthetic organisms performed anoxygenic photosynthesis, using reduced compounds of volcanic origin such as hydrogen or sulphide (Eqs. 4.2 and 4.3) as a source of electrons (Olson 2006; Blankenship 2010). Photosynthesis could then spread to wider areas of the planet by shifting to ferrous ions. In the hydrated form that predominates in solution (FeOH+), ferrous ions have a standard reduction potential (at pH 7) of about +0.15 V, much higher than hydrogen’s (
0.42 V) or sulphide (
0.24 V), thus its utilization as an electron donor in photosynthesis is energetically less convenient. Ferrous ions, however, had the advantage of being relatively abundant in the Archaean oceans because of the absence of free oxygen (Chap. 5). Deposits of oxidized iron known as “banded iron formations” (Fig. 4.2), mainly formed between 3.4 and 1.8 GYA (Bekker et al. 2010) probably reflect an interplay of geochemical (hydrothermal) and biologic processes (Li et al. 2015) including oxidation by photosynthetic bacteria in reactions such as: 4FeOHþ þ CO2 þ 7H2 O ! 4FeðOHÞ3 þ ðCH2 OÞ þ 4Hþ 2FeðOHÞ3 ! Fe2 O3 ðhematiteÞ þ 3H2 O Fe2 O3 þ FeOHþ ! Fe3 O4 ðmagnetiteÞ þ Hþ

ð4:5Þ

Figure 4.3 reports the standard reduction potentials of molecules of biological importance. Oxygenic photosynthesis emancipated life from ancestral dependence on reduced compounds of geochemical origin. The emergence of oxygenic photosynthesis

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Fig. 4.2 Banded iron formations are shale or chert sedimentary rocks made of alternating layers with different iron contents. The reddish layers are rich in hematite (Fe2O3) or magnetite (Fe3O4), possibly produced by Fe++-oxidizing bacteria including photosynthetic bacteria. The alternation of iron-rich and iron-poor layers may reflect fluctuations in iron (Fe++) availability in the ocean, depending on volcanic input. BIF were abundant and widespread in the planet from about 3.7 to 1.8 GYA, but they disappeared almost completely afterwards, due to Fe++ oxidation by oxygen (Chap. 5). (From: http://geologylearn.blogspot.com/2017/01/banded-iron-formations-bifs-evi dence-of.html)

Fig. 4.3 Standard reduction potentials at pH 7 of molecules important in biology. (Data from Hohmann-Marriott and Blankenship 2011)

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coincides with the appearance of cyanobacteria, of which this metabolic pathway is a fundamental apomorphy. Around 900 MYA, oxygenic photosynthesis passed from cyanobacteria to eukaryotes by endosymbiosis (Chap. 9). Oxygen production by cyanobacteria caused the atmosphere/ocean/sediment (AOS) system to shift from anoxic to oxic, a major transition known as the “Great Oxygenation Event” and dated to about 2.40 GYA (Chap. 5). There is evidence that cyanobacteria appeared before that date, yet for some reason there was a delay in oxygen accumulation (Sect. 5.1). Molecular-clock analysis (Box 1.1) dates the first appearance of cyanobacteria to about 3.0 GYA (Schirrmeister et al. 2013, 2015). This estimation is consistent with identification of 2-methylhopanes in sedimentary rocks with an age from 2.78 to 2.45 GY (Brocks et al. 2003; Buick 2008; Waldbauer et al. 2009; Hoshino et al. 2015). Hopanes are molecules produced by spontaneous degradation of bacteriohopanepolyoles, isoprenoid polycyclic hydrocarbons used in cyanobacteria to control the fluidity of the cell membrane (Řezanka et al. 2010). Other investigations cast doubts about these results by suggesting the possibility of contamination from above-lying, more recent layers (Rasmussen et al. 2008; French et al. 2015). Based on evidence that 2-methylhopanes are also produced by anoxygenic phototrophs, Kirschvink and Kopp (2008) inferred a late origin of photosynthesis, suggesting that oxygenic photosynthesis could have evolved close in geological time to the Huronian glaciation, started around 2.4 GYA. Phylogenetic analysis of C-2 hopanoid methylase, a key enzyme of the bacterial 2-methylhopanoid biosynthetic pathway, showed that 2methylhopanoids probably originated in a subset of α-proteobacteria and then transferred to cyanobacteria by horizontal gene transfer (HGT). This suggests that 2methylhopanoids are not reliable biomarkers for cyanobacteria; in addition, because the α-proteobacteria are aerobic, 2-methylhopanoids most likely arose after the oxygenation of the atmosphere, i.e. not earlier than about 2.4 GYA (Ricci et al. 2015). Other lines of enquiry support an earlier origin of oxygenic photosynthesis. One of these is based on the occurrence of traces of gas bubbles in ancient stromatolites. In modern cyanobacterial stromatolites, the oxygen produced by photosynthesis forms bubbles that, if small enough, may remain trapped in bacterial mucilage; during fossilization, the bubbles fills with minerals, producing inclusions that are clearly visible in section. A survey of fossil stromatolites of different ages has shown that those formed before 2.7 GYA do not contain fossilized bubbles, whereas more recent ones do. This suggests that the cyanobacteria appeared around 2.7 GYA, i.e. about 300 MY before the Great Oxygenation (Bosak et al. 2009). Indirect evidence supporting an early origin of cyanobacteria comes from the discovery, in several sites of the planet, of huge carbonaceous deposits (¼ organic carbon) depleted of 13C and carbonate deposits (¼ inorganic carbon) enriched in 13 C, both aged between 3.2 and 2.72 GY (Buick 2008). From Chap. 2, we know that carbonate arises from carbon dioxide in the atmosphere and in the ocean. By preferring 12C to 13C, photosynthesis and other carbon-fixing pathways increase the δ13C of the carbon dioxide pool wherefrom carbonate arises; 13C depletion in carbonaceous deposits and 13C enrichment in coeval carbonate deposits, therefore, is strong evidence for carbon fixation by living organisms. The central point is that Archean deposits of 13C-depleted organic material are so massive and widespread that they could possibly arise only from oxygenic photosynthesis because all other

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carbon-fixing pathways such as anoxygenic photosynthesis and methanogenesis are severely limited by dependence on reducing compounds. More direct evidence comes from geochemical analysis of ancient sedimentary rocks and paleosoils. Local redox conditions at the time of formation affect the amounts, chemical form and isotopic distribution of transition elements such as iron, molybdenum, manganese, chromium and uranium in sedimentary rocks and soil (Chap. 5 for further details). Evidence based on these elements documents transient/local oxygen accumulation in an otherwise anoxic environment from about 3 GYA, suggesting that biological oxygen production started well before oxygen became a permanent component of the atmosphere around 2.4 GYA (Anbar et al. 2007; Crowe et al. 2013; Lyons et al. 2014; Mukhopadhyay et al. 2014; Planavsky et al. 2014). These results are consistent with phylogenomic evidence of a burst of genetic innovation dated to about 3.3–2.8 GYA, referred to as the “Archaean Genetic Expansion”. This window was associated with an expansion in microbial respiratory and electron transport capabilities, including enzymes involved in aerobic metabolism (David and Alm 2011).

4.2

Light, Pigments and Photosystems

Light has the properties of a particle and a wave. It consists of units (light quanta or photons), each conveying an indivisible amount of energy expressed by the relation: E¼hν where h is the Plank constant [6.610
34 Js ¼ 4.110
15 eVs (eV is for electronvolt, 1 eV ¼ 1.610
19 J)], and ν the frequency, namely the number of oscillations through a fixed point in a second. The frequency is equal to the light speed c in the vacuum (about 3108 m/s) divided by the wavelength λ, which is the distance between two successive oscillations. We can therefore write the above equation in the following form: E¼hc=λ which means that the energy conveyed by a single photon is inversely proportional to its wavelength. The constant hc is 4.110
15 eVs  3108 m/s ¼ 1.23  10
6 eVm. For electromagnetic radiations with wavelengths in the micrometre size order (essentially the visible and infrared spectrum), it is convenient to express the constant hc in electronvolt per micrometre (1 μm ¼ 10
6 m): hc¼1:23  10 2 6 eVm¼1:23  10 2 6 eV  106 μm¼1:23 eVμm Therefore, expressing λ in μm:

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107

E¼1:23 eV=λ

ð4:6Þ

Equation 4.6 permits to calculate that a red photon with a wavelength of 0.68 μm (680 nm) conveys a quantum of energy equivalent to 1.23/0.68 ¼ 1.82 eV, namely the standard free-energy variation associated with the movement of one electron across an electric potential difference of 1.82 V. The capture of a photon by a pigment causes a transition in electron configuration from a ground state to a state of higher energy, or excited state. The excited molecule may return to the ground state by one of the following ways: 1. Internal conversion: the energy surplus is entirely dissipated as heat; 2. Fluorescence: the energy surplus is partly emitted in the form of a second photon with a slightly larger wavelength than the photon absorbed, and partly converted into heat; 3. Resonance: the energy surplus is transferred to another molecule, which in turn shifts to an excited state; 4. Charge separation: the energy is converted into chemical energy by transferring an electron to another molecule (as in chlorophyll-based phototrophy), or pumping a ion across a membrane (as in rhodopsin-based phototrophy). The first and second pathways are virtually the only options available to pigments isolated from the biological context. In alcoholic solution, chlorophyll shows an intense red fluorescence due to secondary photons emitted after excitation. The third and fourth pathways are accessible and usually predominate in the cellular context. The reason is that, in living cells, photosynthetic pigments are orderly assembled with proteins into membrane-bound complexes, or photosystems, so they tend to follow interactive pathways. Focusing attention on chlorophylls, these pigments have absorbance peaks in the blue and red or near-infrared region of the spectrum. The absorption of a red (or nearinfrared) photon induces the chlorophyll molecule to shift to the first singlet excited state. The absorption of a blue photon, more rich in energy, induces the transition to the second singlet excited state, a higher-energy state wherefrom the molecule spontaneously decays to the first singlet excited state, emitting the energy difference in the form of heat. The first singlet excited state is the only excited state that persists long enough to permit chlorophyll to convert light energy into chemical energy (path 4). Consequently, the energy available for chlorophyll-mediated photochemistry is never higher than the difference between the first singlet excited state and the ground state, which varies between 1.8 and 1.6 eV according to the type of chlorophyll. A minor part of the chlorophyll molecules present in the photosynthetic apparatus are engaged to form a photochemically active reaction centre (see below). Most chlorophyll molecules and other associated pigments instead form an internal antenna within the photosystems and often also external antennas, functioning in light interception and energy transfer to the reaction centre. In all photosynthetic bacteria except the cyanobacteria, the photosystems and accessory antennas are associated with the cell membrane, which often forms deep invaginations that

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expand its surface extension. In cyanobacteria, the photosynthetic machinery is usually associated with thylakoids, internal membranous compartments physically separate from the cell membrane. The only known exception is the primitive cyanobacterial genus Gloeobacter, in which there are no thylakoids and the photosynthetic machinery lies in the cell membrane. In eukaryotes the photosystems are bound to thylakoid membranes in chloroplasts, organelles derived from an endosymbiotic cyanobacterium (Chap. 9). The photosystems consist of a pigment-protein complex named the reaction centre (RC) and a core antenna made of proteins and a few hundred molecules of chlorophyll and carotenoids. As already mentioned, the photosynthetic machinery usually also include peripheral antenna complexes that interact with the photosystems but are not an essential part of these. The reaction centre contains a special pigment that performs the photochemical reaction of charge separation; this is referred to with the symbol “P” followed by its peak-absorbance wavelength (in nanometres), and usually consists of two closely associated molecules of bacteriochlorophyll a (in anoxygenic bacteria) or chlorophyll a (in cyanobacteria and chloroplasts) and an electron acceptor (A). The antenna pigments function as a highly integrated system so that when any of them captures a photon, the energy absorbed is unidirectionally transferred to the pigment P in the reaction centre. In the excited state (P*) the reaction centre is capable of transferring one of its electrons to the acceptor A. The photosystem, therefore, uses light energy to separate electric charges as follows: Pþhν!P P þA!Pþ þA
The “trick” of the mechanism is that in the excited state P is a stronger reductant than in the ground state, thus it can reduce the acceptor A. The oxidation of P* is an exergonic reaction that occurs spontaneously within a very short time (in the order of picoseconds, 10
12 s), outrunning fluorescence or internal conversion (both energywasting processes). In the reduced state, the acceptor A starts a chain of exergonic reduction reactions associated with PMF-building proton flow across the photosynthetic membrane. F-ATP synthase complexes inserted in the same membrane use the PMF to make ATP (Box 3.1). The photosystems can work in a cyclic or non-cyclic way. In the cyclic way, the electrons return to the reaction centre after flowing through the photosynthetic system. When working in the cyclic way, the photosystems can make ATP but do not reduce NAD(P)+. In the non-cyclic way, electrons flow from the reaction centre to NAD(P)+, which is reduced to NAD(P)H. ATP and NAD(P)H are both necessary to convert carbon dioxide into sugar. In the non-cyclic pathway, of course, an external source of electrons is necessary to replace the electrons transferred from the reaction centre to NAD(P)+ and eventually to carbon dioxide. There are two basic types of photosystem that differ in the nature of the electron acceptor in the reaction center: type-1 (PS1) photosystems use an iron-sulphur protein (iron/sulphur RC), type-2 (PS2) use a quinone (QRC). In both types of

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109

photosystem the reaction centre is a dimeric complex, i.e. it consists of two subunits (Hohmann-Marriott and Blankenship 2011). These are identical (namely the RC is homodimeric) in green sulphur bacteria and heliobacteria, whereas all other photosynthetic organisms have heterodimeric RCs consisting of homologous but not identical subunits (Fig. 4.4). Both RC subunits are functional in electron transport in type-1 photosystems, whereas only one of the two monomers is active in type-2 photosystems. A further important difference is that the photochemically active pigment P of iron/sulphur RCs has an absorbance peak at a larger wavelength and a lower reduction potential than its equivalent in QRCs. This implies that type-1 photosystems can use slightly larger wavelengths than type-2 and can reduce NAD(P)+ to NAD(P)H, whereas type-2 photosystems cannot because the reduction potential gap between their RC and NAD(P)+ is too wide (Vermaas 2002; Bryant and Frigaard 2006; Fig. 4.5). Phototrophic bacteria that use rhodopsin can produce ATP photochemically but cannot convert carbon dioxide into organic matter, so they are obliged to feed on organic compounds from the environment. Figure 4.5 suggests that the same limitation also applies to bacteria with type-2 photosystems such as purple bacteria; actually, these bacteria manage to reduce carbon dioxide using reverse electron flow,

Fig. 4.4 The reaction centre (RC) has a dimeric structure in all known photosynthetic organisms. Purple bacteria and chloroflexibacteria have a type-2 photosystem (PS2); each RC monomer contains two molecules of bacteriochlorophyll a (or bacteriochlorophyll b in certain purple bacteria) associated with a protein forming five transmembrane helices (TMH). Cyanobacteria and eukaryotes have a type 1 (PS1) and type 2 photosystem (PS2). Each RC monomer of the PS2 contains a couple of chlorophyll a molecules associated with a protein (either D1 or D2) forming five TMH; in addition, each RC monomer of PS2 includes a protein (CP43 or CP47) forming six TMH, which is associated with other pigment molecules to form a core antenna. Each RC monomer of the of PS1 in cyanobacteria and eukaryotes contains a couple of molecules of chlorophyll a associated with a 11TMH protein. Green sulphur bacteria and heliobacteria have a type-1 photosystem with a homodimeric RC, each monomer containing a couple of bacteriochlorophyll a (green sulphur bacteria) or bacteriochlorophyll g (Heliobacteria) associated with a 11-TMH protein. In each case, subunits with the same shape but different colours are homologous but not identical (heterodimeric RC), subunits with the same shape and colour are identical (homodimeric RC). (Redrawn from Hohmann-Marriott and Blankenship 2011)

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Fig. 4.5 The three fundamental types of phototrophy. (a) Photoheterotrophy based on the protein rhodopsin: light energy is used to build PMF, which supports ATP production by ATP synthase. The organisms using this form of phototrophy cannot fix carbon dioxide and depend on organic carbon from the environment. (b) Phototrophy based on type-2 photosystems. Cyclic electron flow permits the production of PMF that supports ATP production by ATP synthase. (c) Phototrophy based on type-1 photosystems. Non-cyclic electron flow permits the reduction of carbon dioxide to sugar, with electrons extracted from an external source. Functioning in the cyclic mode, type-1 photosystems can produce PMF/ATP. (Redrawn from Hohmann-Marriott and Blankenship 2011)

a mechanism that transfers electrons to NAD+ against a reduction potential difference, the energy differential being obtained from PMF. Table 4.1 resumes the taxonomic distribution of different types of phototrophic metabolism.

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111

Table 4.1 Distinguishing characters of different phototrophic organisms (RC, reaction centre) Taxonomic group Green and red chloroflexibacteria (Chloroflexales)

Photochemical system Type 2 photosystem (heterodimeric RC)

Non-sulphur purple bacteria (α- and β-Proteobacteriales)

Type 2 photosystem (heterodimeric RC)

Sulphur purple bacteria (γProteobacteriales)

Type 2 photosystem (heterodimeric RC)

Sulphur green bacteria (Chlorobiaceae)

Type 1 photosystem (homodimeric RC) Type 1 photosystem (homodimeric RC)

Heliobacteria (Heliobacteriales)

Candidatus Chloracidobacterium thermophilum (Acidobacteria) Halobacteria (Archaea, Euryarchaeota)

Proteobacteria, Bacteroidetes, Actinobacteria Cyanobacteria

Type 1 photosystem (RC structure unknown) Bacteriorhodopsin or halorhodopsin

Rhodopsin-like proteins Type 1 photosystem (heterodimeric RC)+ Type 2 photosystem (heterodimeric RC)

Metabolic properties In the absence of oxygen: anaerobic photoheterotrophs or photoautotrophs In the presence of oxygen: aerobic heterotrophs As photoautotrophs, they use reverse electron flow to reduce NAD+ In the absence of oxygen: anaerobic photoheterotrophs or photoautotrophs In the presence of oxygen: aerobic heterotrophs As photoautotrophs, they reduce NAD+ by reverse electron flow The photosynthetic system functions prevalently in the cyclic mode Obliged anaerobic photoautotrophs NAD+ reduced by reverse electron flow Obligate anaerobic photoautotrophs

Anaerobic photoheterotrophs The photosynthetic system functions prevalently in the cyclic mode Aerobic photoheterotrophs

In the presence of oxygen: aerobic heterotrophs Under limiting oxygen concentrations, they use light to produce PMF/ATP or to pump chloride ions outside the cells No carbon dioxide fixation Poorly known Primarily aerobic oxygenic photoautotrophs, but metabolically highly flexible

(continued)

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Table 4.1 (continued) Taxonomic group Eukaryotes (Plantae + photosynthetic forms among the Excavata, Rhizaria, Chromista, Alveolata Chaps. 6 and 9)

4.3

Photochemical system Type 1 photosystem (heterodimeric RC)+ Type 2 photosystem (heterodimeric RC)

Metabolic properties Aerobic oxygenic photoautotrophs

The chloroplast derives (directly or indirectly) from a cyanobacterium

Accessory Pigments

As already mentioned, besides the pigments of the reaction centre (usually a couple of molecules of bacteriochlorophyll a, or of chlorophyll a), the photosynthetic machinery utilizes a diversity of accessory pigments to absorb light and transfer the excitation energy to the reaction centres. A part of the accessory pigments is associated with proteins within the photosystem complexes, to form the so-called core antenna. The greater part of accessory pigments is usually located in large external antennas that significantly expand the total amount of radiation absorbed and the wavelength spectrum utilizable. The ecological distribution of photosynthetic organisms largely depends on qualitative and quantitative adjustments in antenna pigments (Bryant and Frigaard 2006; Hohmann-Marriott and Blankenship 2011). Cyanobacterial photosystems have a core antenna made of chlorophyll a and carotenoids, plus large peripheral antennas called phycobilisomes, mainly associated with photosystem 2 and containing protein pigments (phycobiliproteins) capable of absorbing a wide range of wavelengths (Fig. 4.6). Cyanobacteria can adapt to a diversity of light conditions by modulating the amount and type of phycobiliproteins in their peripheral antennas. Green sulphur bacteria and green chloroflexibacteria have peripheral antennas named chlorosomes (Fig. 4.6). Both phycobilisomes and chlorosomes transfer excitation energy to the core antennas of photosystems, wherefrom it flows to the reaction centre. The phycobilisomes and chlorosomes are bound to the surface of photosynthetic membranes; in contrast, peripheral antenna complexes in green plants are integral components of the photosynthetic membrane (Fig. 4.6). Metagenomics analysis of phototrophic mat communities in the Yellowstone National Park (USA) has led to the discovery of a new photosynthetic bacterium, reported as Candidatus Chloracidobacterium thermophilum and placed in the poorly characterized proteobacterial lineage Acidobacteria. This bacterium has a type-1 RC and a chlorosome-like antenna with bacteriochlorophyll a and c; it requires light and an organic substrate to grow in culture, and probably is able to respire aerobically (Bryant 2007).

4.4 The Photochemical Pathway in Anoxygenic Bacteria

113

Fig. 4.6 Diagrammatic representation of major antenna complexes in photosynthetic organisms. Cyanobacterial phycobilisomes are large protein complexes mainly associated with photosystem 2 (PS2). The cyanobacterial PS2 also has two small core antennas (CP43 and CP47). Photosystem 1 (PS1) has two core antennas homologous with CP43 and CP47. In Prochlorophytes, a cyanobacterial lineage lacking phycobiliproteins, both PS1 and PS2 are associated with chlorophyll b-containing peripheral antennas (Pcb). The Heliobacteria have a type-1 photosystem with two small core antennas. Green sulphur bacteria have a type-1 photosystem similar to that of Heliobacteria but associated with large accessory antennas named chlorosomes. Purple bacteria have a type 2 photosystem with small peripheral antennas formed by ring-shaped BChl-a-binding LH1 and LH2 complexes. Red chloroflexibacteria have a type-2 photosystem with a LH1-like antenna. In addition to a LH antenna, green chloroflexibacteria have peripheral antennas similar to chlorosomes of green sulphur bacteria, although of smaller sizes. A chlorosome-like antenna is probably also present in the newly discovered photosynthetic bacterium Candidatus Chloracidobacterium thermophilum. Photosystem 2 in green plants (Viridiplantae Chap. 9) has CP43 and CP47 core antennas and interacts with accessory antenna LHC2 (light-harvesting complex 2). Photosystem 1 has a small core antenna and interacts with accessory antenna LHC1 (and in certain conditions with LHC2 as well). Red plants (Glaucophyta and Rhodophyta Chap. 9) have a photosynthetic apparatus similar to that of cyanobacteria. RC reaction centre. (Adapted from Bryant and Frigaard 2006, licence number 4466670398595)

4.4

The Photochemical Pathway in Anoxygenic Bacteria

Purple bacteria encompass two distinct phyletic lineages, non-sulphur and sulphur purple bacteria, both possessing a type-2 photosystem, thus a quinone-based RC. Non-sulphur purple bacteria are facultative anaerobes. In the presence of oxygen, they behave as aerobic heterotrophs and do not express photosynthetic genes; in the absence of oxygen, instead, they produce the photosynthetic machinery and are able to live as photoheterotrophs or photoautotrophs, depending on the availability of organic matter in the environment. Sulphur purple bacteria are obligate anaerobic photoautotrophs. Neither line is able to reduce NAD+ photochemically, so they must use reverse electron flow when living autotrophically. This mechanism transfers electrons from small organic molecules, sulfur compounds, Fe++ ions or molecular

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hydrogen to NAD+ against a reduction potential difference, with PMF generated by the photosynthetic machinery covering the energy gap. Both sulphur and nonsulphur purple bacteria are able to utilize sulphur compounds as a source of electrons and to oxidize them to elemental sulphur, but they differ in the way they dispose of this by-product: the first accumulate sulphur within the cells, where it is visible as small granules, the latter expel sulphur to the outside. Green sulphur bacteria have a type-1 photosystem, therefore a Fe-S RC, and are able to reduce NAD+ photochemically. As sulphur purple bacteria, they are obligate anaerobic photoautotrophs depending on sulphur compounds, ferrous iron or hydrogen as electron donors. The two following figures schematically illustrate the photochemical pathway in purple (Fig. 4.7) and green sulphur bacteria (Fig. 4.8).

Fig. 4.7 The photochemical pathway in purple bacteria. On the left are values of standard reduction potential at pH 7 (E0 , expressed in volts). P870, the photochemically active pigment of the reaction centre, has a E0 of about – 0.9 V in the excited state (P870*), and about +0.5 V in the oxidized state (P870+). P870* is able to reduce, via intermediate transporters, the ubiquinone UQ (E0 ¼+ 0,10 V). From reduced UQ, the electrons flow through a cytochrome chain (coupled with PMF and ATP generation) and eventually return to the reaction centre. The photochemical apparatus of purple bacteria can also work in a non-cyclic way, involving reverse electron flow (REF) from reduced UQ to NAD+. This endergonic process dissipates a part of the PMF produced in the cyclic pathway. When the photochemical apparatus works in the non-cyclic way, electrons required to fill the positive hole in the reaction centre are extracted from organic or inorganic donors from the environment. BChl bacteriochlorophyll, BPh bacteriophaeophytin

4.5 Evolutionary Interrelationships of Type-1 and Type-2 Photosystems

115

Fig. 4.8 The photochemical pathway in green sulphur bacteria. On the left are standard reduction potentials at pH 7 (E0 , expressed in volt). Green sulphur bacteria have a type-1 photochemical apparatus that operates at lower values of reduction potential than in purple bacteria. P840, the photochemically active pigment of the reaction centre, has a E0 of about
1.25 V in the excited state (P840*), and about +0.3 V in the oxidized state (P870+). P870* reduces an iron/sulphur protein complex (FeS) in the RC. From here, the electrons can return to P870+ through a cytochrome chain, driving PMF/ATP production. Alternatively, the electrons may flow from FeS to ferredoxin (Fd) and eventually to NAD+, which is reduced to NADH. In the latter pathway, the electrons necessary to fill the positive hole in the reaction centre are extracted from simple inorganic compounds (usually sulphides or thiosulphate) from the environment. Chla chlorophyll a, MQ menaquinone

4.5

Evolutionary Interrelationships of Type-1 and Type-2 Photosystems

The distribution of photosynthesis in the bacterial phylogenetic tree is somewhat puzzling, with photosynthetic and non-photosynthetic taxa clumped together within major lineages (Blankenship 2010). For example, purple sulphur and non-sulphur bacteria belong to γ- and α-proteobacteria, respectively, both lineages also encompassing numerous non-photosynthetic lineages. Such a distribution may be explained by assuming that photosynthesis was lost several times independently, giving rise to non-photosynthetic relatives, or was acquired by horizontal gene transfer (HGT) multiple times independently. The genes encoding for the photosynthetic apparatus are aggregated together in few large clusters, which may favour en bloc transfer of the photosynthetic machinery by HGT. Indeed, molecular evidence suggests that the photosynthetic apparatus in different lines of bacteria is a mosaic from multiple events of HGT and subsequent

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adjustments (Raymond et al. 2003; Bryant and Frigaard 2006; Hohmann-Marriott and Blankenship 2011; Gupta 2012). This may help explaining surprising affinities between distantly related lineages. For example, the chloroflexibacteria have a RC and a peripheral antenna closely similar to those of purple bacteria, but some of them (known as “green” chloroflexibacteria) also have peripheral antennas similar to the chlorosomes of green sulphur bacteria. Likewise, the heliobacteria have a similar RC as in green sulphur bacteria, but lack chlorosomes (Fig. 4.6). Phylogenomic analysis suggests that the Heliobacteria (PS1) are the most ancient line of photosynthetic bacteria, followed by Chloroflexibacteria (PS2), Cyanobacteria (PS1 and PS2) and last by green (PS1) and purple (PS2) Proteobacteria (Gupta 2012). Because of prominent HGT, the evolution of the photosynthetic apparatus is not easy to reconstruct. It is generally maintained that an anoxygenic form of photosynthesis (requiring only one type of photosystem) preceded oxygenic photosynthesis (requiring both types of photosystems). Nevertheless, based on current knowledge, it is not possible to choose between a number of alternative evolutionary hypotheses. For example, the cyanobacteria may have primarily had a type-2 photosystem and secondarily acquired a type 1 photosystem by HGT. Alternatively, they originally had one of the two photosystems (either PS1 or PS2) and secondarily evolved the other by duplication and neo-functionalization of the whole gene complex. The derived position assigned to green and purple proteobacteria suggest that these obtained the PS1 or PS2, respectively, by HGT from cyanobacteria and adapted to specialized niches (Hohmann-Marriott and Blankenship 2011). On the other hand, substantial affinities between type-1 and type-2 photosystems are obvious evidence of a common origin. The most likely scenario is that an ancestral form of photosynthesis evolved once and was transmitted (either vertically or horizontally) to several lines of descent, each of which added specific adjustments. The ancestral photosystem probably had a monomeric RC; from this, a homodimeric photosystem with two identical RCs may have evolved by duplication and neo-functionalization of the original gene cluster (Hohmann-Marriott and Blankenship 2011). The dimeric structure possibly protects the RC from the risk of inactivation by an excess of light. The RC of photosystem 2 in cyanobacteria (and eukaryotes) is a heterodimer of the protein D1 and D2, of which only D1 is active in electron transport whilst D2 performs a protective role. A heterodimeric structure may have the additional advantage of reducing the risk that electrons return to the reaction centre, which would seriously reduce the efficiency of the system. This is probably a critical need for oxygenic type-2 photosystem, which operates at particularly high values of reduction potential (Sect. 4.6).

4.6

Oxygenic Photosynthesis

Oxygenic photosynthesis put an end to ancestral dependence on reductants of geochemical origin, thus enabling life to spread virtually all over the planet. Oxygenic photosynthesis is a distinctive property of cyanobacteria, so it is legitimate to

4.6 Oxygenic Photosynthesis

117

Fig. 4.9 Fine structure of the cyanobacterium Synechocystis sp. as seen in thin section by transmission electron microscopy. The cell is packed with thylakoids, membranous structures that host the photosynthetic machinery

assume that its emergence as an integrated metabolic pathway marked the appearance of this bacterial lineage. As already discussed, paleontological and geochemical evidence suggests that the cyanobacteria appeared at least 2.7 GYA, possibly long before. Endowed with a flexible and versatile metabolism, the cyanobacteria occupied a novel evolutionary landscape with virtually no competitors. They spread throughout the planet, maintaining a position of ecological dominance that terminated only in the Phanerozoic, with the global diffusion of photosynthetic eukaryotes. Figure 4.9 shows the cell structure of a typical cyanobacterium. Selection pressure for the evolution of oxygenic photosynthesis had to overcome tremendous biochemical and thermodynamic difficulties. With a standard reduction potential at pH 7 of +0,82 V, water has very little tendency to give up electrons (namely, it is a very weak reductant). To break into electrons, protons and free oxygen, the water molecule must interact with an extremely strong oxidant. In oxygenic photosynthesis this work is done by P680, the photochemically active pigment in the RC of photosystem 2, which in the oxidized state (P680+) attains a reduction potential of about +1.25 V. At the other end of the photochemical chain, a strong reductant is required for reducing NADP+ to NADPH; this difficult task is performed by P700, the active pigment in the RC of photosystem 1, which in the excited state (P700*) has a reduction potential of about
1.25 V, the lowest value attained in the biological world. The energy gap between the two extremes of the oxygenic photochemical pathway is about 2.5 eV, a value that chlorophyll cannot cover in a single excitation event (Sect. 4.1). Oxygenic photosynthesis deploys type2 and type-1 photosystems working in series in order to sum the energy of two photons (Nelson and Junge 2015; Fig. 4.10).

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Fig. 4.10 Diagrammatic representation of oxygenic photosynthesis. In the excited state, P680 (the photochemically active pigment of reaction centre 2) reduces pheophytin a (Pheo, a molecule of chlorophyll a lacking the magnesium ion). In two separate photochemical events, two electrons pass from pheophytin to a plastoquinone molecule strongly bound to photosystem 2 (QA) and then to a weakly-bound plastoquinone (QB), which binds two protons and becomes plastoquinol (PQ). Plastoquinol leaves photosystem 2 and diffuses in the photosynthetic membrane until it reacts with the cytochrome complex Cyt b6f. Electrons from PQ flow through the Cyt b6f complex to plastocyanin (PC), a small soluble protein located in the thylakoid lumen, and eventually replace the electrons lost by the reaction centre of photosystem 1 (P700). The two protons are released in the thylakoid lumen. From P700, electrons flow through a chain comprising the iron-sulfur protein ferredoxin (FD) and reduce NADP+ to NADPH. Photosystem 1 can also operate in a cyclic way, with electrons flowing back from ferredoxin to the complex Cyt b6f and then to P700. The electron flow through the Cyt b6f complex (both in the cyclic and non-cyclic mode) generates PMF, thus enabling an F-ATP synthase to make ATP. In the oxidized state, the active pigment of the reaction centre 2 (P680+) has a reduction potential of about +1,25 V and retrieves the missing electron from a manganese-containing protein complex. After having lost four electrons, this complex returns to the original state by extracting the missing electrons from two water molecules, leaving an oxygen molecule (O2) and four protons (H+) as by-products. The sequential work of the two photosystems permits each electron to move from water to NADP+ using energy from two photons, thus the production of one molecule of oxygen requires the energy of eight photons. (Figure by Somepics – Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid¼38088695)

Photosystems 1 and 2, the cit b6f complex, most of the intermediate electron transporters and ATP synthase are all integral components of the thylakoid membrane (Fig. 4.11). In non-oxygenic photosynthesis, each turnover of the reaction centre working in the non-cyclic mode is coupled with two-electron oxidation of a substrate and oneelectron transfer through the cytochrome chain (Figs. 4.7 and 4.8). In oxygenic photosynthesis, instead, the PS2 is decoupled from the cytochrome chain and its reaction centre interacts with the water oxidizing complex (WOC), a metal-enzyme with an inorganic core containing manganese, calcium, chlorine and bicarbonate,

4.6 Oxygenic Photosynthesis

119

Fig. 4.11 Diagrammatic representation of the photosynthetic apparatus of cyanobacteria as arranged in the thylakoid membrane. The figure shows only a part of the phycobilisome. (Adapted from Allen et al. (2011), licence number 4466680224368)

Fig. 4.12 Electron transfer from water to PS2 oxidized by light, through the water-oxidizing complex (WOC)

with the empirical formula Mn4CaOxCl1–2(HCO3)y. By transferring electrons to the RC of photosystem 2, WOC sequentially generates four oxidation states named S1, S2, S3, S4 (resulting from the loss of 1, 2, 3, 4 electrons, respectively), with S0 indicating the initial, non-oxidized state. In the state S4, WOC is able to oxidize water, retrieving at once the four electrons lost and returning to the S0 state, with a molecule of dioxygen and four protons left as by-products. The protons remain in the thylakoid lumen, where they contribute to PMF building (Barber 2012; Fig. 4.12). With an overall free energy change of 312 kJ per mole of dioxygen, water oxidation is thermodynamically the most challenging multi-electron reaction in biology (Dismukes et al. 2001).

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The WOC was the fundamental innovation that enabled the cyanobacteria to use water as an electron donor. Oxygenic photosynthesis might have evolved through a series of intermediate stages, starting from a photosystem 2-based pathway (Dismukes et al. 2001). A critical step in this scenario was the appearance of photosynthetic forms capable of extracting electrons from manganese ions (Mn+2), which were probably present in relatively high concentrations in the Archean ocean. The couple Mn+4/Mn+2 has a reduction potential of +0.490 V at pH 7, a value already too high for interaction with type-1 photosystems. A second step was replacement of Mn+2 with bicarbonate ions as an electron source; theoretically, this might have already enabled the photosystem to produce molecular oxygen. The appearance of chlorophyll a (a form of chlorophyll that is able to attain particularly high values of reduction potential) was probably the third step. This evolutionary sequence might have occurred in bacteria still possessing only a type-2 photosystem; if so, reverse electron flow was initially needed for NADP+ reduction. Finally, the acquisition of a type-1 photosystem (by either gene cluster duplication or HGT) permitted direct reduction of NADP+ (Fig. 4.13).

Fig. 4.13 Hypothetical steps in the evolution of oxygenic photosynthesis. (a) Chloroflexi-like anoxygenic photosynthesis using organic ions as sources of electrons. (b) The system shifted to using Mn++ ions present in solution as Mn bicarbonate in the Archean ocean. (c) Evolution of a manganese-based oxidizing complex that used bicarbonate ions as a source of electrons and, for the first time, produced free oxygen. (d) Further improvement of the manganese-containing oxidizing system permitted electron extraction from water. The model suggests that bacteriochlorophyll g replaced bacteriochlorophyll a in the photosystem reaction centre during the transition from (b) to (c). The transition from (c) to (d) hypothetically involved replacement of bacteriochlorophyll g with chlorophyll a, and the acquisition of a type-1 photosystem coupled with photosystem 2. D, electron donor (formate, oxalate); P, reaction centre. (Redrawn from Dismukes et al. 2001)

4.7 Pathways of Carbon Photosynthetic Fixation

4.7

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Pathways of Carbon Photosynthetic Fixation

The photosynthetic conversion of inorganic carbon into sugar is commonly known as the “light-independent phase” or “dark phase” of photosynthesis, as it does not involve direct participation of light. This terminology is not particularly appropriate, because carbon dioxide fixation stops almost immediately in the dark due to rapid exhaustion of ATP and NAD(P)H] produced in the “light-dependent phase”. The incorporation of carbon dioxide (or of its hydrated form, the bicarbonate ion) into organic compounds is catalysed by carboxylases, enzymes widespread in the living world from bacteria to fungi, plants and animals including humans. The main metabolic pathways for inorganic carbon fixation in autotrophic organisms are the Calvin, Benson and Bassham cycle, the reductive acetyl-coenzyme A cycle, the reductive citric acid cycle, and the 3-hydroxypropionate cycle with its variants (Rothschild 2008; Fuchs 2011). The Calvin-Benson-Bassham (CBB) cycle is today by far the predominant pathway for autotrophic carbon fixation (Fig. 4.14). The CBB cycle occurs in cyanobacteria and photosynthetic eukaryotes, in purple bacteria and most lytoautotrophic bacteria, but it is apparently lacking in the archaea. Its key enzyme

Fig. 4.14 The Calvin-Benson-Bassham (CBB) cycle is the autotrophic pathway of carbon fixation utilized in cyanobacteria, photosynthetic eukaryotes, purple bacteria and most lytoautotrophic bacteria. The CBB cycle consumes two NADPH and three ATP molecules for each carbon dioxide molecule reduced to sugar. GAP glyceraldehyde-3-phosphate. (By Yikrazuul – Own work, CC BYSA 3.0, https://commons.wikimedia.org/w/index.php?curid¼6918205)

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is ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which catalyses the carboxylation or, alternatively, the oxygenation of ribulose-1,5-bisphosphate (RuBP). The distinctive catalytic and kinetic properties of RubisCO have deeply affected the evolutionary history of the organisms that depend on this enzyme for carbon fixation and, as a result, the whole biosphere (Nisbet et al. 2007). The reductive acetyl-coenzyme A, or Wood–Ljungdahl cycle, converts carbon dioxide into acetate in acetogenic bacteria, or into methane in methanogenic archaea (both non-photosynthetic organisms), utilizing four hydrogen molecules as reductant (Fig. 4.15). This is the only known metabolic pathway that converts carbon dioxide into organic carbon and simultaneously produces ATP thanks to the mechanism known as “electron bifurcation” (Box 3.1). The cycle involves unique enzymes that use tetrahydrofolates as donors/acceptors of single-carbon functional groups, and unique bio-organometallic intermediates (methyl-Co, methyl-Ni, carbonyl-Ni, acetyl-Ni). The reductive citric acid cycle is a reversal of the citric acid (Krebs) cycle, from which it possibly derived. Also known as the Arnon-Buchanan cycle after its discoverers, this pathway converts carbon dioxide into oxaloacetate (Fig. 4.16). For the original citric acid cycle to turn towards the reductive direction, several irreversible steps had to be modified, for example succinate dehydrogenase was replaced by fumarate reductase, NAD+-dependent 2-oxoglutarate dehydrogenase by ferredoxin-dependent 2-oxoglutarate synthase, and citrate synthase by ATP citrate lyase. The reductive citric acid cycle occurs in green sulphur bacteria and several lineages of anaerobic lytoautotrophic bacteria. Unlike the other cycles mentioned above, the 3-hydroxypropionate cycle utilizes the bicarbonate ion instead of carbon dioxide (Fig. 4.17). It occurs in chloroflexibacteria, but some members of this group (for example Oscillochloris trichoides) instead use the CBB cycle, probably acquired by HGT (Tabita 2009).

Fig. 4.15 Reductive acetyl-coenzyme A, or Wood–Ljungdahl cycle. The part of the cycle that leads to methane is found in methanogenic Euryarchaeota (a major archaeal lineage), whereas the part leading to acetate occurs in acetogenic bacteria. It has been suggested that this metabolic dichotomy reflects ancestral divergence of archaea and bacteria from a common progenitor. (Redrawn from Rothschild 2008)

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Fig. 4.16 Reductive citric acid cycle. This metabolic pathway is a reversal of the citric (Krebs) cycle, with some reactions modified in order to push the cycle towards the reductive direction. At each turn, the reductive citric acid cycle converts four molecules of carbon dioxide into oxaloacetate, using eight electrons (from NADH, FADH2 and reduced ferredoxin) and three molecules of ATP. (Redrawn from Buchs 2011)

With the exception of the CBB cycle, all autotrophic carbon fixation pathways described above are strictly anaerobic, as oxygen irreversibly inactivates key enzymes involved. RubisCO, the key enzyme of the CBB cycle, presents a different sort of limitation, being able to function not only as a carboxylase but also as an oxygenase. RuBP oxygenation results in carbon dioxide loss instead of fixation (an effect known as “photorespiration”) and competes with carboxylation in a measure proportional to the ratio between oxygen and carbon dioxide concentration in the proximity to the enzyme. Because of this, oxygen reduces the efficiency of RubisCO in carbon fixation and the yield of the photosynthetic process. Being the key enzyme of autotrophic carbon assimilation in organisms as diverse as cyanobacteria, lytoautotrophic bacteria and plants, RubisCO has most likely been in existence for several billion years (Tabita et al. 2008). Although there is significant natural variation in kinetic parameters of RubisCOs from different organisms, evolution apparently never managed to completely correct the tendency of this enzyme to confuse carbon dioxide with oxygen. The study of the catalytic mechanism of RubisCO helped understanding why, by showing that the carboxylation rate and specificity for carbon dioxide of this enzyme are not independent variables because any change in the active site that improves one, unavoidably worsens the other. The catalytic properties of RubisCO, therefore, appear to be a compromise between

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Fig. 4.17 3-hydroxypropionate cycle. (Redrawn from Rothschild 2008)

opposite evolutionary pressures. In general, the RubisCO extant in nature have much a greater preference for carbon dioxide vs. oxygen, but quite a low carboxylation rate compared with other enzymes (Gutteridge and Pierce 2006; Tcherkez et al. 2006). In spite of sluggish performances, RubisCO and the CBB cycle emerged in cyanobacteria as the best compromise with oxygenic photosynthesis because all alternative autotrophic carboxylation pathways are incompatible with oxygen. To offset low kinetic efficiency, RubisCO is produced in very large amounts, thus being the most abundant protein on the planet. In the cyanobacteria and other bacteria that use RubisCO, the enzyme is almost entirely concentrated in polyhedral bodies named carboxysomes; in photosynthetic eukaryotes, the RubisCO is often concentrated in pyrenoids, dense bodies localized in the chloroplast (Fig. 4.18). Both carboxysomes and pyrenoids act as carbon dioxide concentrating structures that maintain conditions favourable to RubisCO’s carboxylating activity (Raven et al. 2008). Land plants lack pyrenoids (with the exception of hornworts, which reevolved and independently lost pyrenoids several times: Renzaglia et al. 2007), but on several occasions they independently evolved metabolic pathways that concentrate carbon dioxide, for example C4 photosynthesis (Box 12.1). The RubisCO present in cyanobacteria and chloroplasts is classified as “form I” and typically consists of four large and four small polypeptides referred to as rbcL and rbcS, respectively. Other RubisCOs are classified as form II, form III and form IV (Tabita et al. 2008). Form II is an oligomer uniquely comprised of large subunits, found along with form I in some proteobacteria and in dinoflagellates; form III is

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Fig. 4.18 (a) Carboxysome (Cy) in the cyanobacterium Synechocystis. (b) Pyrenoid (Py) surrounded by starch deposits (S) in the chloroplast of the unicellular eukaryote Chlamydomonas reinhardtii. Both carboxysomes and pyrenoids are carbon dioxide-concentrating structures that enhance RubisCO’s carboxylase activity. (a) Kindly provided by Dr. Robert Robertson, Arizona State University, USA. (b) Dartmouth Electron Microscopy Facility, Dartmouth College, http:// remf.dartmouth.edu/imagesindex.html

found only in archaea; form IV is found in gram-positive and gram-negative bacteria including Bacillus subtilis, cyanobacteria and purple photosynthetic bacteria, as well as in archaea. RubisCOs II, III and IV do not perform carbon fixation but instead are involved in subsidiary metabolic pathways, for example archaeal RubisCO III is probably used for disposing of the ribulose 1-5-bisphosphate produced during purine/pyrimidine metabolism (Tabita et al. 2008). RubisCO II possibly participates in the regulation of cellular redox state when the cells have a negative carbon balance, for example in the dark. Molecular phylogeny suggests that RubisCO I derived by horizontal gene transfer from archaeal RubisCO III, although the marginal role that this enzyme performs in archaea appears to be more consistent with the opposite pathway.

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Béjà O et al (2000) Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289:1902–1906 Béjà O et al (2001) Proteorhodopsin phototrophy in the ocean. Nature 411:786–789 Bekker A et al (2010) Iron formation: the sedimentary product of a complex interplay among mantle, tectonic, oceanic, and biospheric processes. Econ Geol 105:467–508 Blankenship RE (2010) Early evolution of photosynthesis. Plant Physiol 154:434–438 Bosak T et al (2009) Morphological record of oxygenic photosynthesis in conical stromatolites. Proc Natl Acad Sci U S A 106:10939–10943 Brasier M et al (2006) A fresh look at the fossil evidence for early Archaean cellular life. Philos Trans R Soc B 361:887–902 Brocks JJ et al (2003) A reconstruction of Archean biological diversity based on molecular fossils from the 2.78 to 2.45 billion-year-old Mount Bruce Supergroup, Hamersley Basin, Western Australia. Geochim Cosmochim Acta 67:4321–4335 Bryant DA (2007) Candidatus Chloracidobacterium thermophilum: an aerobic phototrophic acidobacterium. Science 317:523–526 Bryant DA, Frigaard N-U (2006) Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol 14:488–496 Buick R (2008) When did oxygenic photosynthesis evolve? Philos Trans R Soc B 363:2731–2743 Butterfield NJ (2015) Proterozoic photosynthesis – a critical review. Palaeontology 58:95–972. https://doi.org/10.1111/pala.12211 Crowe S et al (2013) Atmospheric oxygenation three billion years ago. Nature 501:535–538 David LA, Alm EJ (2011) Rapid evolutionary innovation during an Archaean genetic expansion. Nature 469:93–96 Dismukes GC et al (2001) The origin of atmospheric oxygen on Earth: the innovation of oxygenic photosynthesis. Proc Natl Acad Sci U S A 98:2170–2175 French KL et al (2015) Reappraisal of hydrocarbon biomarkers in Archean rocks. Proc Natl Acad Sci U S A 112:5915–5920 Fuchs G (2011) Alternative pathways of carbon dioxide fixation: insights into the early evolution of life? Annu Rev Microbiol 65:631–658 Gupta RS (2012) Origin and spread of photosynthesis based upon conserved sequence features in key bacteriochlorophyll biosynthesis proteins. Mol Biol Evol 29:3397–3412 Gutteridge S, Pierce J (2006) A unified theory for the basis of the limitations of the primary reaction of photosynthetic CO2 fixation: was Dr. Pangloss right? Proc Natl Acad Sci U S A 103:7203– 7204 Hohmann-Marriott MF, Blankenship RE (2011) Evolution of photosynthesis. Annu Rev Plant Biol 62:515–548 Hoshino Y et al (2015) Hydrocarbons preserved in a ~2.7 Ga outcrop sample from the Fortescue Group, Pilbara Craton, Western Australia. Geobiology 13:99–111 Kirschvink JL, Kopp RE (2008) Paleoproterozoic ice houses and the evolution of oxygen-mediating enzymes: the case for a late origin of photosystem II. Philos Trans R Soc B 363:2755–2765 Li W, Beard BL, Johnson CM (2015) Biologically recycled continental iron is a major component in banded iron formations. Proc Natl Acad Sci U S A 112:8193–8198 Lyons TW, Reinhard CT, Planavsky NJ (2014) The rise of oxygen in Earth’s early ocean and atmosphere. Nature 506:307–315 Mukhopadhyay J et al (2014) Oxygenation of the Archean atmosphere: new paleosol constraints from eastern India. Geology 42:923–926 Muller E et al (2017) Primary sulfur isotope signatures preserved in high-grade Archean barite deposits of the Sargur Group, Dharwar Craton, India. Precambrian Res 295:38–47 Nelson N, Junge W (2015) Structure and energy transfer in photosystems of oxygenic photosynthesis. Annu Rev Biochem 84:659–683 Nisbet EG et al (2007) The age of Rubisco: the evolution of oxygenic photosynthesis. Geobiology 5:311–335

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Ohtomo Y et al (2013) Evidence for biogenic graphite in early Archean Isua metasedimentary rocks. Nat Geosci 7:25–28 Olson JM (2006) Photosynthesis in the Archaean era. Photosynth Res 88:109–117 Perez N et al (2013) The potential for photosynthesis in hydrothermal vents: a new avenue for life in the Universe? Astrophys Space Sci 346:327–331 Planavsky NJ et al (2014) Evidence for oxygenic photosynthesis half a billion years before the Great Oxidation Event. Nat Geosci 7:283–286 Rasmussen B et al (2008) Reassessing the first appearance of eukaryotes and cyanobacteria. Nature 455:1101–1104 Raven JA, Cockell CS, De La Rocha CL (2008) The evolution of inorganic carbon concentrating mechanisms in photosynthesis. Philos Trans R Soc B 363:2641–2650 Raymond J et al (2003) Evolution of photosynthetic prokaryotes: a maximum-likelihood mapping approach. Philos Trans R Soc B 358:223–230 Renzaglia KS et al (2007) Bryophyte phylogeny: advancing the molecular and morphological frontiers. Bryologist 110:179–213 Řezanka T et al (2010) Hopanoids in bacteria and cyanobacteria – their role in cellular biochemistry and physiology, analysis and occurrence. Mini-Rev Org Chem 7:300–313 Ricci JN, Michel AJ, Newman DK (2015) Phylogenetic analysis of HpnP reveals the origin of 2methylhopanoid production in Alphaproteobacteria. Geobiology 13:267–277 Rosing MT (1999) 13C-depleted carbon microparticles in 3700 Ma sea-floor sedimentary rocks from West Greenland. Science 283:674–676 Rothschild LJ (2008) The evolution of photosynthesis. . .again. Philos Trans R Soc B 363:2787– 2801 Schirrmeister BE et al (2013) Evolution of multicellularity coincided with increased diversification of cyanobacteria and the Great Oxidation Event. Proc Natl Acad Sci U S A 110:1791–1796 Schirrmeister BE, Gugger M, Donoghue PCJ (2015) Cyanobacteria and the Great Oxidation Event: evidence from genes and fossils. Palaeontology 58:769–785 Schopf JW (2006) Fossil evidence of Archean life. Philos Trans R Soc B 361:869–885 Schopf JW et al (2017) An anaerobic 3400 Ma shallow-water microbial consortium: presumptive evidence of Earth’s Paleoarchean anoxic atmosphere. Precambrian Res 299:309–318 Sugitani K et al (2015) Early evolution of large micro-organisms with cytological complexity revealed by microanalyses of 3.4 Ga organic-walled microfossils. Geobiology 13:507–521 Tabita FR (2009) The hydroxypropionate pathway of CO2 fixation: fait accompli. Proc Natl Acad Sci 106:21015–21016 Tabita FR et al (2008) Phylogenetic and evolutionary relationships of RubisCO and the RubisCOlike proteins and the functional lessons provided by diverse molecular forms. Philos Trans R Soc B 363:2629–2640 Tcherkez GGB, Farquhar GD, Andrews TJ (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Natl Acad Sci U S A 103:7246–7251 Vermaas WFJ (2002) Photosynthesis and respiration in cyanobacteria. Encycl Life Sci. https://doi. org/10.1038/npg.els.0001670 Waldbauer JR et al (2009) Late Archean molecular fossils from the Transvaal Supergroup record the antiquity of microbial diversity and aerobiosis. Precambrian Res 169:28–47

Chapter 5

The Great Oxygenation Event

The evolution of the Earth’s atmosphere is essentially the story of atmospheric oxygen. Virtually every realm of the Earth sciences, biology, geology, geochemistry, oceanography and atmospheric science, is needed to piece together an understanding of the history of oxygen. Catling and Claire (2005)

Abstract Old sedimentary rocks record the history of oxygen in the form of redoxsensitive chemical species such as iron, uranium or cerium ions, and mass-independent fractionation of sulphur isotopes. These proxies show that oxygen became a stable component of the atmosphere around 2.4 GYA, a transition dubbed as the “Great Oxygenation Event” (GOE). Oxygenic photosynthesis is by far the main source of oxygen on Earth. Evidence for transient “oxygen oases” suggests that oxygenic photosynthesis appeared long before the GOE. Methane photolysis in the atmosphere was probably the main oxygen sink preventing stable oxygen accumulation before 2.4 GYA. Around this date, a change in planetary geochemistry permitted average oxygen concentration to rise above a threshold level of about 0.001%; the consequent formation of a thin ozone layer reduced methane photolysis and triggered the transition to an oxic atmosphere. The GOE was coeval with Huronian global glaciations, but the causal link between the two events is uncertain. Oxygen atmospheric concentration stabilized at a low level during most of the Proterozoic. A second rise in oxygen concentration, probably reflecting an increase in global productivity and organic carbon sequestration, started around 800 MYA. The oxygen level was at least 3% 570 MYA and probably exceeded 10% at the beginning of Phanerozoic, thus supporting the evolution of complex life. Water in the ocean depth remained largely anoxic until 600 MYA and accumulated sulphide from biogenic sulphate reduction (euxinic oceans). The GOE profoundly affected biochemistry by promoting the evolution of high energy-yielding aerobic respiration, aerobic lytotrophy and novel biosynthetic pathways involving P450 cytochromes.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_5

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Introduction

The great mountains that we admire in the present world look like immutable, magnificent monuments of the nature, destined to persist forever. In the geological perspective, though, mountains are ephemeral structures destined to be rapidly pulverized and washed away as soon as the forces responsible for their rise cease working. Exposed to the never-ending action of rain and wind, the building rock is chemically attacked by carbon dioxide and water and dissolved in the form of silica and bicarbonate; what remains of the once sturdy framework is broken into fragments that are transported downhill by the rivers and accumulated in sedimentary basins along the coasts. Compressed and chemically modified by the action of their own weight and circulating water, these deposits eventually turn into clastic sedimentary rocks. Other types of sedimentary rocks arise from chemical precipitation of substances present in solution in water basins (chemical sedimentary rocks) or from accumulation of biological material, mainly skeletal structures from unicellular organisms such as radiolarians, foraminifers and diatoms, and from animals (biogenic sedimentary rocks). Sedimentary rocks are even more vulnerable than magmatic rocks (i.e. rocks directly derived from solidified magma) and may undergo repeated cycles of uplifting, erosion, compaction and cementation. Whatever their history, sedimentary rocks eventually get buried at great depths in subduction areas, where they undergo profound chemical and physical transformation due to high pressure and temperature, thus becoming metamorphic rocks. In the course of metamorphosis, only the more resistant parts of the original parent rock, such as zircon crystals (Box 2.2), persist unchanged. There are, however, areas in the planet (the so-called cratons) that fortuitously escaped the ceaseless action of erosion and tectonics and retain extremely ancient, almost intact sedimentary rocks. The reader may now be wondering what have sedimentary rocks to do with the oxygenation of the planet. There is indeed a robust link between the two topics. Sedimentary rocks keep a record of their history, in the form of a diversity of signals such as the size and shape of fragments, the presence or absence of certain minerals, and the isotopic ratios of certain elements. Being the products of interaction of parent rocks with the atmosphere, clastic sedimentary rocks have a lot to say on the past composition of the atmosphere. Some metals such as iron, uranium and cerium can exist in two or more oxidation states that have different solubility in water. For example, iron forms ferrous (Fe+2) or ferric (Fe+3) ions, the former being somewhat soluble in water, the latter forming compounds with very low solubility. Iron in magmatic rocks is mostly in the ferrous form, reflecting the relatively low reduction potential of the mantle. If magmatic rocks undergo erosion in the absence of oxygen, ferrous iron (Fe++) is dissolved and washed away by rainfall, and the sedimentary rocks eventually produced from the fragments will contain very little or virtually no iron. If erosion occurs in the presence of oxygen, instead, ferrous iron is oxidized to the insoluble ferric form (Fe+3) that is incorporated in the ensuing sedimentary rocks.

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Uranium and cerium have an opposite behaviour relative to iron: the minerals formed by less oxidized forms (U+4 and Ce+3) are insoluble, whereas those containing more oxidized forms (U+6 and Ce+4) are soluble and get lost during erosion. Other important redox-sensitive elements are molybdenum, manganese and chromium. The distribution (e.g. presence/absence in sedimentary rocks), chemical form (oxidised/reduced), and isotopic fractionation of these elements are highly sensitive indicators of past levels of oxygen in the atmosphere/ocean/crust (AOC) system. Particularly informative is the distribution of pyrite (FeS2), uraninite (UO2) and siderite (FeCO3) (Catling and Zahnle 2003; Johnson et al. 2014). In the absence of oxygen, the pyrite present in parent rocks is stable and is incorporated as such in sedimentary rocks; in the presence of oxygen, instead, pyrite is rapidly oxidized during weathering, making soluble sulphate that is carried by rivers to the ocean, whereas insoluble iron (III) is retained (Eq. 5.3). A second geochemical record of pyrite oxidation is a rise in chromium concentration in sedimentary rocks (Konhauser et al. 2011). Uraninite and siderite dissolve to form soluble U+6 ions and insoluble Fe+3, respectively, at lower oxygen levels than pyrite (Catling and Claire 2005; Partin et al. 2013; Johnson et al. 2014). Sedimentary rocks formed before 2.4 GYA are usually iron-free (with the exception of banded iron formations, already mentioned in Chap. 4), suggesting that before that date the atmosphere contained very little or no oxygen. In contrast, ferric iron appeared in sedimentary rocks around 2.4 GYA and later became so abundant as to give them a reddish colour, whence the name “red beds” (Fig. 5.1). Much the same signal comes from paleosoils, ancient soils buried under sediments and preserved almost unchanged over long time spans. The most ancient paleosoils currently known, discovered in Australia (Mount Roe) and dated to about 2.8 GYA, have a very low iron content; in contrast, paleosoils formed after 2.4 GYA contain significant amounts of ferric iron. The most sensitive known indicator of oxygen concentration is not from redoxsensitive metals but a type of fractionation of sulphur isotopes that, in contrast to the usual behaviour (Box 2.2), does not depend on mass differences and is therefore known as mass-independent fractionation of sulfur (MIFS). Discovered by Farquar’s group (Farquhar et al. 2000) in Archean sediments, this process is probably due to photolysis by ultraviolet radiation of sulphur dioxide (SO2) from volcanic emissions. MIFS can occur only at atmospheric oxygen concentrations below 10
5 PAL (present atmospheric level, 21%), because higher concentrations produce a UV-shielding ozone layer that stops the process. A MIFS signal regularly occurs in sedimentary deposits throughout the Archean, becomes weaker after 2.4 GYA and disappears about 2.32 GYA (Farquhar et al. 2000; Pavlov and Kasting 2002). MIF is by itself a highly sensitive indicator of oxygen concentration in the AOC system, but its signal may be captured in pyrite and other minerals in sedimentary rocks and transmitted to newly-formed rock independent of oxygen concentration. This ‘crustal memory effect’ implies that repeated cycles of weathering, dilution, burial and uplift beneath an oxygenated atmosphere might have been necessary to erase the MIF signal completely, thus complicating the effort

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Fig. 5.1 Around 2.4 GYA reddish sedimentary formations (red beds) made their appearance on Earth. Their colour is due to the presence of hematite (Fe2O3), a mineral produced by the oxidation of ferrous iron during the erosion of the parent rock. Being insoluble, hematite remained in the sediments and was incorporated in ensuing sedimentary rocks. The picture is an impressive view of the red sandstone formation in the Karijini National Park, Australia

to date the transition (Lyons et al. 2014a). Despite these difficulties, data from a number of sources consistently point to the same scenario: the average level of atmospheric oxygen was below 0.0002% until about 2.4 GYA, with possible temporary oxygen oases developing locally. About 2.4 GYA, oxygen began to build up, causing the complete disappearance of MIF about 2.32 GYA. Oxygen concentration stabilized at a relatively low value for over a billion years after the GOE, yet there is no consensus about the level attained. Chromium isotope analysis of Proterozoic sediments suggests that oxygen atmospheric concentration remained consistently below 0.1% PAL (¼ 0.021%) in the interval between 1.8 and 0.8 GY (Planavsky et al. 2014; Cole et al. 2016). Neither humans nor most animals could survive with such a low oxygen level. Based on the absence of pyrite and uraninite in sedimentary rocks, Johnson et al. (2014) infer that oxygen concentration was never below 1% throughout the Proterozoic. Redox-sensitive metal distribution (molybdenum, uranium and vanadium) suggests that oxygen concentration around 1.4 GYA was about 0.8%, a level sufficient to support respiration of simple animals (Zhang et al. 2016) The transition from an anoxic (oxygen-free) to oxic atmosphere (containing significant amounts of oxygen as a stable component) is known as the Great Oxygenation Event (GOE). Although the name implies a short duration, the event started approximately 2.40 GYA and probably lasted over 200 million years (Bekker 2014a). The oxygenation of Earth surface, including the deep ocean, was complete only at the end of the Proterozoic.

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Fig. 5.2 A planetary lifting. Our planet possibly owns its present blue/white aspect to oxygen accumulation, which drastically reduced the level of methane and methane-derived hydrocarbons in the atmosphere. Before the Great Oxygenation, the Earth probably had the aspect shown on the left, which actually is an image of Titan, one of the satellites of Saturn, whose atmosphere contains some percent of methane and traces of ethane

The Great Oxygenation deeply affected the atmosphere/ocean/continental crust (AOC) system. Elements such as iron and cobalt, which had been abundant in solution in the ocean, disappeared almost completely, others such as copper and zinc became more abundant. The chemical form of some elements underwent a radical change; for example sulphur was present in the ocean mainly as sulphide (S
2) before the GOE, whereas sulphate was the predominant form afterwards. The GOE most likely caused a drastic reduction in the atmospheric concentration of methane (Sect. 5.2), thus allegedly contributing to trigger a succession of global glaciations spanning about 300 million years (Bekker 2014b). Methane removal probably gave our planet its present blue look (Fig. 5.2). The GOE also dramatically affected life. Being a strong oxidant, oxygen is much toxic to non-preadapted organisms. It is common practice, indeed, to treat wounds with hydrogen peroxide (an oxygen-producing chemical) to prevent infection by anaerobic pathogens, notably Clostridium tetani, the causative agent of tetanus. The appearance of oxygen in the atmosphere probably caused the extinction of a great number of life forms. From a menace to life, however, oxygen rapidly turned out to be a great opportunity. New forms evolved, in fact, which not only could withstand oxidative damage by oxygen, but were able to deploy this novel chemical in high energy-yielding aerobic respiration and aerobic lytotrophy (see Martin and Russell 2003 for a review of metabolic variants in prokaryotes).

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Planetary Oxygen Balance

Dioxygen is the obverse of the carbon dioxide coin. For each O2 molecule liberated, a molecule of CO2 is fixed, and the reverse. (Nisbet et al. 2008)

Oxygenic photosynthesis extracts oxygen from water according to the reaction: CO2 þ2H2 OþðhνÞ!CH2 OþH2 OþO2

ð5:1Þ

Water photolysis by UV radiation, a non-biological atmospheric process, produces small amounts of oxygen and hydrogen; the latter is mostly lost to space, leaving the oxygen in the atmosphere (Catling et al. 2001). In accordance with current terminology, we will call sources the processes that cause oxygen to build up in the AOC system. Being highly reactive, oxygen participates in numerous reactions. We will call sinks the processes that remove oxygen. On modern Earth, the main oxygen sinks are aerobic respiration and fire, whose stoichiometry is the opposite of that of oxygenic photosynthesis: CH2 OþO2 !CO2 þH2 Oþenergy

ð5:2Þ

Other important sinks are reactions of abiotic oxidation of reduced compounds emitted by volcanos in gaseous form or as components of magmatic rocks, such as hydrogen sulphide and sulphur dioxide, methane, ferrous iron. A particularly important abiotic sink is the oxidation of pyrite brought to surface by the combined action of tectonics and erosion (Catling and Claire 2005): 3FeS2 þ 3Feþ2 þ 12O2 ! 2Fe2 ðSO4 Þ3 þ 2Feþ3 2Feþ3 þ 2H2 O ! 2ðFe OHÞþ2 þ 2Hþ

ð5:3Þ

[Note that interaction of ferric ions with water produces acid, which possibly facilitates solubilization of other elements such as chromium (Konhauser et al. 2011)] For oxygen to build up in the AOC system, it is necessary that the source exceeds the sink at the global scale, namely: SourceðO2 Þ
SinkðO2 Þ>0 Because oxygenic photosynthesis produces oxygen and organic matter in chemically equivalent amounts (a molecule of oxygen for each carbon atom reduced to carbohydrate), oxygen building up requires that an equivalent amount of organic matter escapes oxidation by oxygen. This occurs prevalently through the burial of organic matter in sediments. Indirect mechanisms of oxygen accumulation are also

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possible, notably the reduction by anaerobic bacteria of sulphate and ferric iron to sulphide and ferrous iron, and subsequent formation of pyrite that is sequestered in sediments on the ocean bottom: 5CO2 þ10H2 OþðhνÞ!5CH2 Oþ5H2 Oþ5O2 ðoxygenic photosynthesisÞ 4CH2 O þ 2SO4
2 ! 4CO2 þ 2S
2 þ 4H2 O ðsulphate
reducing bacteriaÞ CH2 Oþ4Feþ3 þH2 O!CO2 þ4Feþ2 þ4Hþ ðiron 2 reducing bacteriaÞ 2S 2 2 þFeþ2 þ2Hþ !FeS2 þH2 ðspontaneous reaction in anoxic sedimentsÞ The balance of the reactions above is: 2SO4 2 2 þ4Feþ3 þ2H2 OþðhνÞ!FeS2 þ3Feþ2 þ2Hþ þH2 þ5O2

ð5:4Þ

For each mole of pyrite produced biotically and sequestered in sediments, the global system earns five moles of oxygen (Eq. 5.4). Because a fraction of organic matter is continuously buried in sediments (currently about 1% of primary production, Holland 2006), oxygen should have built up to high levels soon after the appearance of oxygenic photosynthesis. The data available depict a different scenario: oxygenic photosynthesis probably appeared long before the GOE but there was a long delay before free oxygen became a stable component of the AOC system (Bekker 2014a; Lyons et al. 2014a; see also discussion in Chap. 4). To further complicate the scenario, oxygen concentration remained substantially stable at a relatively low level for a very long interval after the GOE and started rising again about 800 MYA, reaching nearly modern levels only around 500 MYA (Fig. 5.4). This behaviour suggests that the oxygen level in the AOC system is subject to powerful feedback mechanisms (Sect. 5.3). The total amount of free oxygen in today’s AOC system is equivalent to the photosynthetic production over about 4 million years (Zahnle et al. 2013). The available geochemical evidence suggests that oxygenic photosynthesis appeared at least 200 MY and possibly over 700 MY before the GOE (Crowe et al. 2013; see Sect. 4.1 for further details). The global productivity of Archean Earth was certainly lower than today, yet the level estimated from 13C enrichment of Archaean carbonate deposits (Chap. 4) should have allowed significant oxygen accumulation long before 2.4 GYA (Bekker 2014a). The main gap in the current knowledge of the GOE, therefore, is in explaining why oxygen started rising 2.4 GYA (and not before) and stopped about 200 MY later, to resume only 800 MYA (Kasting 2013). One of the mechanisms initially proposed to explain the delay is biological oxidation of ferrous ions present in solution in the Archean ocean or, in the form

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of minerals, in magmatic rocks exposed to erosion. The formation of red beds clearly shows that iron oxidation did actually occur, at a massive scale, but only after 2.4 GYA. Before that date, banded iron formations (BIFs) were common, yet these probably had nothing to do with oxygen, most likely deriving from the oxidation of ferrous iron of hydrothermal origin by anoxic microorganisms and/or ultraviolet radiation (Bekker 2014c; Crowe et al. 2014; Li et al. 2015). In addition, the oxygen equivalents accumulated in the form of oxidised iron in BIFs, red beds and sulphate deposits in today’s Earth correspond to only 50 MY of oxygen production by photosynthesis at current rates (Zahnle et al. 2013), suggesting that geochemical oxidation is largely insufficient to prevent oxygen accumulation for several hundred million years. In fact, massive worldwide formation of red beds and sulphate deposits after the GOE did not prevent oxygen accumulation. An alternative possibility is that the input of reductants from the mantle to the AOC system and the associated oxygen sink was much higher in the Hadean Earth than today and reduced to a level compatible with oxygen accumulation around 2.40 GYA (Holland 2009). The building up of continental crust may have caused a shift from a prevalently marine to terrestrial volcanism. This, in turn, allegedly determined a change in the composition of volcanic emanations, with a prevalence of hydrogen sulphide before 2.40 GYA and of sulphur dioxide thereafter. Because sulphur dioxide is a less powerful oxygen sink than hydrogen sulphide, the change permitted oxygen to build up (Gaillard et al. 2011). On the same line, Smit and Mezger (2017) propose that the prevalently mafic composition of the exposed crust prevented oxygen accumulation during most of the Archean due to the release of O2scavenging chemicals such as hydrogen sulphide, hydrogen and methane. In this scenario, replacement of the ancestral crust with modern andesitic crust, with a turning point around 2.4 GYA, permitted the AOC system to accumulate oxygen. Although volcanic activity was probably more intense in the Archean than more recently, the GOE appears to be too abrupt to be explained with hypothetical changes in tectonic activity and/or volcanic geochemistry (Lyons et al. 2014a). A third hypothesis focuses on the appearance of multicellular cyanobacteria. According to molecular dating studies, the earliest cyanobacteria appeared in the Middle Archean (around 3.0 GYA), whereas multicellular filamentous cyanobacteria appeared just before the GOE. Ostensibly, by improving the ability of cyanobacteria to form mats both in the aquatic and terrestrial environment, multicellularity may have caused a substantial increase in oxygen production, thus triggering the GOE (Schirrmeister et al. 2013, 2015). This hypothesis does not explain the long gap between the putative appearance of the first cyanobacteria (which by definition already produced oxygen) and the GOE. The same research team also suggests that the heterocysts, nitrogen-fixing cells typical of advanced lineages of multicellular cyanobacteria, evolved after the GOE. A similar hypothesis proposes that benthic microbial communities consumed oxygen from photosynthesis, thus preventing its release in the AOC system; in this perspective, continental landmass expansion triggered the GOE by increasing the areal coverage and photosynthetic efficiency of cyanobacteria (Lalonde and Konhauser 2015). Once again, this model fails to explain the relative rapidity of the GOE and relies on largely theoretical assumptions.

5.3 Methane Was Probably as a Key Driver of Planetary Oxygenation

5.3

137

Methane Was Probably as a Key Driver of Planetary Oxygenation

Excluding changes in tectonics and volcanic geochemistry and biological events, the persistence of an anoxic atmosphere for a long time after the emergence of oxygenic photosynthesis might reflect oxygen removal by a powerful abiotic sink. The “biphasic” model presented by Colin Goldblatt, Tim Lenton and Andrew Watson (Goldblatt et al. 2006) suggests that, after the appearance of oxygenic photosynthesis, the AOC system could set in either of two alternative stable states: an anoxic state with an oxygen concentration below 10
5 PAL, and an oxic state with a concentration above this level. The model holds that the AOC system spontaneously shifts from anoxic to oxic when oxygen rises above 510
3 PAL (about 0.1%), which is the minimum required for the formation of an ozone layer in the stratosphere. The biphasic model focuses on methane. In the anoxic state, life extracts energy from organic matter essentially through fermentation, a process that breaks organic molecules into a more oxidized and a more reduced piece, releasing a small amount of free energy that is used to make ATP. Common products of fermentation are carbon dioxide, molecular hydrogen, methanol, ethanol, acetate. A well-known example is alcoholic fermentation, which converts glucose into two molecules of carbon dioxide and two of ethanol, permitting the synthesis of two molecules of ATP. Under reducing conditions, the bacteria are unable to extract further energy from the products of fermentation. Members of the archaeal lineage Euryarchaeota perform methanogenesis, a metabolic pathway that enables these microorganisms to obtain energy by converting by-products of bacterial fermentation into methane. The archaea exhibit different pathways of methanogenesis. Some reduce carbon dioxide to methane using hydrogen as a reductant: 4H2 þCO2 !CH4 þ2H2 O A second group of methanogenic archaea reduce methanol: H2 þCH3 OH!CH4 þH2 O A third, more extreme group of archaea employ “disproportion” (a form of molecular rearrangement) of acetate: CH3 COO 2 þH2 O!CH4 þHCO3 2 These archaeal lineages are autotrophic because they utilize carbon dioxide as their only source of carbon, converting it into organic carbon through the reductive

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cycle of acetyl-CoA (Sect. 4.5). There are also heterotrophic methanogenic archaea, which disproportionate methanol and utilize intermediates of the methanogenic pathway as a carbon source: 

4CH3 OH!3CH4 þCO2 þ2H2 O ΔG ¼ 2 106 kJ=mol CH4




After the appearance of cyanobacteria, oxygenic photosynthesis was the major source of organic matter: 2CO2 þ4H2 OþðhνÞ!2CH2 Oþ2H2 Oþ2O2 ðorganic matter from photosynthesisÞ

Considering that the eukaryotes most likely appeared in the Mid-Proterozoic and spread only in the late Proterozoic (Chap. 6), it is likely that the food chain in the Archean world was essentially comprised of producers and decomposers, predation (viz. the use of living organisms as food) being rare or almost absent. Whether or not surface ocean water contained traces of oxygen, deeper water and sediments were certainly anoxic and, in the absence of sulphate (see below), the combined activity of fermenting bacteria and methanogenic archaea converted dead organic matter mostly into methane: 2CH2O!CH4þCO2 ðfermentationþmethanogenesisÞ Summing up the two equations above, we obtain: CO2 þ2H2 OþðhνÞ!CH4 þ2O2 ðtotal balanceÞ

ð5:5Þ

Equation 5.5 tells us that ecological interaction of cyanobacteria, fermenting bacteria and methanogenic archaea in the Archean Earth converted carbon dioxide into methane and oxygen in the molar ratio 1:2 (Catling et al. 2007). In the scenario proposed by Goldblatt et al. (2006), for a long while oxygen did not build up in the AOC system because UV-mediated methane photo-oxidation rapidly destroyed it: CH4 þ2O2 þhν!CO2 þ2H2 O ðmethane photo 2 oxidationÞ

ð5:6Þ

By converting methane and oxygen into carbon dioxide and water, methane photo-oxidation did not affect the average redox state of the AOC system; yet, it effectively destroyed oxygen from photosynthesis, thus maintaining its concentration in the AOC system at an extremely low level. Around 2.40 GYA something permitted atmospheric oxygen to rise above the threshold level of about 0.001%. The cause might have been any one of the processes mentioned in the precedent section, or a fortuitous combination of the same. Konhauser et al. (2009) suggest that the culprit was a reduction in the volcanic output of nickel from the mantle; methanogenic archaea require nickel in relatively

5.3 Methane Was Probably as a Key Driver of Planetary Oxygenation

139

large amounts, so a decrease of its concentration from ~400 nM during most of the Archean to ~200 nM about 2.5 GYA might have reduced methane production and favoured oxygen accumulation. At an oxygen concentration above 0.001%, ozone formation is sufficient to inhibit methane photo-oxidation at least partially, thus pushing the system into the oxic state. Goldblatt et al. (2006) suggest that the AOC system stabilized in an oxic state with a low methane level because abiotic oxidation of hydrogen sulphide and sulphur dioxide of volcanic origin led to accumulation of sulphate in the ocean: H2 S þ O2 ! SO2 þ H2 O 2SO2 þ O2 þ 2H2 O ! 2SO4
2 þ 4Hþ

ð5:7Þ

Extremely scarce in the Archean ocean, sulphate allegedly stimulated the evolution/expansion of bacteria performing dissimilatory sulphate reduction, a metabolic pathway that oxidizes organic matter using sulphate as an electron acceptor: 2CH2 OþSO4 2 2 !2CO2 þ2H2 OþS 2 2

ð5:8Þ

In today’s Earth, sulphate-reducing bacteria are the main final decomposers of organic matter in marine sediments, outcompeting methanogenic archaea as long as sulphate-rich ocean water permeates the sediments. Sulphur isotope analysis of sediments of the Transvaal Supergroup (South Africa) documents the build-up of sulphate in the ocean and the disappearance of sulphur MIF from 2.40 GYA (Guo et al. 2009). A second important mechanism underpinning the GOE is probably the oxidation of the AOC system due to hydrogen escape to space (Claire et al. 2006; Kasting 2013; Zahnle et al. 2013). There are numerous hydrogen-producing processes in nature, both biotic and abiotic, for example fermentation, serpentinization of mafic rock, and photolysis of hydrogenated compounds such as water and methane. The latter occurs in the atmosphere and is therefore more conducive to hydrogen loss to outer space. Among several known pathways of methane photolysis, the one reported below converts methane into carbon monoxide and hydrogen through multiple steps driven by ultraviolet radiation: CH4 þH2 Oþhν!!COþ3H2 ðmethane photolysisÞ Methane photolysis still occurs today, but its impact on planetary geochemistry has long ceased to be relevant, because methane is present in trace amounts. Before the GOE, however, methane was almost certainly more abundant and had a longer permanence time because of the lack of oxygen. In the course of over 2 billion years preceding the GOE, methane photolysis has caused the loss to space of substantial amounts of hydrogen. Because of this, the AOC system is now remarkably more oxidized than it should be relative to the mantle, whose redox state probably has not changed much from the beginning (Trail et al. 2011).

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Water photolysis probably also gave a substantial contribution to the oxidation of the planet: H2 Oþhν!H2 þ½ O2 Before the GOE, the same mechanisms that allegedly destroyed oxygen from photosynthesis, did the same with oxygen from water photolysis. Hydrogen escape to space implies that equivalent amounts of free oxygen remained in the AOC system. Zahnle et al. (2013) suggest that the hundreds of millions years elapsed between the appearance of oxygenic photosynthesis and the establishment of an oxic atmosphere were how long it took hydrogen escape to oxidize the Earth surface. Other factors were probably also involved, notably the growth of continental masses, which enhanced the deposition of BIFs, sulphate, carbonate and organic matter. This scenario maintains that the AOC system remained anoxic as long as the sinks produced by continental expansion took pace with hydrogen escape. Hydrogen escape thus acted as a “planetary hourglass” dictating the time of transition. Water photolysis virtually stopped after the formation of an ozone shield, because water condenses in the lower layer of the atmosphere and, unlike methane, is not able to get above the ozone layer, where there is an abundance of highly energetic B and C ultraviolet. Methane can do it but, for reasons mentioned above, its impact on Earth’s geochemistry is currently very low. Today’s rate of hydrogen escape corresponds to the loss of 1 m-deep layer of ocean water per billion years, or equivalently to the production of 0.1 bar of oxygen per billion years (the present inventory of free oxygen in the atmosphere is about 0.21 bar). As a bequest and record of the huge amount of hydrogen lost to space in consequence of methane and water photolysis, an equivalent amount of oxidized matter is now present in the AOC system, mainly in the form of ferric iron and other oxidized metals, sulphate and carbonate. Oxygenic photosynthesis does not affect the global reducing potential of the AOC system, because it produces equivalent amounts of organic matter (a reductant) and oxygen (an oxidant), both remaining on the Earth surface. It is incorrect, therefore, to refer to oxygen accumulation as the “Great Oxidation”. Indeed, a massive oxidation of the planet by water and methane photolysis preceded oxygenation, and virtually stopped because of oxygen accumulation. The GOE was associated with the Lomagundi event, a worldwide excursion of δ13C in carbonates to high positive values between 2.22 and 2.06 GYA, suggesting simultaneous massive burial of organic matter (Bekker 2014c). Conservative estimates for oxygen release coupled to increased organic carbon burial during the Lomagundi Event range from 12 to 22 times the present atmospheric inventory, accounting for the significant increase in oxygen concentration recorded for the same period. The Logamundi event might reflect a transient increase in global productivity due to enhanced phosphorus flux from continental masses to the ocean, in turn caused by acidification consequent to sulphur dioxide oxidation to sulphate (Eq. 5.7).

5.4 The GOE Was Associated with a Long Phase of Climatic Instability

141

Oxygenation initially affected only the atmosphere and superficial ocean water, the deeper water remaining largely anoxic and generating the so-called euxinic oceans, rich in sulphides. Euxinic oceans own their existence to the reduction of sulphate to sulphide by anaerobic bacteria (Eq. 5.8), therefore they did not exist before the GOE. The deep ocean was oxygenated only at the end of the Proterozoic, when oxygen concentration underwent a further substantial increase, and euxinic oceans disappeared since almost anywhere. Some euxinic basins, however, still exist in modern Earth, a well-known example being the Black Sea, where a sharp boundary (the “chemocline”) between 50 m and 100 m depth separates oxic surface waters from anoxic bottom waters rich in sulphides (Canfield 1998; Canfield et al. 2007; Lyons et al. 2009).

5.4

The GOE Was Associated with a Long Phase of Climatic Instability

The Archean Sun was less powerful than today. The early Earth avoided being locked in a perpetual cycle alternating long periods of extreme freezing and spells of extreme warming, thanks to the combined greenhouse action of atmospheric carbon dioxide and methane, then probably much more abundant than today (Chap. 2). According to the model presented in the previous section, as long as methane was the main end product of organic matter decomposition, oxygen from oxygenic photosynthesis was entirely removed by UV-driven methane photo-oxidation. When sulphate-reducing bacteria replaced methanogenic archaea as the major final decomposers of organic matter in sediments, methane production could no longer cope with oxygen production. The ratio of the two gases in the atmosphere rapidly reversed, oxygen becoming abundant and methane a trace component. The evolution of aerobic methanotrophy (Sect. 3.7) furtherly contributed to reducing methane concentration in the atmosphere. Paradoxically, after triggering the GOE, the formation of an ozone layer reduced methane destruction by ultraviolet radiation, permitting this molecule to persist in the atmosphere, albeit at much low levels than before. Thus, methane contribution to planetary warming up by the greenhouse effect (Box 2.1) became much weaker after the GOE. In combination with the lower luminosity of the Sun (only 80% of present luminosity), this may have contributed to trigger the Huronian Glaciations, a succession of global glaciations that took place between 2.4 and 2.1 GYA (Tang and Chen 2013; Bekker 2014b). Compared with Cenozoic glaciations, global glaciations were quite a different story. An ice sheet over 1000 m thick covered the oceans nearly from pole to pole for several million years, and a thinner but equally persistent ice layer covered the continental masses. Each of these events must have caused an extremely severe mass extinction, yet life managed to persist, at least at permissive oases in the equatorial area or near volcanic sources of heat.

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How can geologists know about glaciations that occurred such a long time ago? Massive erosion due to the growth and recession of glaciers produces distinctive patterns of debris deposition that may persist for very long times. The occurrence of large glacial deposits of the same age in continental areas that at the time were at a long distance from each other is considered evidence of global-scale glaciations. A second important indication of past glaciations comes from changes in the 13C/12C ratio in carbonaceous deposits coeval with glacial deposits. Under permissive conditions, photosynthetic activity depletes 12C in the carbon dioxide pool of the atmosphere and ocean, thus coeval sedimentary carbonates (limestones) are enriched in 13C. During extreme glaciations, photosynthetic activity stops almost completely, and this type of fractionation disappears. The data indicate that, between 2.4 and 2.1 GYA, the Earth experienced three events of global glaciation (Sturtian, Marinoan and Gaskiers), each lasting about 10 million years, with long interglacial intervals. Covering the planet surface with a thick layer of ice, global glaciations interrupt the water cycle: due to the cold, very little water vapour passes from the ocean to the atmosphere, and there is not enough vapour condensation to produce rain. Because of this, the global-glaciation condition is unstable. With no rainfall, silicate weathering stops and carbon dioxide from volcanoes accumulates in the atmosphere. Volcanic activity not balanced by silicate weathering may increase atmospheric carbon dioxide by many thousands ppm in a few million years. Due to the greenhouse effect of carbon dioxide, the planet warms up, sea ice melts, the water cycle restarts and silicate weathering resumes working. Because of the inherent inertia of large systems, the planet will experience a “runway greenhouse warming” before conditions return to normality: in a matter of centuries, a brutally hot, wet world supplants the deep freeze. Eventually the planet returns to a more permissive average temperature but, if conditions conducive to global freezing persist, a new glaciation will ensue. This cycle stops and the Earth enters a stable phase of equilibrium when the factor(s) underpinning global freezing are no longer active. After the Huronian Glaciations, the Earth remained warm until about 760 million years ago, when it entered Neoproterozoic Glaciation an even more severe succession of global glaciations (Donnadieu et al. 2014). These terminated around 580 MYA, the return to more permissive conditions paving the way to the first appearance of complex multicellular organisms (the Ediacaran Biota) and, slightly later, to the sudden appearance of numerous animal phyla still in existence today, an event dubbed “Cambrian Explosion”. An important insight from this story is that, because of a combination of factors including astronomical position, size, geological and geochemical properties, and biology, the Earth tends to set itself at an average temperature above the water freezing point, in a range permissive to life as we know it. We may refer to this as the “equilibrium state” (Sect. 2.4). Under special circumstances, though, the Earth may be forced to abandon the equilibrium state and to enter cycles of climatic instability with alternating phases of global freezing and runaway greenhouse warming. No signs of glaciation is visible in rocks older than 2.4 GY, although in earlier times the Sun was even weaker than in the Proterozoic. On the other hand, the level

5.4 The GOE Was Associated with a Long Phase of Climatic Instability

143

Fig. 5.3 The genesis of global glaciations and runaway greenhouse warming according to the runaway ice-albedo feedback model

of oxygen has been very high throughout the last 500 MY, and carbon dioxide plummeted to extremely low levels twice (in the Carboniferous and Cenozoic) without precipitating the Earth into a global glaciation. Thus, besides a drop in greenhouse gases and the “weak Sun”, other factors are probably necessary to force the Earth out of its equilibrium state. According to the runaway ice-albedo feedback model proposed in 1992 by geologist Joseph L. Kirschvink at California Institute of Technology and re-elaborated by Hoffman and Schrag (2002), a fundamental condition conducive to global glaciations is the gathering of continental masses at low latitudes (Fig. 5.3). A minor reduction in the atmospheric level of carbon dioxide, caused for example by an occasional decrease in volcanic outgassing, might induce a slight cooling of the planet and the formation of ice sheets at the poles. Ice has a greater albedo than liquid water, so the development of ice caps at the poles causes a further reduction of the average global temperature. Normally, this immediately slows down silicate weathering in continental masses located at high latitudes, thus increasing the atmospheric concentration of carbon dioxide to the level required to maintain the global temperature within the “equilibrium range”. According to Kirschvink’s model, if the continental masses are gathered in the tropical/equatorial area, they will remain warm enough for silicate weathering to keep going at the usual rate despite a decrease in the average global temperature. This may activate a runaway ice-albedo feedback: the ice caps will keep expanding and increased albedo will further reduce the average temperature. At a point, the process becomes unstoppable, the ice caps get to the equator and the Earth becomes locked in a global glaciation for several million years. There is evidence supporting this scenario for Neoproterozoic glaciations. Around 1.2 GYA the continental masses assembled into a single supercontinent, Rhodinia, localized in the equatorial area. About 825 MYA, Rhodinia started

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breaking up into separate continents, which remained for a long while at low latitudes. According to Kirschvink’s model, this facilitated the start of Neoproterozoic glaciations 760 MYA. The coldest phase, known as the Cryogenian Period, terminated about 635 MYA, when a part of the continents had moved to higher latitudes; one last event of lower intensity (the Gaskier glaciation) occurred in the Ediacaran around 580 MYA (Donnadieu et al. 2014). Interestingly, after a gap of over a billion years, the BIFs temporarily reappeared during the Cryogenian, due to the ice cover isolating deep ocean water from the atmosphere (Lenton and Watson 2011). An additional factor possibly involved in the genesis of Cryogenian glaciation was increased albedo from cloud-condensation nuclei deriving from the activity of photosynthetic eukaryotes, which appeared around 900 MYA and spread in the oceans (Shih and Matzkeb 2013; Feulner et al. 2015). The causes and timing of Huronian Glaciations as well as the extent of ice cover are still a matter of speculation. Archaean plate reconstructions suggest the assembly of a Northern and a Southern Supercontinent, both of which experienced protracted breakup shortly followed by the onset of “icehouse” conditions about 2.4 GYA (Tang and Chen 2013). This suggests that the low-latitude scenario described above is not applicable to the Huronian glaciations, other forces being probably involved. Donnadieu et al. (2014) discuss alternative competing scenarios for the origin of global glaciations. Challenging the classical view that the rise in oxygen concentration was the main cause of global glaciations (see, for example, Kopp et al. 2005), Laakso and Schrag (2017) propose a novel scenario in which the Huronian and Cryogenian glaciations created the conditions for a transient burst in atmospheric oxygen concentration. Allegedly, this triggered the transition from the Archean to Proterozoic state and from the Proterozoic to Phanerozoic state (Fig. 5.4).

5.5

After the GOE, the Earth Stabilized in a Low-Oxygen State for over 1 Billion Years

Traditional models maintain that, after the GOE, oxygen concentration remained stable for over a billion years at a level estimated around 0.8% (Zhang et al. 2016). An alternative model suggest that oxygen rose to this level or even more between 2.4 and 2.2 GYA, but then plummeted to about 0.02% and remained around this level until about 800 MYA (Partin et al. 2013; Planavsky et al. 2012; Lyons et al. 2014a; Cole et al. 2016; Fig. 5.4). The persistence of a low oxygen level during most of the Proterozoic may account for the widespread distribution of anaerobic biochemistry in the eukaryotes (Mentel and Martin 2008; Van der Giezen and Lenton 2012), which most likely emerged and diversified between 2 and 1 BYA (Chap. 6). Oxygen stabilization over such a long time span indicates that powerful feedback mechanisms kicked in after the GOE, preventing both a return to anoxic conditions and further oxygen accumulation.

5.5 After the GOE, the Earth Stabilized in a Low-Oxygen State for over 1 Billion Years

145

Fig. 5.4 The history of atmospheric oxygen encompasses three phases. The pink curve shows a classical, two-step model of atmospheric evolution, while the blue curve shows a more dynamic screenplay emerging from research, with a larger initial increase followed by a plunge to much lower levels for most of the Proterozoic. The following narrative refers to the traditional scenario. Phase I: from the appearance of life to about 2.40 GYA, oxygen concentration was below an upper limit of 0.001% PAL set by the record of mass-independent sulphur-isotope fractionation (MIF). Transient local increases above this level are consistent with some proxies. Phase II: starting about 2.40 GYA, oxygen concentration rose to about 0.8% and remained stable at this level for over a billion year since. Phase III: around 800 MYA, a second rise in oxygen concentration brought the level to about 510
1 PAL (~10%) around 550 MYA; a peak to over 25% is recorded around 300 MYA and a plunge to less than 15% in the Permian; oxygen concentration then rose again, attaining ~21% about 30 MYA and remaining stable since. A lower limit of ~ 0.02% and an upper limit of ~2% for oxygen concentration in phase II is set by MIF and chromium (Cr) isotope data, respectively. A lower and upper limit of about 12% and 33% in the last 420 million years are set by charcoal records (plant material does not burn below 12%) and by the persistence of forest ecosystems (forests would be destroyed by fire above 33%). Oxygen fluctuation during the last 600 MY is shown in detail in Fig. 11.26. Oxygen concentration is expressed as percent of the present atmospheric level (PAL). (Figure adapted from Lyons et al. (2014a), licence number 4464870363372)

As already mentioned, oxygen accumulation in the AOC system reflects the amount of organic matter sequestered in sediments. This parameter depends on net global primary production (viz. the amount of organic matter produced autotrophically on the whole planet each year, net of respiration), which in turn is affected by nutrient availability. The higher is nutrient availability, the higher the primary production, the greater the stock of organic matter sequestered, and the larger the amount of oxygen left in the AOC system, or vice versa. On the other hand, increased sequestration of organic matter in sediments immobilizes greater amounts of nutrients, thus exerting a negative feedback on primary productivity (Fig. 5.5). The picture also needs to incorporate Liebig’s low of the minimum, stating that the growth rate of living organisms does not depend on the total amount of the essential resources available, but only on the scarcest resource, which acts as the limiting factor. In the modern world, nitrogen enters the biological cycle through dinitrogen (N2) fixation by nitrogenase, an enzymatic complex inhibited by oxygen. Organic nitrogen is released as ammonia (NH4+) by decomposition; ammonia can be aerobically oxidised to nitrate (NO3
), which under anoxic conditions can be converted into dinitrogen by heterotrophic denitrifiers or autotrophic ammonia-oxidising

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Fig. 5.5 The level of oxygen in the atmosphere is under the control of both positive (full arrows) and negative feedbacks (dotted arrows) from multiple, intertwined processes. The figure shows only a part of the complex interplay of biological and geochemical processes

(anammox) bacteria (Kartal et al. 2013). The oxygenation of the AOC system in the Late Archean probably reduced nitrogen fixation and stimulated denitrification. This possibly led to a “global nitrogen crisis” that limited primary production until the second oxygen rise in the Late Proterozoic (Falkowski and Godfrey 2008). Phosphate enters the ocean through weathering of apatite-containing rocks and of organic matter on continental masses, and exits the biological chain by sequestration in sediments, where it is reconverted into apatite. Iron persisting in Proterozoic oceans because of low oxygen concentration might have acted as a trap to phosphate, reducing the concentration of its bioavailable fraction to limiting values (Laakso and Schrag 2014; Planavsky 2014). Planetary modelling shows that a combination of enhanced phosphate sequestration in anoxic, iron-rich oceans and a nutrient-based bistability in atmospheric oxygen levels (i.e. the ability to set in a low- or high-level state in response to nutrient availability) is conducive to a lowoxygen world as recorded in the Early to Mid-Proteozoic (Reinhard et al. 2017). The hypothesis implies that the level of dissolved iron during this time span must have been high enough to reduce phosphate bioavailability, but too low to support BIF formation, which virtually disappeared after 1.8 GYA. The development of large areas rich in sulphide ions in the anoxic ocean depth (“euxinia”) might have lowered the availability of other essential microelements, such as manganese, molybdenum and vanadium, which precipitated to the ocean bottom in the form of sulphides (Scott et al. 2008; Lyons et al. 2009). Manganese is essential for oxygenic photosynthesis as a component of the water-splitting complex, molybdenum and vanadium are (alternative) cofactors of nitrogenase. A comprehensive analysis of metal enrichment records supports the possibility of a Mo–N co-limited marine biosphere during many periods of Earth’s history (Reinhard et al. 2013). In conclusion, paleogeochemical inference suggests that the Proteorozoic AOC system set in a state of low primary productivity because of low levels of nutrients in the ocean, which in turn limited oxygen accumulation (Lenton and Watson 2011).

5.6 The Earth Entered a High-Oxygen Phase About 800 Million Years Ago

147

Because of the complexity of the interactions involved and the scarcity of data, however, the geochemical history of the Proterozoic remains poorly understood. Because of prolonged climatic stability and the absence of extreme changes in the atmosphere and ocean, palaeontologist Martin Brasier dubbed the interval between 1.8 and 0.8 GYA as the “boring billion years”. Before this time interval, the Earth had experienced the appearance of life, the evolution of oxygenic photosynthesis, the Great Oxygenation and a succession of global glaciations. After the “boring billion”, the animals made their appearance and rapidly produced an amazing diversity of forms, plants colonized the land, oxygen rose to modern levels and mighty biological radiations alternated with multiple mass extinctions. The “boring billion”, however, was not so boring, having seen three major biological events: the evolution of eukaryotic cells (Chap. 6), multicellularity (Chap. 7) and sexual reproduction (Chap. 8). Even in the absence of major geological or geochemical changes, these innovations are important enough as to make the “boring billion” not so boring.

5.6

The Earth Entered a High-Oxygen Phase About 800 Million Years Ago

After a billion year of stability, at the end of the Proterozoic the level of atmospheric oxygen started rising again, attaining at least 3% 570 MYA (Canfield et al. 2007) and probably exceeding 10% at the onset of Phanerozoic (Berner et al. 2007). Compared with the extremely low levels recorded during the preceding billion years, this was a transition as relevant as the GOE itself. What did happen? In the absence of evidence for major geological events, the most likely cause is an increase in global productivity (Fig. 5.5), yet identifying possible drivers is again an exercise of speculation. Several options are available, including (a) the evolution of nitrogen-fixing planktonic cyanobacteria, (b) an increase in phosphate availability in the ocean, (c) the appearance of photosynthetic eukaryotes, and (d) the colonization of land by phototrophic organisms. Far from being mutually incompatible, these factors may have worked together, pushing the Earth towards the modern highoxygen condition. Nitrogen is an essential nutrient that often limits ecological productivity; on today’s Earth, nitrogen fixation by planktonic cyanobacteria is essential to sustain primary productivity in the open ocean, which receives little nitrogen input from the land. Molecular clock analysis suggests that nitrogen-fixing planktonic cyanobacteria appeared between 900 and 650 MYA, in response to a rise of molybdenum and vanadium concentration in the ocean; this event may have increased primary productivity in the open ocean, thus stimulating oxygen accumulation (Sànchez-Baracaldo et al. 2014). Low concentrations of molybdenum and vanadium in the Proterozoic ocean probably reflected metal sequestration in the form of insoluble sulphides in euxinic ocean waters; an increase in the concentration of

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molybdenum and vanadium requires a rise in oxygen concentration sufficient to oxygenate deep ocean water. Thus, the hypothesis poses a classic chicken and egg dilemma, not considering that there is strong evidence for a worldwide persistence of euxinic conditions up to 600 GYA (Lyons et al. 2009, 2014b). In conclusion, the appearance of nitrogen-fixing planktonic cyanobacteria is unlikely to be the ultimate cause of the Late-Proterozoic oxygen rise, although it probably contributed to sustain oxygen accumulation in the aftermath. Increased sulphur isotope fractionation associated with and immediately following the Sturtian and Marinoan glaciations might reflect increased nutrient load to the ocean from glacial melting. This stimulated primary production and carbon burial, thus increasing atmospheric oxygen level (Canfield et al. 2007). This hypothesis received support from the detection of a worldwide peak of dissolved phosphate in the interval between 750 and 635 MYA (Planavsky et al. 2012). As discussed in detail in Chap. 9, the evolution of the chloroplast around 900 MYA led to emergence of numerous lineages of photosynthetic eukaryotes that spread in internal water bodies and in ocean areas along continental shelves. Unicellular green and red algae most likely colonized terrestrial habitats very soon after their appearance and eukaryotic phytoplankton became dominant over prokaryotic plankton about 800–600 MYA (Planavsky 2014). In association with an increase in nutrient availability, this could have enhanced global primary productivity, thus contributing to the transition from 0.8% oxygen concentration or less in the Mid Proterozoic to about 8% or more in the Late Proterozoic. Lenton and Watson (2011) suggest that a critical event in the Late Proterozoic was land colonization by lichen-like organisms. Present-living lichens are symbiotic associations of multicellular fungi (usually ascomycetes) with cyanobacteria or green unicellular algae (Fig. 5.6a). Free-living fungi live on organic matter from the environment. Lichen-forming fungi grow a vegetable garden in their own body. They produce organic acids (mainly oxalic acid) that chemically dissolve the rock and mobilize mineral nutrients that are transferred to the photosynthetic symbiont in exchange for organic matter (essentially simple sugars). Lichens usually grow very slowly but are able to survive extreme dehydration, freezing cold and scorching heat, and are among the first organisms that colonize newly formed bare rocks, thus giving the start to soil formation. Not surprisingly, lichens are found almost anywhere on land, from Antarctica to tropical rain forests. Modern lichens evolved only in the Phanerozoic, but lichen-like associations might have appeared much earlier, as suggested by fossils from the Doushantuo Formation (China), dated to 551–635 MYA (Yuan et al. 2005). Land colonization by lichenlike organisms might have accelerated the erosion of superficial rocks and increased the output of mineral nutrients from continental masses to the ocean, thus inducing a substantial increase in ocean productivity and the consequent rise of oxygen. A possible example of a “protolichen” is Geosyphon pyriforme (Fig. 5.6b), a primitive fungus that hosts intracellular cyanobacteria and lives autotrophically (Kluge 1994). A second important event marking the end of Proterozoic was the appearance of animals, currently antedated to about 600 MYA or even before. The diffusion of

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Fig. 5.6 (a) Modern lichens are symbiotic associations of multicellular fungi (mostly ascomycetes) and cyanobacteria or green microalgae. Lichens are pioneer organisms that colonize bare rocks exposed to air. The fungal hyphae attack the rock and mobilize mineral nutrients that support the photosynthetic symbiont; in exchange, this hands over organic carbon to the fungal partner. (b) Geosiphon pyriforme is a unicellular multinucleate fungus hosting thousands of cyanobacterial cells in large hyphal swellings. Land colonization by Geosiphon-like organisms in the Late Proterozoic might have contributed to the rise of oxygen by enhancing rock weathering and nutrient input to the ocean

animals accelerated the sinking of organic matter to the ocean bottom, in the form of dead remains and excrements, probably giving a significant contribution to the increase in the oxygen stock (Butterfield 2011). With the appearance of land plants, around 470 MYA, global productivity and organic carbon stocked in sediments rose to levels never attained before, causing a further oxygen rise. The atmospheric oxygen concentration was probably close to PAL (21%) at the end of Devonian (380 MYA), and rose to 25% or perhaps more between the Carboniferous and Permian (around 300 MYA). In concomitance with a catastrophic mass extinction, the oxygen level plummeted to below 15% at the end of Permian, then rising again and attaining the present level about 30 MYA (Fig. 5.6). The fascinating book by Lenton and Watson (2011) gives a detailed analysis of past oxygen fluctuations and underpinning mechanisms.

5.7

The Impact of Oxygen on Biological Evolution

After fluorine, oxygen is the element with the highest reduction potential, meaning that it has a strong tendency to accept electrons and, in doing so, it liberates great amounts of energy (Box 3.1). Importantly, when oxygen accepts electrons, it binds protons to form water, a life-loving molecule. It is not surprising, therefore, that oxygen is the first-choice terminal electron acceptor in the majority of life forms. The oxygen atoms in the bi-atomic molecule O2 are prevalently in a state named “triplet oxygen”. The oxygen molecule may also exist in other states more rich in energy than the triplet state, differing from the latter and from each other in the

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orbital distribution and spin of the four electrons engaged in the molecular bond. The most common high-energy state is “singlet oxygen”, represented with the symbol 1 O2 to distinguish it from triplet oxygen 3O2. Triplet oxygen is scarcely reactive; in order to react with other molecules, even in strongly exergonic reactions such as combustion, oxygen needs to be in the singlet form. The conversion of triplet oxygen into singlet oxygen requires energy in the form of heat or electromagnetic radiation. This is the reason why organic matter does not spontaneously react with oxygen in the air, if not at a negligible rate, and wood does not catch fire at normal environmental temperatures. A local temperature rise, known as “ignition”, is necessary to produce the first singlet oxygen molecules and start combustion, the heat from the reaction then ensuring continuation. Self-supporting combustion of organic matter, even in a very dry state such as paper or hay, requires an oxygen concentration of 17% or higher (Belcher and McElwain 2008). In biological systems, oxygen-using reactions proceed at low temperature and low partial pressure of oxygen thanks to enzymes that lower the activation energy of reaction. On the other hand, several biological redox processes tend to produce singlet oxygen, which is one of the reactive oxygen species (ROS) that damage cellular structures (Lane 2002). The main utilization of oxygen by life is in aerobic respiration. The term “respiration” covers the wide range of metabolic pathways that couple exergonic oxidation of organic matter to ATP synthesis. Life utilizes a great diversity of oxidants. Besides fermentations, a form of respiration in which the final electron acceptor is an organic molecule (Sect. 5.2), there are several forms of anaerobic respiration among the bacteria, which use a diversity of inorganic oxidants including nitrate, nitrite, sulphate, Fe+3, manganese dioxide (MnO2), and compounds of arsenic, selenium, chromium and even uranium. Fermentations produce ATP with the mechanism known as the substrate-level phosphorylation. The other forms of respiration, instead, accumulate energy by chemiosmosis (Box 3.1). In terms of energy, aerobic respiration is the most efficient among extant respiratory pathways. By completely oxidizing glucose to carbon dioxide, aerobic respiration produces energy theoretically sufficient for the synthesis of 36/38 molecules (the real number estimated is 30/32); in comparison, alcoholic fermentation “dismutates” glucose into two molecules of ethanol and two of carbon dioxide, with a net production of energy sufficient to drive the synthesis of only two molecules of ATP. Because of its superior efficiency, aerobic respiration probably evolved very soon after the GOE. Key enzymes involved in biological redox reactions, including photosynthesis, non-fermentative respiration and lytoautotrophic processes, present remarkable structural similarities in organisms as diverse as bacteria, archaea and eukaryotes (Tomiki and Saitou 2004; Schoepp-Cothenet et al. 2013). This most likely reflects rampant involvement of horizontal gene transfer, but also points to shared ancestry. For example, the cytochrome b/c1 complex (or complex III) of aerobic respiration is closely similar to the cytochrome b6/f complex of photosynthesis, and could have evolved from this. The cyanobacteria are able to perform both oxygenic photosynthesis and aerobic respiration, the biochemical machinery of

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photosynthesis being restricted to thylakoid membranes, whereas the enzymes of the respiratory chain are located in both the thylakoid and cell membrane (Vermaas 2001). Interestingly, numerous forms of facultative aerobic bacteria and, in some cases, even mitochondria are able to shift to anaerobic respiration and use nitrate, nitrite or fumarate instead of oxygen as electron acceptors (Mentel and Martin 2008). Aerobic respiration, therefore, might have evolved with relative ease by recombining and modifying pre-existing metabolic pathways (Baymann et al. 2003; Ducluzeau et al. 2009; Schoepp-Cothenet et al. 2013). Once appeared, we do not know yet how many times, when or where, aerobic respiration rapidly spread by horizontal gene transfer, and today it is common both in the bacteria and archaea. Simulations of metabolic networks under anaerobic or aerobic conditions revealed that molecular oxygen underpins over 1000 more metabolic reactions than in anaerobic systems (Raymond and Segré 2006). In addition, because all oxygen-consuming reactions are irreversible, the aerobic metabolism is thermodynamically more efficient. A comparative analysis of the chemical structures, properties and reactions of anaerobic and aerobic metabolites (Jiang et al. 2012) has shown that aerobic metabolism considerably expanded the structural diversity of metabolites by introducing at least 130 novel “molecular scaffolds”. Aerobic metabolism also helped organisms to explore novel “chemical space” by increasing the hydrophobicity and molecular stiffness of metabolites. Because hydrophobic metabolites are fit to modulate membrane functions and to serve as transmembrane signalling factors, these metabolic innovations most likely paved the way to the emergence of complex cellular organization and multicellularity. The establishment of an aerobic metabolism is tightly linked to the evolution of the superfamily of enzymes known as P450 cytochromes or CYPs, containing a covalently bound heme group similar to that in hemoglobin (Nelson 2013). The most common reaction catalysed by P450 cytochromes is the insertion of one atom of oxygen into aliphatic chains. Over 21,000 CYPs are known in archaea, bacteria and, above all, in eukaryotes. For understanding the importance of CYPs, it suffices to note that these enzymes have a crucial role in the synthesis of the following molecules: – Cholesterol and steroids in all eukaryotes; cholesterol controls membrane resistance and flexibility in processes such as phagocytosis, vesicle fusion, ameboidal movement, syngamy. – Cutin and suberin in land plants and analogous hydrophobic coatings in insects, essential for the adaptation of these organisms to terrestrial life. – Lignin, a cell wall-strengthening polymer that enables land plant to attain large sizes and translocate water over long distances (Chap. 11). – Sporopollenin, the complex polymer that coats and protects land plant spores. – A great diversity of secondary metabolites including glycosides, terpenoids and alkaloids, of great importance in plant defence and as a source of drugs for humans. RubisCO, the key enzyme of the Calvin-Benson-Bassham (CBB) cycle, is a major player in the planetary homeostatic system that controls oxygen level.

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Because of its intrinsic carboxylase/oxygenase nature (4.7), RubisCO reacts to increasing O2 or decreasing CO2 concentration by reducing the carboxylation rate, with a negative impact on global primary production. A decrease in primary production in turns reduces organic carbon burial, thus negatively affects oxygen accumulation. Higher CO2 levels or lower O2 levels induce an opposite response (Nisbet et al. 2008). A major consequence of planetary oxygenation and the emergence of an aerobic metabolism was the evolution of the mitochondrion and of modern eukaryotes in the Mid Proterozoic, an event pivotal to a multiplicity of further changes (Chap. 6).

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Chapter 6

Eukaryotes

If you find the story confusing, welcome to the club. Maynard Smith and Szathmàry (1995)

Abstract The eukaryote pangenome has a chimeric structure encompassing genes unique to eukaryotes (~41%), and genes of bacterial (~50% of total) and archaeal ancestry (~9%). Crucially, bacterial sequences far exceed the set acquired from the proteobacterial ancestor of mitochondria. Extant eukaryotic lineages share a common ancestor (LECA) that had all the fundamental traits of eukaryotes including the mitochondrion. The current debate about eukaryote origins revolves around two competing scenarios. The fusion model posits that the eukaryotes derive from the “fusion” of an archaeon and a bacterium, and that the acquisition of the mitochondrion was pivotal to the evolution of other eukaryotic traits. The neomuran model maintains that the archaea and eukaryotes are sister groups devived from a bacterial ancestor, and that fundamental eukaryotic traits including phagocytosis were already in place before the evolution of the mitochondrion by endosymbiosis. Eukaryote placement within the archaea in phylogenomic analysis supports the fusion scenario. The predominance of bacterial sequences in the eukaryote pangenome, the bacterial stereochemistry of eukaryote membrane lipids, and similar trajectories in mitochondrial and chloroplast evolution favour the neomuran scenario. Phylogenomic analysis resolves two major eukaryotic domains, the Amorphea and Diaphoretika, with traditional Excavata being probably paraphyletic. The root of the eukaryote tree remains elusive. Paleontological evidence and molecular clock analysis date the eukaryote lineage to at least 1.5 GYA, the concestor of extant eukaryotes to about 1.2 GYA, and major extant lineages to 900 MYA or less. The chapter includes a review of mitochondrial properties and of locomotor organelles in bacteria, archaea and eukaryotes.

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6 Eukaryotes

Introduction

The origin of eukaryotes (eukaryogenesis) and their evolutionary relationships with bacteria and archaea are a contentious issue of modern evolutionary biology, perhaps even more than the origin of life. The eukaryotes share scores of fundamental characters with the prokaryotic world, but also display a set of unique traits that reflect a deep evolutionary divide (Poole and Neumann 2011). These include phagocytosis and the endomembrane system, a unique cytoskeleton (Box 6.1), the nucleus, mitosis and cell cycle, flagella with 9 + 2 architecture, the supramolecular organization of DNA, unique mechanisms of control of gene transcription and translation and a large number of genes with no homologs in bacteria or archaea. Last, but not the least, the eukaryotes have the mitochondrion, an ATP-producing organelle derived from a symbiotic bacterium. In addition, photosynthetic eukaryotes have the chloroplast, a photosynthetic organelle derived from a cyanobacterium. In both cases, a part of the original genome of the symbiont was lost and a part was transferred to the nucleus and incorporated in the host genome, but a small fraction remains within the organelles and is essential for their functioning. Large-scale sequence analysis has revealed that the eukaryotic pangenome has a chimeric composition (Koonin 2010); besides a number of genes unique to eukaryotes, it encompasses genes with bacterial homologs (only a minor part of which were acquired with mitochondria or chloroplasts), and genes with archaeal homologs (Fig. 6.1). Making the picture even more complex, the genes with bacterial affinity mainly control metabolic functions (“house-keeping” or “operational” genes), whereas those with archaeal affinity are mostly involved in information processing (“informational genes”); eukaryote-specific genes include both operational and informational genes (Embley and Martin 2006; Yutin et al. 2008; Ku et al. 2015). Not surprisingly, such a complex scenario has inspired a multitude of hypotheses on the origin of the eukaryotic lineage. Comparing ribosomal RNA (rRNA) sequences, in the 1970s Carl Woese and his research group discovered a prokaryotic group distinct from the bacteria. Ribosomal RNA has a master role in the assembling of ribosomes; most mutations that affect its three-dimensional structure negatively affect the translation mechanism, thus they are promptly eliminated by natural selection. Consequently, rRNA is an extremely conservative molecule: its sequence differs very little even in far-related groups (Pace 2009). In initial experiments, rRNA sequences from prokaryotes and eukaryotes segregated into two groups, as expected. This orderly behaviour abruptly changed when methanogenic prokaryotes were included in the search. The rRNA from these organisms segregated into a group separate from the other prokaryotes and from eukaryotes. Compared with “conventional” prokaryotes, the novel group presented low internal diversity in rRNA sequence; thus, it was considered an ancient lineage dubbed “archaebacteria”, the rest of prokaryotes being re-named “eubacteria” (Woese and Fox 1976). The two terms are still in use today, but the terms “archaea” and “bacteria” proposed by Woese in 1990 are now more popular (Fig. 6.2).

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Fig. 6.1 Genomic analysis of (a) the fungus Saccharomyces cerevisiae and (b) the red alga Cyanidioschyzon merolae, two distantly related unicellular eukaryotes. From the top, proceeding clockwise, the first two coloured sectors represent genes with archaeal homologs, the following 15 sectors are genes with bacterial homologs, whereas genes with no recognized archaeal or bacterial homolog are in the white sector. The width of each sector is proportional to the gene number indicated. Major archaeal and bacterial groups are colour-coded and denoted 1–18. In both species, over 50% of the genes have a putative bacterial ancestry, but only a minor fraction of these are from the ancestor of the mitochondrion (an α-proteobacterium) or the chloroplast (a cyanobacterium). Competing models of eukaryogenesis give different explanations for the chimerism of the eukaryote genome. (Redrawn from Koonin (2010))

The Archaea rapidly expanded, with the addition of halophilic “bacteria” and other forms from extreme habitats. The discovery of a further set of distinctive properties (Table 6.1) confirmed the idea of the existence of a third Domain of life in addition to bacteria and eukaryotes. Besides a number of divergences, however, homologous traits in rRNAs and 34 ribosomal proteins universally retained in the three domains (Yutin et al. 2012) firmly point to shared ancestry for all extant life. Further investigation in the 1980s and 1990s identified many other archaeal taxa and produced novel insight on the molecular biology of the group, notably DNA replication and gene expression. The result was another surprise: despite their prokaryotic morphology, the archaea present major affinities to eukaryotes than to bacteria (Table 6.2). To account for shared traits, a novel tree in which the archaea and eukaryotes formed a sister clade to the bacteria replaced the tree of life proposed by Woese and Fox in 1990 (Fig. 6.2a, b). The subsequent discovery of important

Fig. 6.2 (a) Early version of the Tree of Life, with three domains diverging from a Last Universal Common Ancestor, or LUCA (Woese and Fox 1976). (b) After the discovery of major affinities between the archaea and eukaryotes, these two lineages were assumed to arise from a shared ancestor and to form a sister clade to the bacteria (Woese et al. 1990). Both model A and model B posit that each of the three domains is a clade, i.e. a monophyletic group (Box 1.1). (c) Eocytic model: the eukaryotes branch within the archaea, forming a clade with an archaeal assemblage originally named “eocytes” (ancient cells) and currently referred to as the TACK (Sect. 6.5 for further details). (d) The latest version of the “neomuran“model by Cavalier-Smith (2014) holds that the Bacteria domain is ancestral to the archaea and eukaryotes, these being sister groups diverged from a gram-positive bacterial ancestor. The eocytic model implies that the Archea is a paraphyletic group; the neomuran model implies that the bacteria is paraphyletic. b, redrawn from Woese et al. 1990; (c, redrawn from Williams et al. 2013). See Pace (2009) for a wider discussion of the topic

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Table 6.1 Unique properties of the Archaea (Cavalier-Smith 2002a). Archaeal taxonomy currently recognizes two major lineages, the Euryarchaeota and Chrenarchaeota, plus several lineages of uncertain rank and position (Petitjean et al. 2015) 1. Isoprenyl-ether (instead of acyl-esther) membrane lipids. 2. Ribosomal three-dimensional structure 3. Non-hollow flagellar shaft of acid-insoluble glycoprotein related to pilin (coniugation pili), not acid-soluble flagellin (Box 6.2). 4. DNA-binding protein 10b promoting negative DNA supercoiling at high temperatures. 5. Unique tRNA modifications, notably archaeosine in position 15 instead of guanine, and absence of queine in the “wobble” position in anticodon. 6. A small protein, LX, in the large ribosomal subunit. 7. Absence of Hsp90 chaperonin (Hsp70 and Hsp90 chaperonins present in bacteria and eukaryotes) 8. The gene for largest RNA polymerase subunit (rpoA) split into two separate genes (but a nonfragmented rpoA gene has been found in two chrenarchaeote species) 9. The gene for largest glutamate synthetase subunit (gltB) split into three separate genes. 10. Reverse gyrase (also present in hyperthermophilic bacteria, probably due to HGT from archaea) Table 6.2 Major properties shared by the Archaea and Eukaryotes but absent in Bacteria (Cavalier-Smith 2002a) 1. Proteins are inserted in, or translocated across membranes only co-translationally, with the participation of a signal-recognition complex (SRP) containing a 7S RNA and a translation-arrest domain that delays the extension of the polypeptide chain until the ribosome/nascent protein complex binds to a SRP receptor anchored in the target membrane. The bacterial SRP complex lacks both the 7S RNA and the delay mechanism. 2. Murein (also known as peptidoglycan, a fundamental component of bacterial cell wall) is lacking. An analogue of murein (pseudomurein) not containing muramic acid evolved secondarily in the Methanobacteriales, an archaeal lineage belonging to the Euryarchaeota. 3. Histones: H1, H2a, H2b, H3 and H4 in the eukaryotes, homologs of H3 and H4 in the Euryarchaeota, none in Chrenarchaeota. A possible homolog of H1 occurs in Actinobacteria. 4. DNA polymerase of the B type, i.e. inhibited by aphidicoline. 5. TATA boxes (repeated sequences of adenine-thymine) initiate transcription (absent in genes transcribed by RNA polymerases I and III in eukaryotes). Sigma factors absent (essential in bacteria to initiate transcription). 6. Several unique DNA-repair enzymes. 7. Similarities in ribosomal RNA and proteins; ribosomes insensitive to chloramphenicol; anisomycin inhibits peptidyl transferase. 8. CCA 30 terminus of tRNA added post-translationally, not gene-encoded. 9. Protein synthesis initiates with methionine, not N-formyl methionine. 10. Multiple origins for chromosome replication.

sequence homologies between the eukaryotes and a subgroup of archaea initially known as the “eocytes” led to the formulation of the “eocytic” tree of life, in which the eukaryotes diverged from within the archaea (Cox et al. 2008), which therefore started being treated as a paraphyletic group (Box 1.1). A more recent formulation of the eocytic model identifies the TACK group (from Thaumarchaeota, Aigarchaeota, Crenarchaeota e Korarchaeota) as the archaeal lineage more closely related to the

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eukaryotes (Williams et al. 2012, 2013; Williams and Embley 2015; Sect. 6.6). Whether the eukaryotes are sister to the archaea (Fig. 6.2b and d) or derived from an archaeal progenitor (Fig. 6.2c), Woese’s notion of three domains of life still stands up, with most genes in archaeal genomes clustering together in phylogenetic trees, away from bacterial or eukaryotic homologs when these exist. Box 6.1 Comparing the Prokaryotic and Eukaryotic Cytoskeleton Prokaryotes and eukaryotes are sharply different in size, cellular organization and morphogenetic abilities. The prokaryotic genome is normally active and ready to transcription, whereas the eukaryotic genome is by default in a nonactive state, DNA being bound to histones and genetically silent. When considering a large range of biological diversity, however, the gap between the two groups becomes less distinct, with several instances of partial overlapping. For example: (a) Histone homologs occur in both bacteria and archaea, besides eukaryotes (Table 6.2). (b) A cytoskeleton is present both in archaea and bacteria and, as in eukaryotes, participates in cell division, cell growth and spatial arrangement of subcellular components (Shih and Rothfield 2006; Michie and Löwe 2008; Shaevitz and Gitai 2010; Wickstead and Gull 2011; Rowlett and Margolin 2015). (c) Some bacteria have internal membrane compartments distinct from the cell membrane, and members of the Planctomycetes (e.g. Gemmata) have a nucleus-like structure (Murat et al. 2010; McInerney et al. 2011). (d) Bacteria such as Thiomargarita or Epulopiscium are so large as to be visible to the naked eye (Levin and Angert 2015). (e) The Actinobacteria synthesize cholesterol and other sterols (CavalierSmith 2002a). There is, however, a property unique to eukaryotes, phagocytosis, the ability to engulf other cells (or just solid particles) within a membranebound compartment known as phagosome. Prokaryotes have evolved sophisticate forms of predation and possess some of the factors that in eukaryotes are involved in phagocytosis (Sect. 6.2), yet phagocytosis is absent and probably never evolved in prokaryotes. Why? To address this important question we might follow the reasoning by Julie Theriot, a cell biologist at the University of Washington, USA (Theriot 2013). Phagocytosis requires cells of relatively large sizes, in order to accommodate their preys. With large cells, diffusion rate becomes an important limiting factor; eukaryotic cells have solved the problem by developing mechanisms of (continued)

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Box 6.1 (continued) intracellular transport based on actin and tubulin. Prokaryotes (bacteria and archaea) lack analogous systems of transport, yet they have a diversity of cytoskeletal structures, some made of homologs of eukaryotic cytoskeletal proteins, others unique to prokaryotes. Prokaryotic homologs of actin and tubulin bind nucleotides (ATP for actin and GTP for tubulin homologs) and hydrolyse them while polymerizing. Quite surprisingly, compared with the diversity of cytoskeletal structures in prokaryotes, the eukaryotes have only two cytoskeletal structures, tubulin microtubules and actin microfilaments, which however perform a vast diversity of functions. This extraordinary versatility is due to accessory proteins, notably nucleation factors and molecular motors (Wickstead and Gull 2011). Nucleation factors (NF) are protein complexes that bind free actin or tubulin and trigger polymerization. For actin, the best-characterized nucleator is the Arp2/3 complex, which has two actin-related proteins as part of the complex and five other proteins holding these together. For microtubules, the best-characterized nucleator is the γ-tubulin ring complex, consisting of 13 molecules of γ-tubulin (a paralog of α- and β-tubulin, Fig. 6.3). Besides specific nucleation factors, eukaryotes have three classes of molecular motors: myosins for actin microfilaments, kinesins and dyneins for microtubules, all lacking obvious prokaryotic homologs. Prokaryotes have motors that act on DNA and RNA but no prokaryotic cytoskeleton-associated motor or nucleator. Prokaryotic cytoskeletal proteins, in fact, polymerize spontaneously. The evolution of nucleators does not appear to be particularly difficult, so their absence in prokaryotes probably reflects a deeper divergence in the functional architecture of the prokaryotic and eukaryotic cell. To control the assemblage of cytoskeletal scaffolds in space and time, prokaryotes do not use nucleators bur merely stabilize or de-stabilize spontaneously polymerizing filaments. The involvement of nucleators/motors imparts the eukaryotic cytoskeleton radically different properties (Fig. 6.4). Prokaryotic and eukaryotic cytoskeletal structures both follow the kinetic pattern known as “dynamic instability”, characterized by simultaneous assembly and disassembly of filaments. Yet, prokaryotic filaments grow and shrink at both ends, whereas eukaryotic filaments can do that only at the free end (named plus end) because the end bound to the nucleating complex (minus end) is much more stable. An important consequence of this apparently minor detail is that nucleators impart directionality to the eukaryotic cytoskeleton, enabling it to perceive, produce and transmit spatial information. (continued)

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Box 6.1 (continued)

Fig. 6.3 (a) Nucleation of actin filaments by Arp2/3. (b) Nucleation of microtubules by the γ-tubulin ring complex. From Theriot (2013), Creative Commons Attribution 2.0 International License

Theriot suggests that differences between the prokaryotic and eukaryotic cytoskeleton may ultimately depend on the way spatial information is generated and transmitted in the two cell types. Bacterial cells usually have a single chromosome bound to the cell envelope in a spatial arrangement that is faithfully reproduced at each cell division (Toro and Shapiro 2010). As a result, genes maintain a specific cellular location across cell generations; this provides spatial information that the bacterial cell can use whenever necessary, for example during cell growth and cell division, without the participation of cytoskeletal structures. In evolving phagocytosis, the progenitor of the eukaryotic cell had to solve a number of intertwined problems: (a) The cell envelope had to be set free from the burden of chromosome spatial organization. (b) The cell envelope had to become flexible enough to form blobs and vesicles. (c) A novel system of spatial sensing had to replace the prokaryotic system based on the cell envelope. (d) Mechanisms for active intracellular transport had to compensate for negative effects of larger cellular sizes. (continued)

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Box 6.1 (continued)

Fig. 6.4 (a) In bacteria, cytoskeletal filaments form spontaneously and aggregate into mixed-polarity bundles (i.e. filaments in the same bundle are oriented in either direction). The best-known example of this type of cytoskeletal structure is ParM filaments in Escherichia coli, an actin homolog. (b) In eukaryotes, cytoskeletal proteins polymerize under the control of nucleators (blue circles) and form assemblages of filaments all with the same polarity. The three-dimensional arrangement of these structures requires the participation of motors besides nucleators, as for example is the case of the mitotic spindle. (From Theriot 2013, Creative Commons Attribution 2.0 International License)

All this was probably obtained with the evolution of nucleators and molecular motors, although the succession of steps is far from clear. (continued)

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Box 6.1 (continued) A long-ranging consequence of the innovations outlined above was the evolution of the nucleus and mitosis. In contrast to the relative simplicity of the prokaryote machinery, chromosome segregation in eukaryotes is driven by a sum of forces created by tubulin (de)polymerization at kinetochores (plusend), depolymerization at spindle poles (minus-ends) and MT-dependent motors bound at kinetochores, chromosome arms, polar microtubule nucleators and cell cortex (McIntosh 2016). The superior efficiency of the mitotic machinery permitted the eukaryote genome to grow by orders of magnitudes and to split into several chromosomes. Theriot’s conclusion is that the evolution of phagocytosis was the primary step in eukaryogenesis, because not only it produced a novel feeding mechanism but also paved the way to the evolution of other fundamental traits of eukaryotic cellular organization. Interestingly, cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesin paralogs that appear to be the only universal cytoskeletal motors in red algae, myosin being absent in most taxa including Porphyra, and dynein motors completely absent. This minimal cytoskeletal set may help explaining why cells and multicellular structures in red algae are more limited in size than in other multicellular lineages (Brawley et al. 2017). Go to https://www.youtube.com/watch?v¼Aa0cvmsD_2Q to see large amoebas feeding on paramecium cells.

6.2

Rise and Fall of the Archaezoan Model of Eukaryogenesis

The hypothesis of an endosymbiotic origin for the chloroplast and mitochondrion was first formulated at the beginning of past century by Russian botanist Konstantin Mereschowsky and French zoologist Paul Portier, essentially based on the apparent ability of these organelles to replicate by fission. For a long time dismissed or ignored, the hypothesis was developed and popularised by Lynn Margulis in the 1960s with novel support from ultrastructural and biochemical data, notably the discovery of two bounding membranes and a genome in both organelles (Margulis 1970). In the 1990s, the endosymbiotic model acquired further strength with the discovery of eukaryotic microorganisms lacking mitochondria, such as the Archamoebae, Metamonada (e.g. Giardia and Trichomonas) and Microsporidia. Molecular phylogeny initially clumped these organisms together at the very base of the eukaryotic tree, so they were portrayed as the surviving descendants of a eukaryotic lineage predating the evolution of mitochondria. Thomas Cavalier-Smith dubbed this putative novel group “Archaezoa” (ancient protists) and incorporated the novel information into his archaezoan model, according to which an ancient member of the Archaezoa engulfed a member of purple non-sulphur bacteria and converted it into a

6.2 Rise and Fall of the Archaezoan Model of Eukaryogenesis

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Fig. 6.5 The classic endosymbiotic model received strong support from the discovery of unicellular eukaryotes apparently lacking mitochondria (the “Archaezoa”). The model lost credit when, at the turn of the century, molecular research showed that the lack of mitochondria was a derived character. The current wisdom is that all present-living eukaryotes arise from a mitochondriate ancestor (LECA, Last common Eukaryotic Ancestor), and that some lineages secondarily lost the mitochondria

mitochondrion, thus giving rise to “mitochondriate” eukaryotes. The Archezoan scenario enjoyed great popularity in the 1980s and early 1990s and was readily incorporated into the standard textbook account of eukaryote origins (Fig. 6.5). At the turn of the century, a first hit to the archaezoan model came from the recognition that the aggregation of putatively amitochondriate eukaryotes at the base of the eukaryote tree did not reflect real phylogenetic affinities, being a consequence of a technical bug dubbed long-branch attraction. With sparse taxonomic sampling and simple tree-building software, taxa with large sequence divergences were artificially attracted to one another and to the base of the eukaryotic tree (Keeling 1998). A second, more serious hit was the discovery that amitochondriate eukaryotes do invariably possess nuclear genes of mitochondrial origin, indicating that the lack of mitochondria is not a primitive trait but the consequence of a loss. More recently it was found out that in most cases the mitochondria have not disappeared completely but have been converted into small organelles that retain a double-membrane envelope and still perform metabolic functions, but are no longer engaged in respiration (Embley and Martin 2006; Sect. 6.7.1). In line with these findings, phylogenetic analysis has re-positioned amitocondriate taxa within primarily mitochondriate eukaryote lineages (Baldauf 2008; Hampl et al. 2008; Wegener Parfrey et al. 2010). Amitochondriate taxa no longer need aerobic respiration, being adapted to live in oxygen-poor habitats such as sediments rich in organic matter or in body cavities of animals including humans (Hjort et al. 2010).

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In summary, novel data emerged after the formulation of the archaezoan model indicate that: (a) the eukaryote genetic system has a chimeric structure; (b) all major eukaryotic lineages primarily possess mitochondria, although some include forms that secondarily lost these organelles; (c) the mitochondria of extant eukaryotes have a monophyletic origin. In this framework, the central issue of eukaryogenesis is the nature of the cell that “accommodated” a bacterium in its inside and converted it into a mitochondrion. We will refer to this organism as the host or the nuclear line, and to its genome as the nuclear genome. It is necessary to note, however, that some eukaryogenesis models propose that the nucleus evolved after the mitochondria and other important eukaryotic traits. A second and no less contentious issue is when the eukaryotes appeared on stage. Multiple hypotheses have been proposed, none of which has gained unanimous consensus and most have been abandoned in the light of novel insight. We will briefly examine a couple of post-archaezoan models that have received special attention, referring to a few excellent reviews for a more exhaustive exploration of the topic (Kurland et al. 2006; Poole and Penny 2006; O’Malley 2010; Lake 2015; Martin et al. 2015; Archibald 2015; Silar 2016).

6.3

Post-archaezoan Models

Most hypotheses formulated after the crisis of the archaezoan model assume that the eukaryotes emerged from the “fusion” of an archaeon and a bacterium, thus they will be referred to as “fusion” or “chimeric” models. Two such models were proposed almost simultaneously in 1998, both inspired by the discovery of communities of methanogenic archaea and sulphate-reducing bacteria living in a close metabolic interaction, or “syntrophy”, in anoxic sediments rich in organic matter. In these environments, the bacteria dismutate organic matter into carbon dioxide and hydrogen, which the archaea use to make methane; by maintaining low concentrations of carbon dioxide and hydrogen, the archaea make the metabolic activity of bacteria thermodynamically more convenient. Moreira and LòpesGarcìa (1998) suggested that an archaeon and a number of sulphate-reducing δproteobacteria merged together, producing a proto-eukaryotic chimeric organism with a nucleus and a primitive endomembrane system (Fig. 6.6). With time, this evolved phagocytosis and acquired the mitochondrion from a α-proteobacterial symbiont, thus giving rise to the progenitor of present-living eukaryotes (LECA, Last Eukaryotic Common Ancestor). A more recent variant of this model incorporates novel insight from phylogenomics and molecular cell biology (Baum and Baum 2014). The “fusion” model by Martin and Müller (1998), later revisited by Martin and Russell (2003) and Lane and Martin (2010), is more simple and direct than the model of Moreira and Lòpez-Garcìa and is currently the mainstream hypothesis (Lane 2015, 2017). It holds that a methanogenic archaeon engulfed a facultatively

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Fig. 6.6 Syntrophic model of eukaryogenesis by Moreira and Lòpez-Garcìa (1998). (a) Syntrophic association of a methanogenic archaeon (ar) and sulphate-reducing anaerobic bacteria (ba). (b, c) The cellular association became more intimate, with the archaeon forming cell projections that increase the contact area between the symbionts, and the bacteria eventually coalescing together. (d, e) The archaeon eventually became the nucleus (n), and the cell membranes of the merged bacteria formed a primitive endomembrane system (em); bacterial genes were transferred to the nucleus. The proto-eukaryote cell thus formed subsequently evolved phagocytosis and acquired the mitochondrion by conventional endosymbiosis. Assuming that the cell membrane and the endomembrane system of the proto-eukaryote are of bacterial origin, this model explains why these membranes are made of acyl-esther, not isoprenyl-ether lipids. The model also accounts for the chimeric nature of the eukaryotic genome. Its weak points are cell fusion, a mechanism unknown in prokaryotes, and a simplistic description of the origin of the endomembrane system and nucleus. (Redrawn from Moreira and Lòpez-Garcìa (1998))

anaerobic α-proteobacterium and converted it into a mitochondrion (Fig. 6.7). This event gave the host access to a much greater amount of energy, powering an evolutionary radiation that gave rise to the diversity of present-living eukaryotic lineages. Martin’s model differs from that of Moreira and Lòpez-Garcìa mainly in assuming that the acquisition of mitochondria was the initial, not the conclusive step of eukaryogenesis. Both models assume that the host was a methanogenic archaeon; methanogenesis is a metabolic pathway only known in the Euryarchaeota archaeal lineage; the two syntrophic models, therefore, are at odd with updated versions of the eocyte tree, with the eukaryotes nested outside the Euryarchaeota (Fig. 6.2c; Gribaldo et al. 2010; Spang et al. 2015). Because methanogenic archaea are strict anaerobes, it is unclear what benefit would the archaeal host obtain from a “proto-mitochondrial” symbiont. Dunn (2017) attempts to overcome this difficulty by proposing that the initial driving force for the maintenance of the symbiont in the archaeal host was heat production. Allegedly, this permitted the thermophilic archaeon to live in cooler habitats, thereby removing obstacles to the evolution of cellular complexity.

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Fig. 6.7 (a-d). The “fusion” scenario as proposed by Martin and Müller (1998) and subsequently revisited by Martin and Russell (2003). (a) Syntrophic association of a methanogenic archaeon and a facultatively anaerobic hydrogen-producing α-proteobacterium. (b, c) The bacterium was engulfed by the archaeon and converted into a mitochondrion (d). With metabolic integration of the two symbionts, the archaeal host shifted from autotrophy to heterotrophy and replaced the original isoprenyl-ether lipids (shaded red) with bacterial acyl-esther lipids (shaded blue). All other distinctive traits of the eukaryotic cell, including an endomembrane system, phagocytosis, a nucleator-dependent cytoskeleton and a nucleus, appeared after the acquisition of the mitochondrion. According to the proponents of the model, the engulfment of the bacterium by the archaeal host did not involve phagocytosis but was simply a consequence of cells “tightly clinging” to each other, much as postulated in the Moreira and Lòpez-Garcìa (1998) model. (e). Phylogenetic diagram based on the fusion model. The Bacteria and Archaea are assumed to have diverged from LUCA at a very early stage. (a-d, adapted from Martin and Russell (2003), Creative Commons Attribution 4.0 International License)

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Expressed as watt (1 W ¼ 1 J/s) per gram of cells, the energy produced by a typical oxygen-respiring prokaryote is around 0.19 W, whereas it is only 0.06 W in eukaryotes. If energy production is referred to the amount of DNA per cell, however, a different picture emerges: prokaryotes turn out to have an average budget of about 0.02 pW (1 pW ¼ 1012 W) per million base pairs (Mb), eukaryotes about 0.38 pW/ Mb, i.e. 19-fold more. When considering only coding sequences instead of total DNA, the difference becomes even more pronounced. Assuming an average of 5000 and 20,000 genes for typical prokaryotic and eukaryotic genomes, respectively, the average energy production per gene is about 0.03 fW (1fW ¼ 1015 W) in prokaryotes vs 57.15 fW in eukaryotes, i.e. 1900-fold more. The recognition that the mitochondria give eukaryotic cells access to a much greater energy budget per gene relative to prokaryotes is considered evidence supporting the hypothesis that eukaryogenesis started with the mitochondrion (Lane and Martin 2010; Lane 2011, 2014). In both prokaryotes and eukaryotes, DNA replication averagely uses only about 2% of the energy budget; in contrast, protein synthesis uses about 75%. Having access to a greater amount of energy per gene, eukaryotes not only can produce larger numbers of different proteins, but also more copies of each protein. In addition, average eukaryotic proteins are longer and encompass more domains than their prokaryotic counterparts, which is probably advantageous in terms of stability and versatility (Brocchieri and Karlin 2005). The larger energy budget permits the eukaryotes to have a genome (total DNA) 10–10,000 times larger and a number of genes one order of magnitude higher (104 vs 103) than in prokaryotes. A further important point is that the average energy per gene increases geometrically with cell sizes; for example, the budget is about 0.9 fW in the minute planktonic alga Ochromonas, 5 fW in the medium-sized flagellate Euglena, and 222 fW in the “giant” amoeba Amoeba proteus. The reason is that cell size increase usually involves an isometric increase in the mass of mitochondria, but a much more modest increase in the number of genes (Box 6.3). According to the interpretation offered by Lane and Martin (2010), the acquisition of mitochondria favoured an increase in cell sizes, thus triggering the evolution of phagocytosis and other eukaryotic traits. The possibility to maintain and express a large number of genes most likely also paved the way to the evolution of a complex multicellular organization, which appeared several times among the eukaryotes but is unknown in prokaryotes. Size increase in prokaryotes involves a proportional increase in the number of genome copies and so it does not improve the energy budget per gene (Fig. 6.8).

6.4

The Neomuran Model

Taking on board the new data emerged in the 1990s, in 2002 Cavalier-Smith proposed a new model of eukaryogenesis that drastically revised the phylogenetic interrelationships of the three domains. This model posits that eukaryotes plus archaea are a clade derived from a gram-positive bacterial ancestor that replaced

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Fig. 6.8 The energy budget in a medium-sized prokaryote (Escherichia), a very large prokaryote (Thiomargarita), and a medium-sized eukaryote (Euglena). The energy produced per gram fresh weight of cells (a) is maximum in Escherichia, minimum in Thiomargarita and intermediate in Euglena. Conversely, the energy per gene (b) or per haploid genome (c) is relatively low in both prokaryote models, and much higher in the eukaryote. The mitochondrial chemiosmotic machinery (Box 3.1) needs constant and rapid interaction with genes that codify for key components. During the process that converted the bacterial symbiont into a mitochondrion, these genes were retained in the organelle, whilst a number of genes necessary for general maintenance were transferred to the nucleus, most of the rest being lost. As a result, each mitochondrion contains a small set of essential genes, but most of the genes servicing all mitochondria in the same cell are present as single copies in the nuclear genome. Lane and Martin (2010) argue that genome streamlining and the transfer of most essential genes to the nucleus converted the mitochondria into a formidable energy-producing machine that increased the host cell energy budget per gene (or per haploid genome) by orders of magnitude. Thiomargarita cells (b) are so big as to be visible by the naked eye; to grow to such extent, this bacterium produces thousand copies of its chromosome, which are distributed at regular intervals along the cell membrane (represented as blue bodies in the drawing), with a giant vacuole filling the inside of the cell. Thus, despite size increase, the amount of energy per gene (or haploid genome) remains in the prokaryotic range. (Adapted from Lane and Martin (2010), licence number 446491044575)

the original cell wall of murein (peptidoglycan) with a more flexible envelope of N-glycoproteins linked to the cell membrane (Figs. 6.2d and 6.9). To emphasize the evolutionary relevance of this change, Cavalier-Smith named the putative EukaryoteArchaea clade Neomura (from the Latin murus, wall), whence the name given to his model. An updated version of the neomuran model appeared more recently (CavalierSmith 2014).

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Fig. 6.9 Neomuran model of the tree of life and eukaryogenesis. The Eukaryotes and Archaea are considered as sister groups forming the Neomura clade. The putative neomuran ancestor was a gram-positive bacterium (possibly an actinobacterium) that replaced the murein cell wall with a flexible pellicle of N-glycoproteins bound to the cell membrane. Ancestral Neomura emerged as forms adapted to moderately hot and acid conditions. The Archaea and Eukaryotes diverged from ancestral Neomura by specializing as extreme thermoacidophyles and phagotrophs, respectively. (Redrawn from Cavalier-Smith (2006))

In the neomuran scenario, the Neomura arose from a bacterial ancestor that had lost the murein wall, possibly due to casual mutation of an essential gene (as occurs in the so-called “L forms” of gram-positive bacteria: Errington 2013), or in response to environmental pressures such as antibiotics inhibiting murein synthesis, or acidic conditions de-stabilizing murein cross links. The substitution of a flexible pellicle of N-glycoproteins for the rigid murein wall, an event that Cavalier-Smith calls “neomuran revolution”, permitted the mutant cell to survive and adapt to normally unfavourable conditions. In line with this model, the enzymes responsible for Nglycosylation of integral membrane proteins in eukaryotes and archaea (dolicholdependent glycosyl transferases) present clear sequence homologies with each other and also with undecaprenol-dependent glycosyltransferases involved in murein synthesis in gram-positive bacteria, suggesting evolutionary continuity between the two pathways (Cavalier-Smith 2014). A second important innovation postulated by the neomuran model was the adoption of histones for DNA packaging in substitution of bacterial DNA gyrase. A part of present-living archaea (within the Euryarchaeota) have proteins similar to histones H3 and H4; in addition to H3 and H4, the eukaryotes have histones H1, H2a and H2b. A possible reason for the evolution of histones was adaptation to high but not extreme temperature, which might have required more stable DNA packaging. According to Cavalier-Smith, the use of histones for DNA packaging in the Neomura accelerated the evolution of the part of the genome that controls gene duplication and expression (informational genes) but had a minor effect on

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operational genes, which therefore retained ancestral affinities with their bacterial counterparts. Two sister lineages diverged from a neomuran ancestor followed sharply different but equally innovative adaptive pathways. One is the Archaea, which retained a prokaryotic cellular organization, reinforced the cell envelope with novel solutions, replaced ancestral acyl-esther G3P lipids with isoprenyl-ether G1P lipids, evolved reverse gyrase (a novel DNA-handling enzyme probably derived by fusion of a DNA topoisomerase and DNA helicase: Gribaldo and Brochier-Armanet 2006), and adapted to conditions of anaerobiosis, low reducing potential, extreme temperature and acidity. Most likely, the primary habitat of the archaea was anoxic sediments rich in organic matter, where they flourished by extracting energy from by-products of bacterial fermentation (Valentine 2007). Seizing the opportunity offered by a flexible cell envelope, the other lineage evolved phagotrophy, i.e. the ability to ingest other cells and use them as food, thus giving rise to the eukaryotes (Valas and Bourne 2011). Phagotrophy gave the novel lineage access to entirely new niches and deeply modified the flow of matter and energy in ecosystems. Being a complex and potentially dangerous process, phagotrophy could not emerge suddenly from scratch. As mentioned in Box 6.1, several entwined innovations were necessary, including a novel type of cytoskeleton, mechanisms for controlled vesicle budding and fusion, and a radical change in the topology of DNA and cell division (Sect. 6.5). By retaining the notion that the first step of eukaryogenesis was the evolution of phagocytosis, the neomuran model leaves the archaezoic scenario almost unchanged, yet it introduces radically innovative phylogenetic inferences. In summary, the neomuran model holds that: (a) The bacteria (which Cavalier-Smith calls “eubacteria”) are near the root of the Tree of Life, whereas the archaea and eukaryotes form a clade (the Neomura) derived from a gram-positive bacterial progenitor. (b) The distinctive properties of the informational system of the Neomura reflect rapid adaptation to moderately hot and acid habitats. (c) Being sister to each other, the eukaryotes and archaea have the same age; thus, in contrast to mainstream belief, the archaea must be younger than the Bacteria. (d) The Archaea are not a new Domain of life but rather a third kingdom of Bacteria, better referred to under the name “archaebacteria”. (e) Unique properties of the archaea (Table 6.1) reflect ancestral adaptation to extreme thermoacidic habitats from which the eukaryotes and most bacteria were excluded. Founded on a combined analysis of molecular and cellular data (the latter usually ignored in conventional phylogenomic analysis), the neomuran model is the better substantiated and more articulate hypothesis of eukaryogenesis hitherto presented, especially in the version published by Cavalier-Smith in 2014, yet in several respects antithetic to dominant bio-molecular orthodoxy (Lake 2015; Lane 2015). A strong point of the neomuran model is the possibility to explain in terms of vertical inheritance (from the hypothetical bacterial ancestor to Neomura) why

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bacterial genes in the eukaryotic pangenome are three times more numerous than archaeal genes and only a minor fraction of them is of mitochondrial origin (Fig. 6.1). The weakest point is that independent phylogenomic analyses place the eukaryotes within the Archaea (Sect. 6.6). Critics see a second major weakness of the neomuran model in the assumption that phagocytosis and other distinctive traits of the eukaryotic cellular organization appeared before mitochondria. They point out that phagotrophy requires cells of larger sizes and greater complexity than prokaryotic cells, with an energy cost ostensibly unaffordable without mitochondria (Lane and Martin 2010). In fact, most amitochondriate protists (e.g. the Microsporidia, Metamonada, and the stramenopile Blastocystis) are parasites or symbionts of other organisms (mainly animals), have lost phagotrophy and live as osmotrophs; others however, including the parasite Entamoeba histolytica and ciliates living in anaerobic sediments, retain a free phagotrophic lifestyle. In addition, the neomuran model suggests that the bacterial progenitor of Neomura was a fully aerobic organism, as are actinobacteria and other extant gram-positive bacteria. According to CavalierSmith, the acquisition of mitochondria was possible because of phagocytosis, not viceversa, and was probably a late step of a fast evolutionary sequence whose intermediate forms rapidly disappeared. It is worth noting that chloroplasts were undisputedly acquired by phagocytosis (Chap. 9); postulating a different origin for mitochondria and chloroplasts unparsimoniously sets aside striking similarities in the evolutionary trajectories of the two organelles.

6.5

The Origin of the Nucleus

The nucleus is the trait that gives eukaryotes their name. Far from being a simple bag for DNA, the nucleus is a highly specialized organelle whose properties are central to the functioning of the eukaryotic cell. The nuclear envelope bears specialized pores that control the traffic of molecules and keep gene duplication and transcription (intranuclear) separate from translation (cytoplasmic). During the interphase, the chromosomes are bound to the nuclear envelope, each occupying a discrete territory (Speicher and Carter 2005). As discussed more in detail in Box 6.3, the nucleus-tocytoplasm volume ratio is an important parameter affecting the metabolic activity of the cell. Why and at which stage the eukaryotic cell evolved a nucleus is a matter of speculation. By analogy with the mitochondrion and chloroplast, the doublemembrane structure of the nuclear envelope prompted hypotheses of a symbiotic origin (reviewed in Poole and Penny 2006, and Martin et al. 2015), now dismissed in favour of an endogenous origin (Jékely 2008; Cavalier-Smith 2010b). The nuclear envelope is a specialized area of the endoplasmic reticulum, thus it probably evolved in cells that already possessed an endomembrane system. If the primary function of the endomembrane system was phagocytosis, it is possible that the nuclear envelope evolved as a replacement for the cell envelope in providing support to DNA when ancestral eukaryotes specialized as phagotrophs. In bacteria,

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the cell envelope has a central role in chromosome spatial organization, replication and segregation, and a cell wall-bound FtsZ complex controls cell division (Toro and Shapiro 2010). At an early stage of eukaryote evolution, a system of internal membranes and of microtubules (the mitotic spindle) took charge for DNA handling, and the FtsZ complex was replaced by the ESCRT complex (“Endosomal Sorting Complexes Required for Transport”) in the control of cell division (Cavalier-Smith 2010b, 2014). If the necessity to free the cell envelope from the chromosome burden may account for the internalization of the genetic system and the evolution of a dedicated cytoskeletal system in the eukaryote cell, it remains to explain why the genetic system was sequestered within the nuclear envelope. One of the hypotheses put forward is that the nuclear envelope evolved to prevent harmful ribosome chimerism after the transfer of ribosomal protein genes from the proto-mitochondrion to the host genome (Jékely 2008). Without a nuclear envelope, host rRNA might bind to bacterial ribosomal proteins, producing faulty ribosomes. The nuclear envelope permits the host rRNA to be retained in the nucleus, where it correctly associates with host ribosomal proteins (synthesized in the cytosol and traslocated to the nucleus across nuclear pores), whereas mitochondrial ribosomal proteins are synthesized in the cytosol and translocated to the mitochondrion by specialized translocons (Sect. 6.7.1). An alternative hypothesis (Martin and Koonin 2006) proposes that the nuclear envelope evolved in response to invasion of the host genome by group II introns, self-replicating sequences from the mitochondrial symbiont. These sequences replicated extensively and inserted randomly within host genes, producing spliceosomal introns, non-coding sequences that presently account for a significant fraction of total genome in eukaryotes (Box 6.3). In the absence of a repair mechanism, the insertion of foreign sequences within host genes would seriously disrupt genetic information. The evolution of the nuclear envelope introduced a spatial and temporal separation between transcription and translation, thus permitting the removal of introns from transcripts before these bind to ribosomes. Known as RNA splicing, this important operation is performed by the spliceosomes, complexes of small nuclear ribonucleoproteins (Irimia and Roy 2014). There are no spliceosomes in prokaryotes, RNA splicing is rare and mostly affects non-coding RNAs. The mechanism of cell division in prokaryotes generally requires that the genome is carried in a single chromosome with a single replication origin (Toro and Shapiro 2010; Egan and Vollmer 2013).1 In contrast, mitosis is perfectly compatible with genomes split into several chromosomes, each with more than a single replication origin (McIntosh 2016). This permitted genome expansion without impairing the precision of chromosome segregation or the rate of replication. In a concatenation of

1 Besides the main chromosome, the bacteria may have one to many accessory chromosomes of small sizes, named plasmids, which carry non-essential genes and are replicated and transmitted independently of the main chromosome. Instances are known of plasmid segregation mediated by a membrane-independent cytoskeletal system (Box 6.1).

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causes and effects, initial inefficiency of the ancestral mitotic machinery might have increased the frequency of partial or total genome duplication, which in turn compensated for imprecise chromosome segregation by increasing the probability for daughter cells to receive an entire copy of the genome (Cavalier-Smith 2010b). Genome duplication not only produced genetic redundancy, thus favoring the appearance of new genes, but also permitted cells to increase genome size (Box 6.3). By improving the energy budget, the acquisition of mitochondria also was conducive to an increase of genomic and cellular sizes, and favoured the birth of new genes (Lane 2011, 2014).

6.6

Was the Host a Primitive Eukaryote or a Complex Archaeon?

Metagenomic analisis (Box 1.1) of marine sediments from Loki Castle, a site near the Mid-Atlantic Ridge in the Arctic Ocean, has revealed a group of psychrophilic (cold-adapted) Archaea, dubbed Lokiarchaeota, whose composite genome encompasses sequences homologous with distinctive eukaryotic genes including the replication initiation complex, ubiquitin, histones, actin, tubulin, and ESCRTIII (Spang et al. 2015; Klinger et al. 2016). The Loki genome is also the first prokaryotic genome found to encode large numbers of proteins showing clear homology with small GTPases that in eukaryotes play key functions in the regulation of the cytoskeleton, cell motility, compartment identity, and intracellular trafficking. Phylogenetic analysis of sequences from bacteria, eukaryotes and archaea, including Loki sequences, produced a tree of life in which the eukaryotes branched within the Lokiarchaeota, and these in turn nested within the TACK group (Figs. 6.10 and 6.11a). Phylogenomic analysis of archaeal sequences extracted from aquatic sediments worldwide led to the recognition of a novel archaeal group, named Asgard superphylum, which encompasses the Lokiarchaeota along with other novel lineages. The Asgard archaea affiliate with eukaryotes in phylogenomic analysis and some of them display homologues of eukaryotic membrane-trafficking machinery components (Zaremba-Niedzwiedzka 2017). Sequence homologs of important eukaryotic genes also occur in TACK archaea not belonging to the Asgard lineage, including genes for three ribosomal proteins, two sub-units of RNA polymerase, and for the transcription factor Elf1 (Saw et al. 2015). Cavalier-Smith (2014) classifies the Archaea into the Euryarchaeota and Filarchaeota, the latter including the Chrenarchaeota plus the “Thaumarchaeota” and “Korarchaeota”. A shared character of the Filarchaeota is the occurrence of genes for actin-like proteins and ESCRT filaments. So defined, this group should also encompass the Lokiarchaeota and probably the other “Asgard” lineages. The occurrence of eukaryotic genes in some – but not all – Archaea is consistent with the eocyte scenario (Cox et al. 2008) and even suggests that the putative

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Fig. 6.10 The genome ofTACK archaea includes sequence homologs of numerous “eukaryotic signature” proteins (blue rectangles). The asterisks in white rectangles means that homologs were found only in a part of the taxa examined. (From Saw et al. (2015), Creative Commons Attribution 4.0 International License)

archaeal progenitor of eukaryotes possessed a primitive form of phagocytosis (Koonin 2015a). This, however, should not be taken as an incontrovertible validation, because as of yet it cannot be ruled out that the above mentioned genes were inherited from a common ancestor of Archaea and Eukaryotes and conserved in the TACK and Lokiarchaeota, but lost in the rest of Archaea (Cavalier-Smith 2002a, 2014). Moreover, functional correspondence of archaeal sequences with eukaryotic counterparts remains vague, and no member of the Lokiarchaeota or their Asgard relatives has yet been isolated, imaged, or cultured. Many small GTPases in eukaryotes are covalently bound to membrane lipids; this membrane anchoring plays a fundamental role in linking the GTP–GDP cycle to membrane identity, dynamics, and compartmentalization. Vesicle traffic in eukaryotic cells depends on a complex molecular machinery including COPII coats/scaffolds to curve membranes and dynamins to mediate membrane scission events. No homologs of GTPase lipid-binding enzymes, COPII or dynamins have so far been detected in archaea, including the Loki genome. Despite these obvious caveats, the results from metagenomic analysis inspired an updated version of the fusion model pointing to the acquisition of the mitochondrion by a putative (loki)archaeal host as the starting event in eukaryote evolution (Dey et al. 2016). An alternative approach to metagenomics in the effort to circumvent difficulties in the isolation and culture of microorganisms from nature is “single-cell genomics”, consisting in the amplification and sequencing of DNA extracted from single cells.

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Fig. 6.11 (a) In agreement with the eocyte model, phylogenomic analysis by Spang et al. (2015) and Saw et al. (2015) placed the eukaryotes within the archaeal lineage Lokiarchaeota, which in turn nested within the TACK. (b) In contrast, the phylogenomic analysis by Rinke et al. (2013) resolved the eukaryotes as the sister group to the Archaea with a boostrap of 100%. Using the same data, a great number of alternative trees can be produced; a clade with a bootstrap of 100% (or 1) means that it is present in all possible trees, so it is considered to be more reliable than alternative topologies with a lower bootstrap; a bootstrap below 70% (or 0.70) is usually considered insufficient. Maximum likelihood algorithms use bootstrap values to select the most likely tree(s). (c) The analysis by Raymann et al. (2015) places the eukaryotes on the branch linking the Euryarchaeota to the TACK clade. This result is compatible with different phylogenetic topologies, depending on the position of the root (the origin) of the archaeal tree. If the root lies within the TACK superphylum, the eukaryotes (in blue) are sister to the Euryarchaeota (1). A root within the Euryarchaeota implies that the eukaryotes are sister to the TACK superphylum (2). A root between the Euryarchaeota and the TACK superphylum would produce topologies (3, 4) compatible with 1 and 2, but also one in which the eukaryotes are sister to the whole of Archaea (5). A, redrawn from Spang et al. (2015). B, from Rinke et al. (2013) licence number 4466611208627. C, redrawn from Raymann et al. (2015). DPANN: Diapherotrites, Pararchaeota, Nanoarchaeota, Nanohaloarchaeota, Aenigmarchaeota

In contrast with the metagenomic data reported above and exemplified in Fig. 6.11a, a recent phylogenomic analysis of sequences from archaea, bacteria and eukaryotes, including sequences obtained by single-cell genomics of uncultivated taxa, resolved the eukaryotes as the sister group of the archaea with strong statistical support (Rinke

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et al. 2013; Fig. 6.11b). These results have been criticized as possibly due to the use of a “poorly fitting phylogenetic model and contamination of the eukaryotic data set with genes of mitochondrial and plastid origin rather than nucleocytosolic versions for some of the eukaryotes analysed” (Williams and Embley 2014). Interestingly, a recent analysis explicitly reporting “a robust sister relationship of eukaryotes with the TACK superphylum”, actually places the eukaryotes on the branch linking the TACK superphylum to the Euryarchaeota (Raymann et al. 2015), which is compatible with a sister relationship of eukaryotes and the whole of Archaea, although alternative topologies are also possible (Fig. 6.11c). Over a total of 5922 functional genes present in the genome of the unicellular fungus Saccharomyces cerevisiae, 2442 (about 41%) are eukaryotic-signature genes, i.e. genes with no recognizable homologs in archaea or bacteria, 537 (about 9%) have archaeal homologs, and 2943 (~50%) bacterial homologs (Fig. 6.1). Of the genes with bacterial homologs, those with homologs in the α-proteobacteria (i.e. of likely mitochondrial origin) are only 254 (about 8.6% of bacterial genes and 4.3% of total genes). Similar results were also obtained for the unicellular red alga Cyanidioschyzon merolae (Koonin 2010). In line with these data, the mitochondrial proteome has a highly chimeric composition: mitochondrial proteins encoded by genes with homologs in α-proteobacteria (thus probably acquired from the bacterial endosymbiont) are a minority (about 10–20% of total) relative to proteins encoded by genes with homologs in other bacterial lineages and by eukaryote-only genes (Gray 2015). The data above indicate that: (a) Eukaryote evolution involved the appearance of a great number of new genes. (b) The genetic relatedness of eukaryotes to bacteria is much stronger than expected if it were predominantly due to mitochondrial HGT. (c) The genetic relatedness of eukaryotes to archaea, although as strong as to αproteobacteria, is less robust than expected if the eukaryotic nuclear line were directly derived from an archaeal host as assumed by eocytic models. A way to explain the predominance of bacterial genes in the eukaryote genome is assuming that these were acquired over the time by horizontal gene transfer from engulfed bacterial prey (Doolittle 1998). Although inter-domain lateral gene transfer events probably occurred throughout the history of eukaryotes, it is now clear that genes so acquired do not persist in the long term, unless they are associated with endosymbiosis (Katz 2015). Eukaryotic genes with bacterial affinity, yet not traceable back to mitochondria, chloroplasts or other endosymbioses are thus better explained in terms of vertical inheritance. Whether the nuclear line was a complex archaeon or a primitive eukaryote is not merely a matter of semantics. The archaea have membranes made of G1P isoprenylether lipids, whereas the eukaryotes have G3P acyl-ester lipids as in bacteria (Sect. 3.8). Supporters of the eocytic scenario suggest that the putative archaeal-derived nuclear line acquired the ability to make acyl-ester lipids by gene transfer from the proto-mitochondrion. Ostensibly, the transition from one type to the other of membrane lipids occurred in parallel with the evolution of the mitochondrion, in

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response the necessity to harmonize membrane biochemistry in the host and the symbiont (Dey et al. 2016). This sort of transition, necessarily gradual, is theoretically possible because, in contrast with former belief, membranes containing both types of lipids are stable and functional (Shimada and Yamagishi 2011). The hypothesis recently received support from the discovery that the Lokiarchaeota and several uncultured Euryarchaeota possess the genetic potential for the synthesis of chimeric membrane lipids, namely di- or tetraether-linked isoprenoid lipids with G3P stereochemistry, or lipids with one ether-linked isoprenoid chain at position sn1 of a G3P backbone and one ester-bound fatty acid at position sn-2 (Villanueva et al. 2016). The neomuran model assumes that the putative cenancestor of eukaryotes and archaea had G3P ester lipids, which were retained in the eukaryote lineage and replaced with G1P ether lipids in archaea due to adaptation to thermoacidic conditions. The taxonomic distribution of other membrane components, notably phosphatidylinositol, sterols and cardiolipin, is problematic as well. Neither the archaea nor proteobacteria can synthetize phosphatidylinositol (PI), a fundamental component of eukaryotic membranes. PI 3-phosphate is essential for phagocytosis and endosomal trafficking, PI 4-phosphate regulates trans-Golgi secretion, PI 4,5bisphosphate is involved in exocytosis, PI 3,5-bisphosphate participates in late endosome/multivesicular body trafficking. The metabolic pathway for sterol biosynthesis is absent in the archaea and is present only sporadically in proteobacteria (Summons et al. 2006). In contrast, phosphatidylinositol, sterols and cardiolipin are ubiquitous components of the cell membrane in actinobacteria (Cavalier-Smith 2009, 2014). Taking on board these and other lines of evidence from cell and molecular biology, the neomuran scenario suggests that the Neomura arise from an actinobacterial ancestor or share a common ancestor with actinobacteria (CavalierSmith 2014). Vertically inherited from the bacterial ancestor, phosphatidylinositol and sterols were conserved in the eukaryotes, whereas they were lost in the archaea when these substituted G1P isoprenyl-ether for G3P acyl-esther lipids. The unique molecular architecture of membranes in the archaea suggests that this lineage is primarily adapted to extreme temperature and that mesophilic and psychrophilic archaea (Cavicchioli 2006) are derived. Interestingly, phylogenomic analysis of the archaea has disclosed clear signs of genome streamlining, i.e. genome reduction (Csűrös and Miklòs 2009; Wolf et al. 2012); during their evolutionary history the archaea seem to have lost a significant proportion of the genes present in their progenitor, possibly in consequence of adaptation to low energy input and high environmental stress (Valentine 2007). In contrast with this view, Williams et al. (2017) infer a moderate increase in gene content, via gene duplication and horizontal gene transfer, throughout archaeal history from a common ancestor that had a relatively small genome. The discovery of eukaryotic sequences in archaea, notably in the novel group known as Lokiarchaeota, supports to the eocyte model, but it does not contradict the neomuran scenario either, because this postulates that eukaryotes and archaea share a common ancestor, thus predicting that they share parts of their genomes.

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Similarities in the DNA replication machinery of eukaryotes and archaea, for example, is taken as evidence that the archaeal/eukaryotic cenancestor, independently of its basal or derived position in the phyletic tree (cf. Fig. 6.11a and b), possessed a DNA replication apparatus that was as complex in its main features as in modern eukaryotes (Makarova and Koonin 2013). Despite the strength of arguments advocating caution, the idea that eukaryotes derive from a symbiosis between an archaeal host and a bacterial lineage, the former giving rise to the cell proper and the latter to the mitochondrion, currently dominates the scientific mainstream (Williams et al. 2013; Williams and Embley 2015; Guy et al. 2014; Koonin and Yutin 2014; Koonin 2015a, b; Roger et al. 2017). Based on data from phylogenomics, the archaeal host is often modelled as an organism already endowed with fundamental eukaryotic properties including a cytoskeleton and phagocytosis (but see Dey et al. 2016). Leaving out the membrane issue, such an organism would easily fit the protoeukaryote postulated by the neomuran model. As Keeling (2014) acutely observes, “if the first prokaryotes known were the archaea, and the bacteria had been discovered afterwards, most likely the bacteria would have been considered primitive instead of the Archaea, despite the lack of conclusive proof for either inference”. Likewise, if the discovery that all extant eukaryotes are primarily mitochondriate had been made 10 years earlier (in the 1980s rather than the 1990s), the “mitochondrion-first” models linking the rise of eukaryotes to the acquisition of mitochondria most likely would have never seen the light.

6.7

The Mitochondria

In prokaryotic cells, the chemiosmotic machinery coupling vectorial proton transport to ATP synthesis is located in the cell membrane (and in the thylakoid membrane as well in cyanobacteria). In the eukaryotes, the cell membrane hosts a diversity of carriers and pumps, but chemiosmotic coupling is restricted to the mitochondria (and the chloroplasts in photosynthetic eukaryotes). The mitochondria usually have oblong shape and sizes in the range of one to few micrometres, in special cases much larger, and are outlined by an inner and outer membrane that are derived from the inner and outer membrane of the gram-negative bacterial endosymbiont, respectively. The inner boundary membrane contains the respiratory chain and ATP synthase complexes, and forms the invaginations known as mitochondrial cristae (Figs. 6.12 and 6.13), whose morphology (lamellar, tubular or discoidal) has taxonomic relevance (Adl et al. 2012, 2018). The inner membrane is a tight diffusion barrier to all ions and molecules. These can only get across with the aid of specific protein trasporters, each selective for a particular ion or molecule. Because of its ion selectivity, an electrochemical potential of up to 180 mV builds up across the inner mitochondrial membrane. The outer membrane is freely crossed by ions and small, uncharged molecules through pore-forming integral proteins (porins). Cellular localization by means of antibodies has revealed that the respiratory chain complexes reside in the cristae, whereas protein transporters are located in the

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Fig. 6.12 Diagrammatic representation of a mitochondrion. The part of the figure marked “TEM” is a detail of a mitochondrion as seen in ultrathin section with a transmission electron microscope

Fig. 6.13 The chemiosmotic machinery in the inner mitochondrial membrane consists of five large membrane protein complexes. Complex I transfers electrons from NADH to coenzyme Q, pumping four protons from the matrix into the crista lumen. Complex II transfers electrons from succinate to coenzyme Q and does not directly contribute to building proton gradient. Complex III transfers electrons from reduced coenzyme Q to cytochrome c, pumping one proton in the process. Complex IV transfers electrons from cytochrome c to oxygen and contributes to the proton gradient by removing protons from the matrix to make water. Complex V (an F-ATP synthase) consumes the proton gradient to make ATP. Complexes I, III and IV tend to aggregate together to form a supercomplex (Kühlbrandt 2015)

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part of the inner boundary membrane not forming cristae (Vogel et al. 2006). A multisubunit protein complex known as MICOS (mitochondrial contact site and cristae organizing system) located at the junction of cristae with the inner boundary membrane maintains the lateral segregation (Fig. 6.14) and forms contact sites with TOM and SAM protein-importing complexes in the outer boundary membrane (Fig. 6.15). The internal compartment, named mitochondrial matrix, is rich in soluble proteins (up to 500 mg/ml) and contains a complete system for DNA duplication, transcription and translation. The mitochondrial matrix is homologous with the bacterial cytoplasm, from which it distinguishes for a pH of about 8. The alkaline pH of the mitochondrial matrix contributes to build the trans-membrane protonmotive force (PMF Box 4.1) that drives ATP synthesis (Kühlbrandt 2015). Mitochondrial ribosomes are membrane-attached, their only products (at least in human cells) being hydrophobic proteins that integrate into the inner membrane cotranslationally. The mitochondrial F-ATP synthase (Box 3.1) is the most conspicuous protein complex present in the cristae. The ATP synthase complexes form linear arrays of dimers located in the most tightly curved regions along the ridges of flattened cristae (Fig. 6.14) or along the longitudinal axis of narrow tubular cristae. Dimerization depends on subunits e and g in yeast. ATP-synthase dimerization is essential for membrane bending and crista development in mitochondria, as demonstrated by the observation that the mitochondria in yeast strains lacking subunits e and g lack

Fig. 6.14 ATP synthase forms V-shaped dimers (yellow) arranged in rows along the ridge of cristae; the other complexes of the respiratory chain (green) reside in the flattened part of cristae. ATP-synthase dimerization and arrangement in rows determines membrane bending. The segregation of respiratory complexes in the cristae maintains a high proton concentration (red dots) in the proximity of ATP synthase. (Adapted from Kühlbrandt (2015), http://creativecommons.org/ licenses/by/4.0/)

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Fig. 6.15 Most of the proteins necessary for the maintenance of mitochondria are encoded by nuclear genes, synthesized in the cytoplasm, and translocated to one of the five topological districts in the mitochondria: inner (IM) and outer membrane (OM), intermembrane space (IMS), crista membrane, matrix. The mitochondrial translocation machinery comprises five major protein import pathways. Presequence-carrying preproteins are imported by the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM23). TIM 23 inserts proteins with a hydrophobic sorting signal into the inner membrane, whereas it transfers hydrophilic proteins to the matrix with the help of the TIM23-associated motor (PAM). A mitochondrial processing peptidase (MPP) removes the presequences. Proteins destined to the intermembrane space (IMS proteins) contain cysteine-rich motifs and are imported by TOM in cooperation with the mitochondrial IMS import and assembly (MIA) system, which catalyses the formation of disulfide bonds in the imported proteins. The precursors of β-barrel proteins are translocated through TOM to small TIM chaperones of the IMS and are then inserted into the outer membrane by the sorting and assembly machinery (SAM). The precursors of metabolite carriers of the IM are imported via TOM, small TIM chaperones, and the carrier translocase TIM22. A number of α-helical OM proteins are imported by the mitochondrial import (MIM) complex. The membrane electrochemical potential across the IM drives protein translocation by TIM23 and TIM22 complexes. The occurrence of TOM and TIM complexes with the same core structure throughout the eukaryotes is considered evidence for the single origin of mitochondria. A sixth complex, known as the oxidase assembly (OXA) translocase (not shown) inserts proteins from the matrix into the inner membrane. Protein insertion in the crista membrane involves interaction of TOM and SAM with the MICOS (mitochondrial contact site and cristae organizing system) complex located at the junction of cristae with the boundary inner membrane (see Fig. 6.14). The mitochondrial protein import machinery is in part of eukaryotic ancestry (TOM and TIM), in part bacterial (SAM and OXA). (From Wiedemann and Pfanner (2017), Creative Commons Attribution 4.0 International License)

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cristae and have only about half the membrane potential measured in the wild type (Kühlbrandt 2015). This suggests that the cristae do not merely amplify membrane surface area, as generally thought, but help maintain a high proton gradient that enhances ATP synthase activity (Fig. 6.14). Bacterial F-ATP synthases lack dimerspecific subunits and are unable to form dimers; consistently, bacterial cells do not form structures comparable to mitochondrial cristae. The mitochondria are highly dynamic organelles: in the living cell they change shape and size continuously, by merging together or splitting into smaller organelles. Mitochondrial fusion is mediated by eukaryotic proteins belonging to the dynamin family. Mitochondrial fission also depends on dynamin proteins in several eukaryotes including animals, fungi and land plants (van der Bliek et al. 2013; Lackner 2014). In other lineages, for example the unicellular red alga Cyanidioschyzon merolae, mitochondrial fission utilizes homologs of FtsZ and Min proteins, which control cell division in bacteria (Rowlett and Margolin 2015). This suggests that dynamin paralogs partially or completely replaced the ancestral proteobacterial divisome several times independently (Leger et al. 2015; Arimura 2018).

6.7.1

Facts About Mitochondria

1. The mitochondrial genome (mt-genome or mt-DNA) is usually in the form of a single circular chromosome, but instances are known of single linear (the green alga Chlamydomonas, the malaria agent Plasmodium, some ciliates) multiple circular (the chitrid Spizellomyces punctatus) or multiple linear chromosomes (the green alga Polytomella parva). The sizes of the mt-genome vary from a minimum of 6 Kb in Plasmodium gallinaceum to several thousand Kb in the Kinetoplastida (Excavata). The mt-genome of Naegleria gruberi (Heterolobosea) forms a circular chromosome of about 50 Kb and comprises 67 functional genes, including a complete set of tRNA genes. The human mtgenome forms a circular chromosome of about 16 Kb and contains 37 genes, of which 13 genes encoding proteins of the electron transport chain, 22 genes for tRNA, and 2 genes for rRNA. The most gene-rich mt-genomes known to science occur in the Jakobida (97–99 genes), the most simple in the Apicomplexa (5 genes, 3 for proteins and 2 for RNA in Plasmodium). Each mitochondrion contains 1 to 10 genome copies. Despite its small dimensions, the mt-genome may account for a major fraction of total cellular DNA, as each cell may contain hundreds or thousands of mitochondria. In extremely large cells, for example egg cells, the mt-genome may be over 99% of total DNA (Barbrook et al. 2010; Burger et al. 2013; Smith and Keeling 2015). 2. Some angiosperms have mitochondria with an enormous genome fragmented into several circular chromosomes. The mt-genome in Amborella is 3.9 Mb and in two species of Silene about 6.7 e 11.3 Mb, sizes larger than most bacterial genomes. The enormous size of the mt-genome in Silene is due to multiple duplication of the original genome (Wu et al. 2015). In Amborella, instead, the

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3.

4.

5.

6.

185

mt-genome expanded by incorporating “alien” mt-genomes from green algae, mosses and other angiosperms. The mechanism suggested is cellular rupture, mitochondrial fusion with foreign mitochondria and cellular healing, followed by incorporation of the modified cell or one of its derivatives into an apical meristem. The size increase of the mt-genome does not involve an increase in the net number of active genes (49 in angiosperms) because the alien sequences incorporated are partial and probably non-functional (Rice et al. 2013). Molecular analysis points to the rickettsiales, a α-proteobacterial lineage of obligate intracellular parasites including several human pathogens, as the closest bacterial relatives of mitochondria (Wang and Wu 2015; Ball et al. 2016; Roger et al. 2017). Most of the proteins necessary for the maintenance of mitochondria (over 1000 different proteins in mammals) and, in some cases also a part of the tRNAs used in intra-organelle protein synthesis are encoded by nuclear genes and imported from the cytoplasm (Lithgow and Schneider 2010; Gray 2015). Mitochondrial proteins synthesized on cytoplasmic ribosomes, sometimes referred to as extrinsic mitochondrial proteins, are translocated to the different topological districts of the mitochondrion by specialized translocons (Fig. 6.15). About 60% of extrinsic proteins contain amino-terminal positively charged presequences of 10–100 amino acid residues (mitochondrial targeting sequences) that are essential for their import into the mitochondrion; the rest use presequence-independent import pathways (Wiedemann and Pfanner 2017). The Translocase of the Outer mitochondrial Membrane (TOM complex) translocates most extrinsic mitochondrial proteins to the intermembrane space. The Mitochondrial Import and Assembly (MIA) machinery oxidizes and folds the proteins destined to remain here. The proteins destined to other topological districts use one of the following pathways: (1) insertion into the outer membrane by the Sorting and Assembly Machinery (SAM) complex; (2) insertion into the inner membrane by the TIM22 insertase; (3) translocation across the inner membrane to the mitochondrial matrix by the TIM23–PAM complex. Proteins translocated into the matrix remain here or are inserted into the inner membrane by the OXA complex, which probably permits the insertion of proteins with an inverted topology relative to that mediated by TIM22. The OXA complex also mediates the insertion of inner membrane proteins encoded by the mitochondrial genome (intrinsic mitochondrial proteins). Mitochondrial targeting presequences are generally cleaved off by a mitochondrial peptidase after translocation (Lithgow and Schneider 2010; Wiedemann and Pfanner 2017; Fig. 6.15). About 10–15% of nuclear genes engaged in the maintenance of mitochondria come from the original proteobacterial endosymbiont, a minority from other bacteria (e.g. chlamydias), the rest originated by duplication and neofunctionalization of pre-existing eukaryotic genes. This suggests that symbioses with other types of bacteria preceded the evolution of the mitochondrion and left a number of genes in the host genome that were co-opted in mitochondrial maintenance (Valas and Bourne 2011; Gray 2014, 2015; Roger et al. 2017).

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7. Besides a variable number of functional genes, the mt-genome contains noncoding sequences; functional mitochondrial genes code for components of the mitochondrial genetic system (tRNA, rRNA and ribosomal proteins) and of the chemiosmotic machinery (ATP synthase, cytochrome c oxidase, cytochrome b, NADH dehydrogenase). Non-coding sequences are generally a minor part of the genome but, as an exception, the Kinetoplastida (a flagellate lineage including trypanosome parasites) have a very large mitochondrial genome prevalently made of non-coding sequences; this large mt-genome is replicated into thousands of copies that aggregate to form a dense body named kinetoplast (Barbrook et al. 2010). 8. Of 54 ribosomal proteins in α-proteobacteria (L1 to L33 in the large subunit and S1 to S21 in the small subunit), mitochondrial ribosomes lack S21 in all presentliving eukaryotes and additionally S1 in the Amorphea (Sect. 6.8 for eukaryote systematics). In return, mitochondrial ribosomes contain 19 novel proteins (20 in the Amorphea) with no homolog in bacterial or eukaryotic (cytoplasmic) ribosomes. These proteins probably evolved de novo with the mitochondrion (Desmond et al. 2011). 9. Several eukaryote lineages adapted to oxygen-poor or anoxic habitats lack mitochondria but retain double-membrane-bounded mitochondrion-related organelles, or “anaerobic mitochondrial derivatives” (AMR) including hydrogenosomes and mitosomes (Stairs et al. 2015; Leger et al. 2017). The hydrogenosomes ferment pyruvate to CO2, H2 and acetate, producing ATP by substrate-level phosphorylation; the only documented function of mitosomes is the production of FeS clusters used as precursors of essential prosthetic groups of redox enzymes. The occurrence of the protein Tom40 (a component of the TOM protein import system) in the outer membrane of hydrogenosomes and mitosomes is the strongest evidence for a mitochondrial origin of these organelles, ruling out an independent origin from α-proteobacteria. The reduction of mitochondria to AMR occurred independently in several eukaryote lineages, usually involving mt-genome loss, whereas the TOM and TIM complexes were retained in a simplified form. The hydrogenosomes in Nyctotherus ovalis, a ciliate living in the hindgut of cockroaches, and in Blastocystis hominis (Stramenopila), a common intestinal parasite of humans, retain a small genome (Hjort et al. 2010; Stairs et al. 2015). Novel species of the animal phylum Loricifera living in an anoxic habitat at the bottom of the Mediterranea Sea lack mitochondria; instead of mitochondria, the cells of these minute animals contain hydrogenosome-like organelles associated with bacterial endosymbionts (Danovaro et al. 2010). The metamonad protist Monocercomonoides lacks mitochondria as well as all nuclear hallmark of mitochondrial sequences; the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in all eukaryotes, was replaced in this organism by a cytosolic sulphur mobilization system acquired by lateral gene transfer from bacteria (Karnkowska et al. 2016). Likewise, the AMRs present in Entamoeba histolytica and Mastigamoeba balamuthi (Archamoebae, Amoebozoa) have a sulphate activation pathway

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10.

11.

12.

13.

14.

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not found anywhere else in eukaryotes, whose key enzyme (ATP sulphurylase) has been acquired laterally from bacteria (Pánek et al. 2016). There is no clear-cut explanation for the persistence of a genetic system in the mitochondria (and chloroplasts). The maintenance of an active genome in organelles is energetically expensive. The synthesis of the 13 proteins encoded by mitochondrial genes in the human species requires a machinery involving over 100 different proteins, of which 70–80 for building mitochondrial ribosomes and 20 aminoacyl-tRNA transferases, besides at least 22 different RNAs (rRNA and tRNA). The CORR (CO-location for Redox Regulation of gene expression) hypothesis, originally proposed by John Allen in 1993 and expanded in more recent work (Allen 2003), stems from the consideration that the proteins encoded by mitochondrial genes are fundamental components of the chemiosmotic machinery (Fig. 6.13). The CORR hypothesis proposes that these genes were retained in the mitochondria to ensure a tight coordination between their expression and the redox state of the organelle, as originally was the case in the free-living proteobacterial ancestor of mitochondria. Variations in the redox state are promptly corrected by modifying the expression of mitochondrial genes. According to the CORR hypothesis, this mechanism ensures the maintenance of conditions favourable to ATP synthesis and prevents the establishment of harmful redox conditions. The CORR hypothesis is consistent with the loss of the genome in mitosomes and hydrogenosomes, which no longer perform chemiosmotic coupling. In all eukaryotes, except the Jakobida and Metamonada (ExcavataSect. 6.7), a RNA polymerase of viral origin replaced the mitochondrial RNA polymerase of bacterial origin (Barbrook et al. 2010). Cytochrome c (an important mitochondrial protein) in the Euglenozoa (Euglenida, Kinetoplastida and Diplonemida Sect. 6.7), contains only one cysteine residue in the heme-bound domain; in all other eukaryotes there are two (Allen et al. 2008). Electron-transfer reactions in the respiratory chain generate reactive superoxide radicals as side products, which are dangerous to mitochondrial proteins and DNA, as well as to other cellular components. Scavenging enzymes such as superoxide dismutase or catalase permit cells to reduce oxidative damage by oxygen radicals. Damaged or dysfunctional mitochondria are either complemented with undamaged parts of the mitochondrial network by fusion, or sorted out for destruction in lysosomes (mitophagy). During ageing, fission overpowers fusion and the mitochondrial network fragments. This prevents the complementation of damaged mitochondria by fusion and accelerates their deterioration (Kühlbrandt 2015). In most eukaryotes with sexual reproduction, the mitochondria are inherited uniparentally, i.e. only from one, usually maternal parent. Uniparental mitochondrial transmission may have evolved in parallel with sexual reproduction to facilitate purifying selection against defective organelles (Chap. 7). The predominance of maternal inheritance may reflect higher mutational load in metabolically more active paternal gametes (Greiner et al. 2014). The diversity of mechanisms underpinning uniparental mitochondrial transmission in different

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lineages suggests multiple origin (Radzvilavicius et al. 2017) as well as repeated reversal to biparental transmission due to susceptibility of uniparentally-transmitted mitochondria to accumulate deleterious mutations, a process known as Muller’s ratchet (Greiner et al. 2014). 15. Beside energy conversion to synthesize most of the ATP needed in cells, the mitochondria perform numerous metabolic processes, including the biosynthesis of amino acids, lipids, heme, and Fe-S clusters, and play crucial functions in cellular signalling pathways, quality control, and programmed cell death. The mitochondrial iron-sulfur cluster (ISC) machinery exports a FeS-containing moiety from the mitochondrial matrix to the cytoplasm, for use in cytoplasmic FeS protein biogenesis. The ICS machinery is the only known function of mitosomes (see point 9). Defects of mitochondria in humans lead to severe diseases of the nervous system, heart, muscles, and other tissues (Wiedemann and Pfanner 2017).

6.8

The Last Eukaryote Common Ancestor (LECA) Possessed a Full Set of Fundamental Eukaryotic Traits

After the appearance of life and photosynthesis, the birth of the eukaryotic cell was probably the third most important biological transition in the history of life, encompassing the appearance of over 60 novel characters, many of which extremely complex (Table 6.3), and at least 2000 gene “families” (sets of genes traceable to the same ancestral sequence). As already said, about 40% of the genome of eukaryote models such as Saccharomyces or Cyanidioschyzon consists of sequences lacking recognizable homologs in bacteria or archaea. This fraction rises to 60% when considering only the eukaryote core genome, i.e. genes present in all major eukaryote lineages (Koonin 2010). Of particular phylogenetic relevance are proteins involved in the construction of cytoskeletal structures. Eukaryotic actin has structural affinities with the bacterial proteins MreB and ParM, which play a central role in chromosome segregation, cell polarization and cell shaping (MreB) and plasmid segregation (ParM) in Escherichia coli. Eukaryotic tubulin is akin to FtsZ, a bacterial protein that controls the formation of the dividing septum after chromosome replication, and with the FtsZ-related protein TubZ that controls plasmid segregation in Bacillus subtilis. Homologs of MreB e FtsZ are also present in archaea. The molecular motors kinesin and dynein (which use microtubules as tracks) and myosin (which uses actin microfilaments) are ubiquitous in eukaryotes (Box 6.1) but lack homologs in prokaryotes, barring weak sequence homologies of kinesin and myosin with a bacterial GTPase and of dynein with a ATPase (Shih and Rothfield 2006; Michie and Löwe 2008; Shaevitz and Gitai 2010; Rowlett and Margolin 2015). If eukaryotes evolved at an extremely fast rate, as seems to be the case from the paleontological record, most phylogenetic signals might have been lost, thus

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Table 6.3 Main distinctive characters (apomorphies) of extant eukaryotes. The universality of these characters (save instances of loss or reduction) implies inheritance from a common ancestor. The relative order by which the characters appeared during early eukaryote evolution is uncertain. The list only includes traits that presumably evolved before extant eukaryote lineages diverged, thus excluding later additions such as the chloroplasts or intermediate filaments, and is probably not exhaustive. (Adapted from Cavalier-Smith (2009)) 1. Phagocytosis 2. Actin and actin-related proteins (Arps) functioning as actin nucleators (Box 6.1) 3. Endomembrane system and biochemical machinery for vesicular transport 4. Myosin, microfilament mediated intracellular transport and ameboidal movement 5. α-, β-, γ-tubulin 6. Molecular motors associated with microtubules (dyneins and kinesins) 7. Flagella with an inner skeleton of 9 + 2 microtubule pairs (Box 6.2) 8. Peroxysomes 9. Sphingolipids 10. Phosphatidylinositol 11. Calmodulin 12. Ubiquitin and polyubiquitin labelling system 13. 26S proteasomes with 19S regulatory subunit 14. Genome amplification by addition of large non-coding sequences 15. Spliceosomal introns and spliceosomes 16. mRNA capping by 7-methyl-guanosine-triphosphate at 50 end 17. Three RNA polymerases (I, II, III) with distinct functions 18. RNA-interference machinery 19. Cell cycle resetting by anaphase proteolysis 20. Nuclear envelope and chromatin (nucleosomes) 21. Nucleolus 22. Telomers and telomerase 23. Mitosis, centromers, kinetocores 24. Cell division mediated by actin, not FtsZ 25. Meiosis and synaptonemal complex 26. Syngamy and sexual reproduction 27. Mitochondria

explaining the absence of prokaryotic homologs for scores of eukaryotic genes. The crucial point, however, is not so much the origin of unique eukaryote genes, but the way bacterial and archaeal sequences entered the eukaryotic genome. Did the nuclear line (the host) vertically inherit them from a bacterial or archaeal progenitor or obtained them by horizontal gene transfer? This brings us back to the yet unsolved dispute between the archaezoan/neomuran model and the eocytic model. Genome sequencing and notation of Naegleria gruberi (Fig. 6.16), a member of the Discoba (Sect. 6.9) has revealed that about 4100 genes over a total of 15,727 occur in at least one of the other major eukaryotic lineages (Fig. 6.17). This is evidence that (at least) 4100 genes present in the eukaryote pangenome were directly

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Fig. 6.16 The fresh-water and soil protist Naegleria gruberi (Heterolobosea) has a unique threestage life cycle encompassing ameboidal cells, biflagellate cells that quickly develop from amoebas in response to starvation, and dormant cysts developing in response to dehydration. Upon rehydration, the cysts form new amoebas. (Adapted from Fritz-Laylin et al. (2010), licence number 4464930241794)

inherited from LECA, which thus was probably a relatively complex organism (Fritz-Laylin et al. 2010; Koumandou et al. 2013). Perhaps even more significant is the recognition that fundamental traits of present-living eukaryotes, such as the endomembrane system, the phagocytosis machinery, the nuclear lamina and the mitochondrion (Table 6.3), can confidently be traced back to LECA, thus making the absence of primarily amitochondriate eukaryotes phyletically irrelevant (Poole and Neumann 2011; Schlacht et al. 2014; Koreny and Field 2016).

6.9

Eukaryote Phylogeny and Systematics

Molecular phylogeny has revolutionized the traditional eukaryote systematics based on Whittaker’s five-kingdom system. The relative rarity of horizontal gene transfer (HGT) in eukaryotes facilitates the identification of phylogenetically informative hortologous genes and the reconstruction of intra-group phylogenetic relationships. Based on the absence/presence of a gene fusion (dihydrofolate reductase and thymidylate synthase), Stechmann and Cavalier-Smith (2002, 2003) split the eukaryotes into two groups, the Unikonta (with separate genes) and Bikonta (with the two

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Fig. 6.17 Phylogenomic comparison of free-living eukaryotes. About 4100 genes present in the genome of Naegleria also occur in at least one of the other eukaryotic lineages investigated (marked by dotted circles), suggesting that they were inherited from the last common ancestor (LECA). See Fig. 6.18 for further details on eukaryote systematics. (From Fritz-Laylin et al. (2010), licence number 4464921217421)

genes merged together and translated in a single bifunctional protein). The denominations proposed were based on the assumption that the Unikonta ancestrally had a single basal body (Box 6.2) and the Bikonta two, now considered to be almost certainly incorrect (Roger and Simpson 2009; Cavalier-Smith 2014; also see Fig. 6.18). The term Unikonta is currently being replaced with Amorphea (Adl et al. 2012), a term referring to the frequency of ameboidal forms in the group (Fig. 6.17); the term “Bikonta” is still in use although the biflagellate condition is now considered ancestral in all extant eukaryotes (Box 6.2). Barring the position of several groups that probably evolved at a particularly fast rate and consequently are prone to “long branch attraction” (Sect. 6.2), the current eukaryote tree encompasses two major branches, or “eukaryotic domains”: the Amorphea (from Greek amorphos, formless) and Diaphoretika (from Greek diaforetikos, diverse) (Roger and Simpson 2009; Burki 2014; Burki et al. 2016; Adl et al. 2012, 2018). Alternative denominations are Podiates for the Amorphea (Cavalier-Smith et al. 2016) and Corticata for the Diaphoretika (Cavalier-Smith (2010b), with reference to microfilament-supported pseudopods and membranebound cortical alveoli.

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The larger group that does not fit within either eukaryotic domain is the Excavata, a protist taxon that owns the name to a ventral feeding groove present in some members (e.g. the Jacobida) and used for the capture of food (Cavalier-Smith 2002b). Molecular analysis by Hampl et al. (2008) resolved the Excavata as a monophyletic group encompassing two sister clades, one encompassing the Jakobida, Euglenozoa and Heterolobosae and referred to as the Discoba or JEH, the other embracing several amitochondriate lineages (Parabasalida, Oxymonadida, Diplomonada) collectively known as Metamonada, or POD. Subsequent phylogenomic work resolved three excavate clades, the Discoba, Metamonada and Malawimonada, with the Malawimonada more closely related to the Amorphea and the Discoba and Metamonada to the Diaphoretika (Derelle et al. 2015; Brown et al. 2018). Taking on board these results, the phylogenetic classification of eukaryotes by Adl et al. (2018) uses the informal denomination “excavates” instead of the formal name Excavata introduced by Cavalier-Smith in 2002. In the original definition by Stechmann and Cavalier-Smith (2003), the Bikonta included the organisms now placed in the Diaphoretika plus the “Excavata”; in its current use, the Bikonta encompasses the Diaphoretika, the Discoba and perhaps the Metamonada but excludes the Malawimonada (Derelle et al. 2015). The Amorphea includes the Opisthokonta (essentially comprising the Fungi, Animals and Choanoflagellates) and the Amoebozoa (Tubulinea and Micetozoa) (Smirnov et al. 2011; Adl et al. 2012; Paps et al. 2013; Cavalier-Smith et al. 2014, 2016). A number of eukaryotes of uncertain position (collodictyonids, rigifilids, and Mantamonas) have recently been recognized as a sister clade to the Amorphea, named “CRuMs” (Brown et al. 2018). The Diaphoretika encompasses the Archaeplastida (or Plantae), the Stramenopila or Heterokonta (brown algae, diatoms, oomycota, xanthophytes, chrysophytes), the Alveolata (Dinoflagellates, Apicomplexa, Ciliophora) and the Rhizaria (Cercozoa, Foraminifera and Radiolaria). The assemblage of Stramenopila, Alveolata and Rhizaria has been resolved as a clade named SAR from the initials of the three subgroups (Burki et al. 2008, 2016), or Harosa (Cavalier-Smith et al. 2015). Despite extensive phylogenomic work, the position of the root (origin) of the eukaryotic tree of life remains elusive. Current hypotheses include placement: 1. Between the Opisthokonta and all remaining Eukaryotes (Katz et al. 2012; Katz and Grant 2015). 2. Between the Amorphea (Unikonta) and Bikonta (Stechmann and Cavalier-Smith 2003; Richards and Cavalier-Smith 2005; Roger and Simpson 2009; Derelle and Lang 2012; Derelle et al. 2015; Fig. 6.18). 3. In the Excavata or between the Excavata and the rest of eukaryotes (He et al. 2014). 4. Within the Euglenozoa, or between the Euglenozoa and the rest of Eukaryotes, collectively named “neokaryotes” (Cavalier-Smith 2010a). Cavalier-Smith (2013) lists 19 major apomorphies of neokaryotes relative to the Euglenozoa, including Tom40 subunit of TOM mitochondrial complex strongly divergent from the euglenozoan homolog and 14 novel ribosome proteins.

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Figures 6.18 and 6.19 show two eukaryote trees published in 2009 and 2016, respectively. For further details on eukaryote systematics and phylogeny, see Adl et al. (2007, 2012, 2018), Burki (2014), Cavalier-Smith et al. (2014, 2016), CavalierSmith (2016), Burki et al. (2016), Archibald et al. (2017), Simpson et al. (2017). Ruggiero et al. (2015) provide a nomenclatural and taxonomic review of all organisms. Metagenomic analysis of 18 S ribosomal DNA from planktonic samples collected in the photic zone of the ocean throughout the world suggests that protists (viz. unicellular eukaryotes) account for over 85% of total eukaryotic diversity (de Vargas et al. 2015). The same probably holds true for other marine, freshwater, and terrestrial ecosystems (Pawlowski et al. 2012). Yet, as for bacteria and archaea, there is no agreement on how to define a species in protists and no single species concept can be applied unequivocally to all protist groups. Database expansion from protist metagenomics will probably warrant significant change in the phylogenetic scenario outlined above. AMORPHEA

DIAPHORETIKA

Archaeplastida Apusomonads (= Plantae) Cryptomonads + Amoebozoa Breviata Haptophytes Slime moulds Stramenopiles Archamoebae 0 Phalansterium 1 2+ Alveolata Multicilia 2 2 1/2 2 1 1 2 2 Lobose Rhizaria amoebae 2 0

Opisthokonta (animals and fungi)

2

2*

JEH

“excavates”

LECA EUKARYOTES

POD

PROKARYOTES

Bacteria

Archaea

Fig. 6.18 Eukaryote phylogenomics resolves two megagroups: the Amorphea (¼ Unikonta or Podiates) and the Diaphoretika (¼ Corticata), plus the “excavates, a group currently treated as paraphyletic. Different colours distinguish main clades within each megagroup: the Opisthokonta and Amoebozoa in the Amorphea, the Archaeplastida (¼ Plantae), Cryptophyta, Haptophyta, Stramenopila, Alveolata and Rhizaria in the Diaphoretika. The Excavata show a basal separation of a line retaining the mitochondria (Discoba or “JEH”, from the initials of Jakobida, Euglenozoa, Heterolobosea), and a line without mitochondria (the Metamonada, or “POD” from the initials of Parabasalida, Oxymonada, Diplomonada), plus a small group named Malawimonada. The topology shown in the figure tentatively places the root of the eukaryote tree between the Diaphoretika/ excavates and the Amorphea. The numbers in the circles indicate the putative ancestral number of basal bodies (Box 6.2). The symbol 2+ indicates the presence of more than two basal bodies in some members of the Breviata lineage; the symbol 2* indicates that the single flagellum in the Opisthokonta is associated with an axoneme-forming basal body plus a free basal body. (Adapted from Roger and Simpson (2009), licence number 4464930535863)

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Fig. 6.19 Eukaryote tree by Burki et al. (2016). Major novelties relative to the tree shown in Fig. 6.18 are: (1) The Stramenopila, Alveolata and Rhizaria cluster in the SAR clade, whose sister group is the Haptista, a lineage encompassing the Haptophyta and the Centrohelida. (2) The Cryptista, a lineage encompassing the Cryptophyta and the Katablepharidae, is the sister clade to the Archaeplastida. (3) The Opisthokonta clusters with the Apusomonada and Breviata in the clade “Obazoa”. All clades, except the Archaeplastida (dotted triangle), have high bootstrap support. The sizes of the triangles are proportional to the number of species investigated. (Redrawn from Burki et al. (2016))

Box 6.2 Locomotor Organelles in the Three Domains of Life The term flagellum (plural flagella) in cell biology indicates a heterogeneous ensemble of organelles that propel the cell in liquid media or generate a flow of liquid in the proximity of immobile cells. Thus defined, flagella are present in bacteria, archaea and eukaryotes, with major structural and functional differences clearly pointing to independent origins. As primary determinants of cell polarity and directional motility, the flagella permit cells to tactically respond to environmental cues. (continued)

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Box 6.2 (continued) The Bacterial Flagellum The flagellum of bacteria is a sophisticated nanomachine composed of about 40 different proteins, of which 25 are structural components, in amounts ranging from a few molecules to more than 10,000. It consists of a base firmly inserted in the cell envelope, an intermediate hook, and a hollow filament several micrometres in length (Berg 2003; Chen et al. 2011). Figure 6.20 shows a schematic representation of the flagellar base in gram-negative bacteria. Bacterial flagella generate propulsive force by rotating along their axis. The motive force of rotation is proton inflow across the base (or sodium ions in some bacteria from marine or alkaline habitats). Chemiosmotic coupling with redox reactions (Box 3.1) or ATP-dependent proton (sodium) pumping builds the underlying electrochemical gradient. In some species, the flagella can rotate both counter-clockwise (CCW) and clockwise (CW) from the perspective of an observer looking at the cell from the free end of the filament; in others, the flagella rotate only CCW or only CW (Fig. 6.21). Temporary transition from CCW to CW rotation or from active movement (mediated either by CCW or CW rotation) to pause permits the cells to change direction. By modulating the frequency of shifting from one to the other state, bacteria are able to move coherently towards chemical or physical attractors such as food, oxygen or light (Jarrell and McBride 2008). When Long Is Long Enough The flagellar filament is a hollow tube made of the protein flagellin (also known as FliC), with a molecular mass ranging from 30 to 60 kDa. The filament grows by addition of flagellin units to the distal end; protein units move along the inner channel, about 2 nm in diameter, and bind to the free end of the filament. Flagellar filaments broken by mechanical shearing are reconstituted to a species-specific length range by injection of flagellin through type-III secretion system associated with the flagellar base and powered by proton-motive force. Flagellin molecules passively move along the filament channel toward the distal end, where they polymerize. The growth rate is higher at the beginning, when the filament and the queuing line of flagellin molecules are still short; the rate progressively decreases as the filament grows, and eventually stops at a final length primarily determined by the strength of proton motive force and the mass of flagellin molecules. Flagellar length varies in a species-specific manner but, because of this distinctive growth mechanism, also depends on environmental variables such as pH and the type of substrate (Hughes 2017). (continued)

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Box 6.2 (continued)

Fig. 6.20 Flagellar base in gram-negative bacteria, represented in full (left) and with a part of base components removed (right). The base consists of a series of rings tethered to the cell envelope and interconnected by a rod. The L and P rings act as a bushing (in mechanical engineering a bushing is a ring-like structure that constrains the movement of parts, in this case the rotating rod). The active part of the flagellar machine consists of a stator (in brown) and a rotor (yellow circle), both inserted in the cell membrane. The stator is a ring of MotA protein subunits tethered to the rigid peptidoglycan layer by the protein MotB (not shown); the rotor consists of a C ring, a M/S ring and an intermediate disk made of the protein FliG (in orange). The filament is a hollow structure, only 20 nanometres in diameter but usually over 10 micrometres long, made of the protein flagellin. The bacterial flagellum is a highly efficient molecular machine. Proton flow from the outside to the inside of the cell induces a shape change in MotA, exerting a torque (rotary force) on the FliG disk. The MS ring transmits the torque from FliG to the rod, and this in turn transmits rotary motion to the filament via the flexible hook. The protein epsE can rapidly stop rotation by decoupling the rotor from the rod. The flagellar base in gram-positive bacteria differs is some respects from that of gram-negative bacteria, notably it has no L ring due to the lack of an outer membrane. (From: http://cronodon.com/BioTech/Bacteria_motility.html)

ä Fig. 6.21 (continued) the flagellum to relax in a slowly rotating spiral; resumption of CCW pushes the cell in a different direction. There is no CW rotation. (c) Bacteria with two polar and two lateral flagella (lophotrichous flagella, e.g. Agrobacterium tumefaciens): CW rotation pushes the cell forward, a brief pause permits the cell to change direction, no CCW rotation. (d) Bacteria with flagella attached throughout the cell surface (peritrichous flagella, e.g. Escherichia coli and Bacillus subtilis): CCW rotation causes the flagella to aggregate in a bundle that pushes the cell forward; CW rotation causes the flagella to disentangle and the cell to wander with no direction (tumbling); resumption of CCW rotation pushes the cell in a different direction. (e) Bacteria with a tuft of flagella at each pole (amphitrichous flagella, e.g. Spirillum volutans): CCW rotation of tail flagella pushes the cell forward. Head and tail swapping causes the cell to change direction; no CW rotation. Receptors sensitive to chemical or physical attractors modulate the frequency of transition from one state to the other. (Adapted from: http://cronodon.com/files/Prokaryotes_1.pdf)

(continued)

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Box 6.2 (continued)

Fig. 6.21 Modes of operation of flagella in bacteria. (a) Bacteria with a single polar flagellum (e.g. Pseudomonas): CCW rotation pushes the cell forward, CW rotation pulls the cell backward. (b) Bacteria with a single lateral (monotrichous) flagellum, e.g. Rhodobacter sphaeroides: CCW rotation pushes the cell forward, a pause in rotation causes

(continued)

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Box 6.2 (continued) The Archaeal Flagellum The archaea have flagella superficially similar to bacterial flagella, consisting of a base inserted in the cell membrane and a long rotating filament (Fig. 6.22). Closer examination, however, reveals profound differences pointing to an independent origin. Because of this, the archaeal flagellum was given a specific denomination, the archaellum (Ghosh and Albers 2011; Jarrell and Albers 2012). Major distinctive traits of the archaeal locomotor machinery include: – Protein components lack sequence homologies with flagellar proteins in bacteria; notably, the archaeal filament consists of two proteins named archaellins (flaA e flaB) distinct from flagellin but akin to type-IV bacterial pilin. – Flagellar rotation depends on ATP hydrolysis, not proton (or sodium) inflow. – The filament is 12–15 nm in diameter and elongates by addition of archaellin subunits at the base, not the tip. – In Halobacterium salinarum CW rotation pushes the cell forward, CCW pulls the cell backwards; as in many bacteria, archaeal flagella aggregate into a loose bundle, but the shifting from CW to CCW rotation does not cause bundle dissociation nor tumbling (cf. Fig. 6.21d). It is not known if these properties are unique to H. salinarum or are more widespread in the archaeal lineage. Gene sequence analysis suggests that the bacterial flagellum evolved by modification of type-III secretion system, a multiprotein complex involved in the secretion of certain proteins (Abby and Rocha 2012). The archaellum, instead, appears to be a rotating variant of type-IV pili, structures used in bacteria as grappling hooks to adhere to solid surfaces, with the addition of components from type-II secretion systems; it is unclear whether the archaea acquired these systems vertically or by horizontal gene transfer from bacteria. Most of the proteins that make the archaellum are encoded by 7–13 genes in a single locus named fla operon (or “archaellum operon”), whose expression is controlled by environmental factors. With a swimming speed of 400–500 body lengths per second, hyperthermophylic archaea are the fastest organisms on Earth, to a relative scale. It is not yet understood how a relatively simple structure as the archaellum may produce such a great power (Albers and Jarrell 2015a, b). In the Neomuran scenario (Cavalier-Smith 2014), the putative concestor of archaea and eukaryotes lost the original (bacterial) flagellum, possibly because of structural instability in moderately hot/acid conditions, and subsequently each lineage independently evolved a novel type of flagellum. (continued)

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Box 6.2 (continued)

Fig. 6.22 Diagrammatic representation of the flagellum in the two main lineages of archaea. The proteins that make the flagellum in the archaea have significant homologies with proteins of type IV pili and type-II secretion systems. (Adapted from Ghosh and Albers (2011), STM Agreement)

The Eukaryotic Flagellum The flagellum of eukaryotes is sharply different from and more complex than its prokaryotic counterparts. This organelle has been early recognized as a distinctive feature (apomorphy) of the eukaryotic domain, and a specific denomination has been proposed on several occasions. American biologist Lynn Margulis called it undulipodium but the term did not radicate in the literature; others (Jékely and Arendt 2006; Cavalier-Smith 2013; Mitchell 2016; Adl et al. 2012, 2018) use the term cilium (plural cilia), which is certainly the most sensible option. The denomination “cilia” has traditionally been employed for relatively short flagella present in large numbers in animal multiciliate cells and ciliate protists, reserving the term “flagella” to the other instances. Ciliar motility typically consists of an effective stroke followed by a recovery stroke, whereas flagellar motility usually consists of successive waves originating at the base or the tip of the flagellum and propagating along its length (Dawe et al. 2007). Probably because of these differences, the mainstream literature retains the generic term “flagellum” for the typical locomotor organelle of eukaryotes. For the sake of simplicity, we also do so in this book, adding the attributive “eukaryotic” when appropriate. Lynn Margulis suggested that, much like mitochondria and chloroplasts, eukaryotic flagella derived from an endosymbiotic bacterium, namely a spirochaete; this hypothesis is now abandoned in favour of an endogenous origin. (continued)

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Box 6.2 (continued) The eukaryotic flagellum is a cytoplasmic protrusion surrounded by an extension of the cell membrane and internally supported by an axial cytoskeleton (the axoneme) made of nine peripheral pairs of partially fused microtubules (doublets) connected by rays to two central microtubules. At the base of the flagellum is a basal body (named kinetosome or blepharoplast by protistologists), a structure made of nine triplets of microbules tethered to the axoneme through a transition zone (Fig. 6.23). Each axonemal doublet consists of a complete 13-protofilament A-microtubule (like most cytoplasmic, singlet microtubules) and a partial 10-protofilament B-microtubule, which assemble off the ends of the A and B-tubules of the basal body triplets, respectively (Fig. 6.23). The A microtubule of each doublet bears a pair of dynein arms; by hydrolysing ATP, dynein arms force microtubule doublets to reversibly slide along each other, causing an undulatory or whip-like movement. The arrangement of axonemal microtubules in doublets permits the A microtubule to function as a dynein cargo and the B microtubule of the adjacent doublet to function as track, so that unidirectional bends form between doublet pairs. The cell membrane enveloping the flagellum is a cell surface domain separate from the rest (Mitchell 2016).

Fig. 6.23 The eukaryotic flagellum consists of an outer shaft supported by a cytoskeleton of 9 + 2 microtubule pairs, and a basal body of 9 microtubule triplets. This structural design is ubiquitous in eukaryotes and is a major apomorphy of the group

(continued)

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Box 6.2 (continued) Over 660 genes take part in the building, maintenance and functioning of the eukaryotic flagellum, many of which have no recognizable homologs in bacteria or archaea (Carvalho-Santos et al. 2011; Moran et al. 2014; Mitchell 2016). In addition to α, β and γ tubulins, which are universal in eukaryotes, flagellar assembly involves δ, ε and ζ tubulins, which are tubulin variants present only in eukaryote lineages with flagellate cells (Findeisen et al. 2014). Most eukaryote lineages are flagellate or form flagellate cells at some stage in their life cycle. Flagella are lacking in higher fungi (Dikarya), in several lineages within the Amoebozoa including cellular slime molds (Dictyostelida), in the red algae and in the angiosperms. These non-flagellate lineages diverged from flagellate progenitors and independently lost the flagella during their evolutionary history (Adl et al. 2018). It is not clear whether the mitochondrion evolved before or after the flagellum, although the high energy requirement of flagella favours the latter possibility. The last eukaryotic common ancestor (LECA) clearly possessed both. Most common among extant eukaryotes is the biflagellate condition, yet almost all major lineages include taxa with only one flagellum, e.g. most opisthokonts, some amoebozoa, trypanosomids and centric diatoms, or more than two flagella, e.g. three in the Foraminifera, four or eight in diplomonads and prasinophytes, to hundreds or thousands in parabasalids, ciliates and ferns (Yubuki and Leander 2013; Fig. 6.24). Comparison of the flagellar apparatus in different eukaryote lineages is somewhat difficult because researchers working in different fields (e.g. protozoology, phycology, zoology, botany) tend to use different terms and notations. With reference to the direction of cell movement, the flagella may be anterior (i.e. attached at the forward-directed cell extremity) or posterior. In some biflagellate protists such as the Euglenozoa, both flagella are attached anteriorly but one is directed forward and the other backwards, thus somewhat inconsistently they are referred to as the anterior and posterior flagellum, respectively; in cells with a dorsal-ventral symmetry the flagella may be dorsal or ventral, depending on the relative position of their basal bodies (Fig. 6.24). Beside an axoneme-bound basal body, at the base of each flagellum there can be a free basal body (Fig. 6.25a). Flagellar basal bodies, either linked to an axoneme or free, are named kinetosomes, but they are often also referred to as “centrioles”, a denomination better reserved to the paired structures localized in the centrosome (see below). Adl et al. (2012) refer to axoneme-bound and free kinetosomes as kinetosome C and kinetosome X, respectively. (continued)

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Box 6.2 (continued)

Fig. 6.24 Types of flagellate cells in eukaryotes.(a) Anisonema acus (Euglenozoa), a phagotrophic euglenid, has two flagella stemming from an apical pocket, or reservoir, one directed forward (anterior or dorsal flagellum), the other directed backward (posterior or ventral flagellum). Anisonema and other non-photosynthetic phagotrophic euglenids use the flagella to glide forward and backward on solid substrates and to capture preys. The anterior flagellum has also a sensorial function. From Leander (2012), with permission. (b) The green unicellular alga Chlamydomonas reinhardtii has two anterior flagella about 12 μm in length, which propel the cell in the direction indicated by the arrow by beating in opposite directions with an asymmetric ciliary waveform (something like a breast stroke) with a frequency of approximately 60 beats s1. In response to environmental stimuli such as excessive light intensity, the flagella briefly switch to a symmetric flagellar waveform (resembling a sinusoidal pattern) to move the cell backward, and then resume normal movement in a different direction (https://vimeo.com/5592484). The flagella of Chlamydomonas are also able to mediate gliding on solid surfaces or at the air/water interface. (c) The sperms of animals (Amorphea, Opisthokonta) swim by means of a single posterior flagellum. (d) The choanoflagellates (Amorphea, Opisthokonta) are sessile protists with a single flagellum surrounded by a collar of 30–40 actin-supported microvilli. Flagellar beating produces a water current that transports small preys to the collars, where they are captured by phagocytosis (see animation at http://www.biology.ualberta.ca/facilities/multimedia/uploads/zool ogy/choanoflagellate.html). (e) The ciliates are phagotrophic protists with numerous flagella arranged in rows called kineties. Being short relatively to the cell size, these flagella are traditionally called “cilia”, whence the name of the group

(continued)

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Box 6.2 (continued)

Fig. 6.25 (a-c) Morphology of the flagellar apparatus in a typical excavate (a), a chytrid (b), and a choanoflagellate (c). K1, K2, kinetosome 1 (old) and 2 (young, see text); N, nucleus; R1, R2, R3, flagellar roots. (d) The kinetid in Breviata anathema (Amorphea) has a flagellate kinetosome (C) and a free kinetosome (X). (e) The kinetid in the unicellular green alga Chlamydomonas reinhardtii (Viririplantae, Archaeplastida) has two kinetosomes (BB), each forming a flagellum. (a-c) Redrawn from Azimzadeh (2014). (d) From Roger and Simpson (2009), licence 4464930535863. (e) Picture by William Dentler (Kansas University, USA)

The kinetosomes function as microtubule-organizing centers (MTOCTs) that control the formation of flagellar axoneme(s) and flagellar roots. The latter are cytoskeletal structures made of microtubules and fibrous proteins, which anchor the flagella to the cellular body and contribute to cellular shaping and scaffolding, a function of critical importance in the absence of a rigid external envelope (Yubuki and Leander 2013). The ensemble of kinetosome(s) + root forms the kinetid, or mastigont. (continued)

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Box 6.2 (continued) Kinetid morphology is conserved enough to infer homology over large phylogenetic distances, and variable enough to distinguish eukaryotic lineages within major clades (Adl et al. 2018). Comparative analysis of this complex trait, however, requires highly dedicated ultrastructural work, so it is not surprising that, nearly 70 years after the introduction of routinary use of transmission electron microscopy, the information currently available covers only a minor part of the overall taxonomic spectrum, not to mention developmental stages. In different lineages, there can be one, two or more kinetids per cell, and each kinetid may be associated with one, two or rarely more flagella. Adl et al. (2018) refer to kinetids with one or more than one kinetosome as unikont or dikont kinetids, respectively. Most excavates have a single dikont kinetid per cell, with an anterior and posterior kinetosome each associated with a flagellum, and three main microtubular roots plus a fourth smaller one (Yubuki and Leander 2013; Azimzadeh 2014; Fig. 6.25a). Among the Diplomonada, Chilomastix has one dikont kinetid with four kinetosomes and four flagella whereas Giardia has two dikont kinetids, each with four kinetosomes, and usually four flagella (Dawson and House 2010; Cavalier-Smith 2013). Within the Amorphea (formerly Unikonta), the Opisthokonta have a dikont kinetid consisting of a flagellate kinetosome, a free kinetosome and a single conical root of microtubules radiating in the cytoplasm around the nucleus (Fig. 6.25b, c), or no conspicuous root at all in the animals (Yubuki and Leander 2013; Azimzadeh 2014). The Breviatea, a group associated with the Opisthokonta (Burki et al. 2016), have a dikont kinetid with a single flagellum, two basal bodies and two roots (Walker et al. 2006; Fig. 6.25d). A dikont kinetid with a single flagellum, two basal bodies and three or four roots is probably the ancestral condition in the Amoebozoa, but many amoebozoan taxa completely lack flagella (e.g. the Dictyostelida and the whole of Discosea). Others, such as the Archamoebae, Phalansterium and the multiflagellate lobosean amoeba Multicilia, have unikont kinetids (CavalierSmith 2002b; Paps et al. 2013; Yubuki and Leander 2013; Pánek et al. 2016; Adl et al. 2018). The green alga Chlamydomonas has a typical dikont kinetid with two kinetosomes, each bearing a flagellum (Fig. 6.25e), and four microtubular roots arranged in a cruciate pattern (Silflow and Lefebvre 2001). The uniflagellate sperms of centric diatoms (Stramenopila) have a unikont kinetid with a conical root enveloping the nucleus (Idei et al. 2013). Particularly impressive is the flagellar apparatus in ciliates, with several hundreds or thousands of short flagella (called cilia) arranged in longitudinal rows, each emerging from a unikont kinetid with three roots (Pearson and Winey 2009). The numerous variants in the flagellar apparatus of extant eukaryote lineages (continued)

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Box 6.2 (continued) possibly derived from a complex excavate-like architecture by independent loss of ancestral traits (Yubuki and Leander 2013). The flagella afford the cells two types of movement: sliding on solid substrate (or in some cases at the air/water interface) and free swimming in liquid medium. Numerous protists, for example the Euglenozoa, the Apusozoa (Amorphea), filose amoebas (Cercozoa, Rhizaria) and Katablepharidae (Cryptista) use the flagella mainly for sliding (Cavalier-Smith 2013). Forward and backward flagellar sliding is mediated by specific forms of dynein and kinesin, respectively, which are bound to integral complexes in the flagellar membrane and use axonemal microtubules as tracks. Sliding flagella may have sensorial functions as well, and are able to explore the environment with undulatory movements (Mitchell 2007; Carvalho-Santos et al. 2011; Moran et al. 2014). In some protists, for example in phagotrophic euglenozoa, sliding flagella are also used for capturing and carrying the prey to the cellular body by kinesin-mediated transport (Cavalier-Smith 2013). The flagella specialized in free swimming display fast undulatory, helicoidal or whip-like movements mediated by dynein arms on axonemal microtubules (Carvalho-Santos et al. 2011)). Sessile protists as well as many filterfeeding animals utilize the flagella/cilia to create water currents that facilitate prey capture (Fig. 6.24d). The eukaryotic flagellum possibly evolved from microtubule-supported cytoplasmic projections (Mitchell 2004, 2016; Jékely and Arendt 2006; Carvalho-Santos et al. 2011). Ancestral flagella were probably employed to capture prey and transport it to the main cell body for phagocytosis; initially, motility may have been gliding, followed by flagellar beating to generate feeding currents and eventually by free swimming in liquid medium (Fig. 6.26). It is not clear whether the 9 + 2 axonemal structure evolved before the appearance of free-swimming locomotion or afterwards, though the presence of flagella with a 9 + 2 axoneme in phagotrophic euglenids favours an early origin. Mitchell (2007) suggested that motility regulation by the central microtubule doublet provided a selective advantage and that the geometry of this regulatory mechanism favoured an outer cylinder of precisely nine microtubule doublets, with the distance between doublets determined by the reach of dynein arms. The axoneme ability to bend does not necessarily require a 9 + 2 microtubule arrangement. For example, the flagellar axoneme in sperm cells of centric diatoms lacks the central microtubule pair and its basal body contains 9 microtubule doublets instead of triplets, yet the flagellum is perfectly functional (Idei et al. 2013). In general, however, alterations in axonemal structure negatively affect flagellar motility and have been tolerated in nature only under special conditions. In some parasitic protists and in the sperms of some animals with internal fertilization, for example, the axoneme lacks the central (continued)

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Box 6.2 (continued)

Fig. 6.26 Hypothetic model of autogenous origin of flagella. The model posits that the flagellum evolved in a eukaryote that already had a microtubular cytoskeleton radiating from a microtubule-organizing center (MTOC) as well as a nucleus, mitosis and an endomembrane system. The flagellum started as a cytoplasmic protrusion supported by microtubules nucleated by the MTOC during interphase. The microtubular skeleton then evolved a 9 + 2 symmetry. Microtubule-associated molecular motors mediated anterograde (directed towards the distal, plus microtubule end) or retrograde movement (towards proximal, minus microtubule end). As in extant sliding flagella, particles bound to membrane receptors of protoflagellar projections were transported to the cell body for phagocytosis by retrograde movement. Primitive flagella initially worked as sensorial/raptorial structures, then adapted for cellular gliding and last for swimming in liquid medium. (Redrawn from Carvalho-Santos et al. 2011)

microtubule pair and the rays, and the flagella only produce a helicoidal movement suitable for propulsion in high-density liquid media. Sensorial cilia in animals lack the dynein arms, the central pair and the rays, and are immobile (Mitchell 2007). Expansion of the axonemal dynein family in some organisms permitted the assembly of flagella with specific traits, such as the different cilia types present in the oral zone, body wall, and caudal region of ciliates, or the cilia in the epithelia of airways, brain ventricles, fallopian tubes and embryonic node in mammals (Mitchell 2016). Whereas the core structure of eukaryotic flagella is ancient and highly conservative, a multitude of ancillary variants is known, encompassing variations in the number and distribution of flagella, changes in the geometry of the roots, the addition of hairs, membranous projections (vanes) and reinforcing structures. Flagella lacking surface projections, as those present in the Opisthokonts, are referred to as “smooth” or “whiplash” flagella. In most eukaryotic lineages the flagella bear superficial structures of varying morphology, for example thin non-tubular hairs (Euglenida, Dynoflagellata, a part of (continued)

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Box 6.2 (continued) the Haptophyta) or stiff tubular hairs with a bipartite (Cryptophyta and Prasinophyta) or tripartite structure (Stramenopila). The flagella of Kinetoplastida and Parabasalida are partly attached to the cell membrane, forming the structure known as the “undulating membrane” (Briggs et al. 2003). The kinetids are often linked to the nucleus by means of root structures to form a morphological unit named caryomastigont. The flagellar shafts usually disassemble before mitosis, but the kinetids persist and may function as spindle polar bodies (SPB) mediating the nucleation and positioning of the mitotic microtubule apparatus. In biflagellate cells, each daughter cell receives one SPB, which regenerates the flagellar apparatus in the subsequent interphase (e.g. Chlamydomonas). In several biflagellate protists, for example the Euglenozoa, Diplomonada and Katablepharida, the flagella are not disassembled during cell division, but duplicate before mitosis so that each daughter cell receives a pre-built flagellar apparatus (Leander 2012). Some eukaryotes possess a pair of kinetosome-like organelles in all cells, including cellular types or stages that lack flagella. These structures, named centrioles, are arranged at a right angle to each other and are surrounded by dense pericentriolar material containing microtubule nucleators (MTOC) and regulators of the cell cycle. The complex of centrioles and pericentriolar material is known as the centrosome. A centrosome is present in the animals (with exceptions), in basal fungi and some members of the Amoebozoa (e.g. the Myxogastria, or plasmodial slime molds), in the brown algae and in the Plasmodiophora, cercozoan parasites of plants (Azimzadeh 2014). The centrosome lies near the nucleus and duplicates once at each cellular cycle, immediately before mitosis, by assembling two new centrioles perpendicular to the old ones; each newly formed centrosome receives one old and one new centriole. Much like kinetids, after duplication the two centrosomes function as spindle polar bodies, each daughter cell inheriting one. Thus, the essential difference between centrosomes and kinetids is that the former occur in non-flagellate cells. When necessary, the centrosome mediates the development of a flagellar apparatus by migrating to the cell periphery and turning into a kinetid, with one or both centrioles nucleating an axonemal complex. In flagellate cells of brown algae (sperms and zoospores), both centrosomal centrioles are active and produce an anteriorly and a posteriorly directed flagellum. During spermatogenesis in the animals, only the older centriole of the pair is active and produces a flagellum, the other becoming a free kinetosome at the base of the flagellum. The free kinetosome enters the egg cell during fertilization and here it develops into a founder centrosome wherefrom all the centrosomes and flagella of the adult body will arise. Interestingly, all cells in the animal body initiate the formation of a flagellum when entering differentiation. Known as the “primary cilium”, this is nucleated by the older (continued)

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Box 6.2 (continued) centriole of the centrosome and remains a rudimentary structure with no function in most cellular types; in some types of sensorial neurons (e.g. retinal cones and rods, and olfactory cells), the primary cilium develops into a nonmotile sensorial structure. In animal cells bearing numerous cilia, for example ciliate cells in the bronchial epithelium of mammals, hundreds of new centrioles develop from the initial centrosome; these singly migrate to the cell periphery, dock at the cell membrane and nucleate a ciliary axoneme (Dawe et al. 2007). A remarkable exception among animals is the worm Planaria (Platyhelminthes), whose cells lack a morphologically recognizable centrosome, the basal bodies necessary for flagellar development apparently developing de novo (Azimzadeh 2014). An acentriolar pathway of basal body formation has also been reported in mammals (Dawe et al. 2007). The centrosome is considered to be an ancestral character in the Amorphea, implying that a centrosome was present in the last concestor of the group and that those amorphean lineages lacking a centrosome have in fact secondarily lost it, either along with the flagella (e.g. the Dikarya, Discosea and Dictyostelida) or independently (Choanoflagellates, Fig. 6.27). The absence of a centrosome in the other major lineages, instead, suggests that this is not an ancestral trait of eukaryotes. A centrosome probably evolved independently in the Amorphea, brown algae and Plasmodiophora. A centrosome-like structure also occurs in apicomplexan parasites (Francia and Striepen 2014). In land plants, somatic cells lack flagella and centrioles, but male gametes are bi- or multiflagellate (with the exception of most seed plants, whose male gametes lost the flagella). Despite this, land plants do not have centrosomes, the kinetosomes necessary for the formation of flagella developing de novo in the cell line destined to form male gametes. De novo kinetosome formation also occurs in centric diatoms during the development of male gametes, and in the heterolobosean amoeba Naegleria during the transition from the amoeboid to flagellate form (Carvalho-Santos et al. 2011). Non-centriolar spindle polar bodies are an obligate choice in mitotic cells lacking a centrosome and flagella (Figs. 6.27 and 6.28). Examples include the “spindle pole bodies” in dikaryan fungi (Fu et al. 2015), “nuclear-associated bodies” in Dictyostelida (Gräf 2015), “microtubule centers” and “polar complexes” in diatoms (De Martino et al. 2009), “nucleus-associated organelles” in red algae (Scott 1986), and “polar organizers” in liverworts, a basal lineage of land plants (Brown and Lemmon 2011). On the other hand, the kinetids take no part in the development of the mitotic microtubule apparatus in several flagellate protists. For example, the kinetids control the segregation of the mitochondrion and its kinetoplast in trypanosomes (Drechsler and McAinsh 2012), and replicate in specific cellular areas before micronucleus division in ciliates, but in neither case do they participate in mitosis (Pearson and Winey 2009). Figure 6.28 shows examples of mitosis variants in different lineages. (continued)

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Box 6.2 (continued)

Fig. 6.27 Distribution of flagellate and amoeboid forms and of discrete spindle polar bodies (SPB) in eukaryotes. The flagellate condition is ancestral, whereas the amoeboid form is probably derived and evolved independently multiple times. Flagellate eukaryotes generally use kinetids as SPBs, but several eukaryotes that form flagellate cells only in a phase of their life cycle (e.g. the Opisthokonta and brown algae) use centrosomes instead. Exceptions include the diatoms, which use non-centriolar SPBs in vegetative cells and form a unikont kinetid de novo in sperm mother cells, and the choanoflagellates, whose flagellate cells use non-centriolar SPBs during division. A centrosome-like structure was reported in the apicomplexan parasites Eimeria and Toxoplasma. The SPB of the apicomplexan Plasmodium lacks morphologically identifiable centrioles, but contains plaques positive to antibodies against the centriolar protein centrin 1 (Francia and Striepen 2014). The apparent predominance of centriolar SPBs is most likely due to taxonomic bias from dataset. (Redrawn from Azimzadeh 2014)

Comparative analysis suggests that early flagellate eukaryotes had a unikont kinetid with a single flagellum and a single kinetosome, yet LECA probably was a biflagellate organism with an anterior and a posterior flagellum emerging from a dikont kinetid as in Euglenozoa (Cavalier-Smith 2014; Fig. 6.25a). This implies that present-living eukaryotes are primarily biflagellate, the uniflagellate and non-flagellate condition having evolved multiple times by reduction. In all known instances of uniflagellate eukaryotes, for example centric diatoms (Stramenopila), trypanosomids (Euglenozoa) and opisthokonts, the conserved flagellum is the anterior one in anatomical terms; relative to the direction of movement of the cell body, however, the flagellum lies anteriorly in diatoms but posteriorly in trypanosomids and opisthokonts. (continued)

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Box 6.2 (continued)

Fig. 6.28 Mitosis variants in eukaryotes. (a) Open mitosis in animals: the nuclear envelope and other nuclear structures (e.g. the nucleolus and nuclear lamina) break down at the onset of the process and reassemble at the end. (b) Semi-open mitosis (e.g. Plasmodiophora brassicae and Chlamydomonas reinhardtii): the nuclear envelope opens only at the poles and microtubules from spindle polar bodies (SPBs) radiate into the nucleus, where they capture the condensed chromosomes. The nucleolus (nu) persists during mitosis. (c, d) Semi-closed mitosis (e.g. ascomycetous fungi and trypanosomids): the SPBs are transiently (Schizosaccharomyces pombe) or permanently (Saccharomyces cerevisiae) inserted in the nuclear envelope, where they nucleate the mitotic spindle. In the unicellular parasite Trypanosoma brucei (d), the kinetids control the duplication and partition of the flagella and of the mitochondrion, but the assembly of the mitotic spindle is under the control of an independent, non-centriolar SPB bound to the nuclear envelope. (e, f) Closed mitosis (e.g. Dinoflagellata and Parabasalida): the nuclear envelope remains intact, but forms one to several pole-to-pole invaginations called “nuclear channels”; mitotic spindle microtubules emanating from cytoplasmic SPBs enter the nuclear channels and connect with chromosome kinetochores bound to the nuclear envelope, thus driving chromosome segregation to nuclear poles. (e) In the Parabasalida (Metamonada, Excavata) and some members of the Dinoflagellata (Alveolata) such as Crypthecodinium cohnii there are several nuclear channels, the kinetids take no part in mitosis and the SPBs are non-centriolar amorphous structures. (f) In other dinoflagellates such as Oodinium, Amphidinium and Syndinium there is a single nuclear channel, the SPBs arise from kinetids and contain a pair of centriole-like structures. In summary, the SPBs may arise from centrosomes (in centrosome-possessing eukaryotes) or kinetosomes (in numerous flagellate eukaryotes), in both cases containing a couple of centrioles; in other cases, the SPBs lack any discrete structure (e.g. dinoflagellates or land plants), or have a structured organization but lack centriole-like structures (e.g. higher fungi). (Adapted from Drechsler and McAinsh (2012), Creative Commons Attribution 4.0 International License)

(continued)

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Box 6.2 (continued) The notion that the common progenitor of extant eukaryotes was flagellate suggests that centriolar SPBs (kinetids, centrosomes or whole flagella) are ancestral to non-centriolar SPBs, which therefore should have evolved multiple times in separate lineages (Fig. 6.27). Yet, the scenario is probably more complex, because the flagella probably derived from a non-centriolar MTOC functioning as a polar spindle body (Fig. 6.26), suggesting that mitosis preceded flagella. Current knowledge of the architectural dynamics of the flagellar apparatus is restricted to a minority of taxa. Further information is paramount to a better understanding of the evolution of the flagellar apparatus and its relatioships with mitosis.

6.10

Dating Eukaryote Appearance and Diversification

As for almost everything about the evolutionary history of eukaryotes, the chronology of eukaryote appearance and diversification is an area of yet unresolved controversy. Proposed scenarios range from “eukaryote-first” hypotheses depicting the eukaryotes as the earliest domain of life from which the bacteria and archaea derived by reduction (reviewed by Mariscal and Doolittle 2015), to currently dominating eukaryote-last scenarios (Roger et al. 2017 and references therein). Molecular-clock analysis (Box 1.1) has produced estimates ranging from over 2.3 GYA (Hedges et al. 2004) to about 1.8 GYA (Douzery et al. 2004; Weneger Parfrey et al. 2010; Eme et al. 2014) for the first eukaryote appearance, and of about 1.2 GYA for the appearance of the mitochondrion (Shih and Matzkeb 2013). These dates suggest that eukaryotic fossils older than 1.2 GYA represent pre-LECA lineages (stem eukaryotes), whereas younger fossils are from post-LECA lineages and may include the progenitors of extant eukaryotes (crown eukaryotes). The core of the problem is how to discriminate eukaryotes from prokaryotes in the fossil record. This not only affects paleontological inference, but also impacts on molecular dating, which depends of calibration from paleontological data. As we will see in Chap. 8, eukaryotes have a strong propensity to evolve multicellular forms with pronounced cellular differentiation; the first complex eukaryotes, however, appeared only about 600 MYA with the so called “Ediacaran Biota”, followed by the first animals about 40 MY later. No one doubts that ediacaran fossils were eukaryotic nor that eukaryotes must be older than 600 MY. More uncertain is the nature of a filamentous fossil reported as Bangiomorpha pubescens, dated to over 1.2 GYA and tentatively interpreted as a red alga close to present-living Bangia (Knoll 2014). Based on the observation that filamentous forms are common among bacteria, including some exhibiting a degree of cellular differentiation, CavalierSmith (2002b) argues that there is no compelling reason for ruling out that Bangiomorpha is a filamentous bacterium. Indeed, barring Bangiomorpha, the red algal lineage appears in the fossil record only from the Late Proterozoic, about

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800 MYA, and starts diversification only from 570 MYA. Recent molecular clock analyses concur in suggesting that the Bangiomorpha fossil date or taxonomic assignment is problematic (Weneger Parfrey et al. 2010; Eme et al. 2014). Likely eukaryotic signatures in microfossils are cell wall ornamentations or other features that reflect the existence of a eukaryotic cytoskeleton (Box 6.1). Applying this criterion, the oldest undisputable eukaryotic fossils are the acritarchs, unicellular organisms appeared about 0.8 GYA, whereas more doubtful is the interpretation of fossils such as Grypania (1.4–1.8 GYA) and Tappania (1.5 GYA) (Knoll 2014). A third line of evidence is from biomarkers, molecular fossils that prove the existence of metabolic pathways unique to specific lineages. The oldest proposed biomarker evidence for eukaryotes is the report by Brocks et al. (1999) of 2.7-GYold steranes, which are breakdown products of sterols including cholesterol. Subsequent investigation showed that these sterol biomarkers were younger than the rocks hosting them, probably being contaminants from above-lying more recent rock (Rasmussen et al. 2008). Further study confirmed the occurrence of steranes in Transvaal Supergroup sediments dated from 2.67 to 2.46 GYA (Waldbauer et al. 2009), yet no significant amounts of steranes were found in cores extracted from the 2.6 GY-old Pilbara Craton in Australia (French et al. 2015; also see review by Knoll 2014). An environmental weakness on this kind of evidence is that sterol biosynthesis requires molecular oxygen, yet geochemical data indicate that the Archaean atmosphere and oceans were essentially anoxic until 2.4 GYA (Chap. 5). Presentliving eukaryotes primarily possess mitochondria, which require substantial levels of oxygen to function. The palaeochemical evidence of Archean steranes, if definitely confirmed, would probably point to putative eukaryotic forms using sterols in their membranes but still lacking mitochondria, a notion apparently at odds with “mitochondrion-first” models of eukaryogenesis. It is also to be considered that some bacteria including myxobacteria, actinomycetes and planctomycetes are able to make steroids (Summons et al. 2006; Cavalier-Smith 2014). Cavalier-Smith (2002b, 2013, 2014) argues that eukaryotes evolved at extremely high rates at the onset of their existence, because they occupied completely new ecological niches as phagotrophic predators. Accelerate evolution causes an amplification of molecular divergence that erroneously sets back the molecular clock. Based on a critical re-examination of fossil and molecular evidence, Cavalier-Smith (2014) now places the birth of the eukaryote lineage around 1.2 GYA. This should also be the likely birth date of archaea, which Cavalier-Smith considers the sister group of eukaryotes. The discovery of novel macrofossils from the 1.56 GY-old Gaoyuzhuang Formation, North China (Zhu et al. 2016) probably requires the origin of eukaryotes to be antedated. Preserved as carbonaceous compressions, these fossils have linear to lanceolate shapes up to 30 cm long and nearly 8 cm wide, suggesting an origin from benthic multicellular eukaryotes. In spite of paleontological evidence of an earlier origin, eukaryotic fossils antedating 800 MYA are rare and poorly diversified, becoming more abundant and diverse between 800 and 720 MYA, as documented by the detection of 17 novel “species” of vase-shaped protists distributed worldwide, and 26 siliceous microfossils in Canada (Knoll et al. 2006; Knoll 2014). The palaeontological record from this interval encompasses likely representatives of numerous extant eukaryotic

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clades, including red algae, green algae, heterokonts, amoebozoans, cercozoans and fungi. After a long pause concomitant with the coldest phase of Neoproterozoic glaciations (Chap. 5), a multitude of eukaryotic microfossils reappeared from 660 MYA. A worldwide shift to higher Zn/C ratio in marine sediments about 800 MYA may be evidence for eukaryotic phytoplankton replacing cyanobacteria as the main primary producers, an inference based on the fact that modern eukaryotic phytoplankton has higher Zn demands and higher Zn/C ratios relative to cyanobacteria (Isson et al. 2018). Neoproterozoic eukaryotic diversification is also documented by “molecular fossils”. Steranes begin to be regularly present in sedimentary rocks after 800 MYA; notably, the occurrence of C28 e C29 sterans (made of 28–29 carbon atoms) in rocks aged about 750 MY suggests that by that time green and red algae had become major primary producers on continental shelves (Kodner et al. 2008). Gammacerane, a derivative of tetrahymanol found in 750-million-year-old rocks, is possible evidence for the existence of ciliates. A C28 sterane tentatively identified as 26-methylcholestane, was detected in sedimentary rocks from Australia, Sweden and USA and tentatively assigned to basal sponges (Brocks et al. 2015). Calibrated molecular-clock analysis estimated the origin of fungi at 1060– 760 MYA, and the origin of Ascomycota at 650–500 MYA (Lücking et al. 2009). The Ediacaran biota, enigmatic forms appeared around 600 MYA and flourished until the end of the Proterozoic, are the first multicellular complex organisms. The Phanerozoic started 541 MYA with the “Cambrian explosion”, a relatively short interval of about 20–25 MY, during which most present-living animal phyla made their appearance, and several other eukaryotic lineages went through a major diversification (Knoll 2014). As discussed in Chap. 5, about 800 MYA the level of oxygen in the atmosphere started rising again after over a billion year of stability, causing the oxygenation of deep ocean waters until then rich in sulphides. This change was coeval with eukaryote diversification, although the cause/effect relationship of the two events is yet to be ascertained (van der Giezen and Lenton 2012). Combined together, paleontological evidence and molecular clock data suggest that the eukaryote domain appeared at least 1.5 GYA, the concestor of extant eukaryotes evolved around 1.2 GYA, and major extant lineages diverged after 900 MYA. Box 6.3 Nuclear DNA: Between Paradox and Enigma The eukaryotes have not yet finished surprising us. Following the demonstration of DNA as the depositary of biological information in 1943 and the definition of its molecular architecture ten years later, DNA became the focus of intense investigation throughout the world. The first systematic data revealed that eukaryotes store much more DNA in their cells than prokaryotes. This caused no wonder: because DNA carries biological information, organisms with a more complex organization were expected to have more DNA than (continued)

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Box 6.3 (continued) simpler ones. When, in 1961, research demonstrated that the informational unit of DNA is a triplet of nucleotides, simple mathematics led to believe that eukaryotes had a much higher number of coding sequences, or genes, than prokaryotes. After the introduction in 1983 of the technique known as “polymerase chain reaction” (PCR), researchers began to sequence DNA from a growing number of species. Once the sequences were determined, special software permitted the identification of genes. The result was now a big surprise: eukaryote genomes do encompass a larger number of genes than prokaryotic genomes, but the difference is of only one order of magnitude, around tenfold, whereas the average difference in total DNA is of three orders of magnitude, i.e. one thousand times. Clearly, most eukaryotic DNA consists of non-coding sequences. A second unexpected feature of the eukaryotic genome was its very wide size ranging: spanning from the smallest to largest known genome, there is a difference of about 1000 times in plants, 3300 times in animals, and perhaps as much as 300,000 times in protists. Such a vast variability does not reflect the number of genes, which varies from some thousands in smaller genomes to some tens of thousands in larger ones, and even less organismal complexity. As an example, because of the widespread occurrence of intra-genic noncoding introns, the amount of DNA in 30 typical human genes is equivalent to the whole genome size of a typical prokaryote containing several thousand genes. This is known as the “C-DNA paradox”, where “C-DNA” (or C-value) indicates the total DNA amount in the haploid genome of eukaryotes (Gregory 2005; Elliott and Gregory 2015). The size of C-DNA is huge relative to the encoding fraction of it and, quite surprisingly, is an extremely variable trait even among relatively akin taxa. A classic example of the C-paradox is the genome of the pufferfish (Tetraodon nigroviridis) and the African lungfish Protopterus aethiopicus, both belonging to the vertebrate superclass Osteichthyes. The first has the smallest C-value among vertebrates (390 Mb, 1 Mb ¼ 1 million base pairs), the latter the largest (130,000 Mb), despite the two animals being fully comparable in terms of anatomic complexity. The human genome consists of 3235 Mb and contains 19,000–20,000 protein-coding genes (the number of genes is usually referred to protein-coding sequences, the amount of DNA sequences transcribed into functional RNAs but not translated into protein being relatively constant in each domain). Shockingly, not only this means that about fivefold as many genes as in a ordinary bacterial cell are sufficient for making a human being, but that over 98% of the human genome is non-coding DNA. It is now clear that both the C value and gene number are very weakly linked to complexity in eukaryotes (Fig. 6.29). (continued)

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Box 6.3 (continued) Mammals Birds Reptiles Frogs Salamanders Lungfishes Teleost fishes Chondrostean fishes Cartilaginous fishes Jawless fishes Non-vertebrate chordates Crustaceans

Insects Arachnids Myriapods Molluscs

Annelids

Echinoderms Water bears (Tardigrada) Flatworms (Platyhelminthes) Rotifers Red algae (Rhodophyta) Green algae (Chlorophyta) Brown algae (Phaeophyta) Flowering plants (Angiosperms) Non-flowering seed plants (Gymnosperms) Ferns (Monilophytes) Club mosses (Lycophytes) Mosses and kin (Bryophytes) Roundworms (Nematodal) Cridarians Sponges (Porifera) Fungi Protozoa Bacteria Archaea

–1

0

1

2 3 Log10 C-value (Mb)

4

5

6

Fig. 6.29 Genome size ranges in archaea, bacteria and selected eukaryotes. (From Gregory 2005, licence number 4466610154288)

Prokaryotes have a sharply different genomic arrangement. The typical size of a prokaryotic gene is about 1200 base pairs, the total genome in bacteria is 1.5–8 Mb and encompasses 1500–8000 genes, with only about 20% of noncoding DNA. There are bacteria with particularly tiny genomes, for example the mycoplasmas (obligate intracellular parasites of plants and animals), and bacteria with relatively large genomes overlapping with the smaller eukaryotic genomes, for example the cyanobacteria and actinobacteria, but the average order of magnitude remains 6 (1 Mb) for bacteria and 9 (1 Gb) for eukaryotes (Fig. 6.29). In mathematical terms, the number of genes in bacteria and archaea is linearly correlated to genome size, in eukaryotes it is not. The (continued)

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Box 6.3 (continued) percentage of the eukaryote genome composed of protein-coding sequences decreases as genomes grow larger, with protein-coding regions contributing a very small fraction in all but the smallest eukaryotic genomes. This differs markedly from the situation in prokaryotes, in which protein-coding gene number is the primary determinant of genome size diversity (Elliott and Gregory 2015). Non-coding DNA in Eukaryotes Non-coding DNA in eukaryotes consists of non-repetitive and repetitive sequences. The first are present singly or as few copies, the latter occur in numerous identical copies scattered in the genome or forming tandemlyrepeated arrays (Fig. 6.30). Non-Repetitive Sequences: – Genes for ribosomal RNA (rRNA), transfer RNA (tRNA) and a diversity of small RNAs with regulatory functions. These are about 0.5% of total in the human genome. – Cis and trans regulatory elements: sequences that, with a diversity of mechanisms, control the transcription of specific genes. These are a minor part of human genome. – Introns (from “intragenic region”): intra-gene sequences transcribed into RNA and subsequently cut out to produce functional RNA. Introns account for about 26% of human genome. – Pseudogenes: sequences derived from redundant gene copies by mutation and loss of function. The number of pseudogenes shows remarkable variation among lineages; for example, there are about 19,000 pseudogenes in the humane genome but only 51 in the chicken genome. – Extragenic unique sequences, of uncertain function. Repetitive Sequences: • Simple-sequence repeats: short tandemly repeated sequences, mostly located in non-coding parts of the genome; these include the telomeres, located at the chromosome ends and essential to prevent chromosome deterioration/fusion, and microsatellites scattered in the genome. Function-less microsatellites tend to accumulate mutations retained across generations, thus producing the variability exploited for DNA fingerprinting. • Satellite DNA: tandemly-repeated sequence arrays forming chromosome centromers and constitutive heterochromatin (see below). (continued)

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Box 6.3 (continued)

Fig. 6.30 Human genome composition. About 98.5% of the human genome consists of non-coding DNA. (From Gregory 2005, licence number 4466610154288)

• Transposable elements or transposons: sequences of likely viral origin that move from a site to another of the genome using either the “past and copy” (class I transposons, or retrotransposons) or the “cut and copy” procedure (class II transposons or DNA transposons). Only a minor part of transposable elements in eukaryotic genomes are still mobile, most being degenerate transposons that have lost mobility and gone through major changes in the original sequence. Transposons and transposon-derived sequences account for about 45% of the human genome and about 85% of maize genome. They encompass the genome regions known as long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), and long terminal repeats (LTRs). Spliceosomal introns (the most common type of introns in eukaryotes) also derive from transposable elements of viral origin (Sect. 6.5). It has long been thought that non-coding DNA was just “junk” from degenerate sequences (pseudogenes) or genetic parasites (“selfish” DNA) inserted in the genome and serially replicated using cellular resources, but lacking any useful function. Systematic analysis of a larger number of genomes, however, has revealed that several lineages (e.g. the yeast Saccharomyces, the malaria agent Plasmodium, the Microsporidia) have drastically downsized their genomes not only reducing the number of genes but also (continued)

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Box 6.3 (continued) eliminating most non-coding DNA (Cavalier-Smith 2005). This proves that, under appropriate selection pressure, eukaryotes are perfectly able to get rid of non-coding DNA. On the other hand, both genome size and the % of noncoding DNA tend to increase with morphological complexity. For example, within the Opisthokonts (Amorphea, see Fig. 6.18) the percent of non-coding DNA increases from about 25% in Saccharomyces, to 58% in Capsaspora owczarzaki (a unicellular relative of the animals) and 93% in the Cnidaria (Sebé-Pedrós et al. 2017). The fact that most eukaryotic genomes retain large chunks of non-coding DNA is evidence that this performs essential functions. In fact, there is evidence that several human genetic diseases depend on alterations in repetitive-DNA sections of the genome. In addition, the introns, another major class of non-coding DNA, permit eukaryotes to use alternative splicing to make multiple proteins from the same gene. Alternative splicing allows the human genome to direct the synthesis of many more proteins than expected from gene number, and abnormal variations in splicing are implicated in genetic disorders (Carey 2015). Involvement in gene expression and alternative splicing, however, cannot account for the staggering amount of non-coding DNA present in most eukaryotic genomes. Is there a more general function for non-coding DNA? This is the so-called “C-value enigma” (Elliott and Gregory 2015). For over a hundred years, it has been known that the nuclear to cytoplasmic volume ratio, or caryoplasmic ratio, is constant in metabolically active cells. In other words, cells with large cytoplasmic volumes (setting aside the vacuoles and other large-sized storage compartments) have large nuclei, and vice versa (Fig. 6.31). Cellular and nuclear sizes are linearly correlated with genome size but not with gene number. Let us add to the picture that, having primarily evolved as a phagotrophic predators, the eukaryotes tend to have larger cells than prokaryotes. Numerous eukaryotic lineages lost phagotrophy as a way to get food, but they generally retained large cells for morphogenetic purposes, for example to form long filaments that explore the substrate (e.g. fungal hyphae and plant rhizoids), or cells specialized in long-distance transport of materials (e.g. vascular cells in land plants and brown algae) and signals (e.g. neurons in animals). It is also useful to remember that mitochondriate cells get great advantage from being large, because this increases the amount of energy per gene (Sect. 6.3). (continued)

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Box 6.3 (continued)

Fig. 6.31 (a) The sepal epidermis of Arabidopsis, a flowering plant, consists of randomly scattered large and small cells. (b) Fluorescence microscopy after staining with a DNAbinding fluorochrome shows that the nuclei (visible as green spots) in large cells are much larger and contain more DNA than in small cells. DNA amplification arises from endoreduplication of the whole genome during cellular development. (From Marshall et al. (2012), Creative Commons Attribution 2.0 International License)

Why do large cells need large nuclei? A greater amount of cytoplasm requires more protein and more ribosomes. The cell may get this simply by elongating the cell cycle, namely by taking more time for growth before each division; this however may excessively reduce reproductive efficiency. Another way might be an increase in the concentration of the enzymes of the transcription machinery; yet, macromolecular concentration in the nuclear matrix is already close to saturation, so a kinetically significant increase is hardly possible. A third possibility is an increase in the number of coding genes present in the genome, attainable by gene duplication. This might have two negative consequences: (a) gene copies would mutate independently, thus impairing puryfying selection of functional alleles (Loewe 2008), (b) the increase of the energy budget per gene afforded by larger cellular size would reduce or nullify (Sect. 6.3). The skeletal DNA hypothesis by Cavalier-Smith (2005) proposes that nuclear DNA, besides carrying genetic information, is a fundamental component of the nuclear matrix, a molecular scaffold needed for the spatial (continued)

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Box 6.3 (continued) arrangement of chromosomes in the interphase nucleus. In this perspective, an amplification of non-coding DNA would permit the formation of more nuclear matrix, namely more space to host greater amounts of enzymes and products, thus accelerating gene transcription and RNA maturation without increasing the number of genes. In response to evolutionary pressure for larger cells, eukaryotes amplified non-coding DNA by “tolerating” the spreading of transposable elements, or duplicating the whole genome and retaining degenerate redundant genes (pseudogenes). Under the effect of natural selection, the process tended to an equilibrium condition characterized by an optimal cost/ benefit ratio. The skeletal functions of DNA might depend on the densely arranged ionisable phosphate groups that give the molecule a pronounced anionic polyelectrolyte character. Depending on the basic molecular structure of DNA, not nucleotide sequence, the skeletal function is a property shared by both coding and non-coding DNA. The skeletal DNA model predict that the amount of non-coding DNA scales roughly isometrically with cell size, a condition generally confirmed by available data (Marshall et al. 2012; Amodeo and Skotheim 2016). Comparative genomics has shown that different mammal species have similar genome sizes but different types of transposable elements; for example, Alu sequences account for about 40% of the human genome, and are similarly abundant in other primates but not in other mammal orders. This suggests that non-coding DNA is continually lost and replaced with novel elements in a dynamic equilibrium that tends to maintain optimal genome sizes independently of the type of DNA employed. The correlation between genome, nuclear and cellular sizes applies to actively dividing cells, but not to resting cells. Likewise, the increase in cellular size obtained with the formation of a vacuole is a functional artifice that improves the cell surface to cytoplasmic volume ratio but does not affect the caryoplasmic ratio. Importantly, DNA occurs in two different states in the interphase nucleus, euchromatin and heterochromatin. DNA in heterochromatin is more densely packed than in euchromatin and is not transcribed into RNA. There is a facultative and constitutive heterochromatin; the first does not change during the cell cycle nor in different cellular types within the same organism; the latter consists of sequences that may be eu- or heterochromatic according to the cell cycle phase or cellular type. Telomers and other types of satellite DNA are typical components of constitutive heterochromatin. For the same amount of DNA, heterochromatin is probably less efficient than euchromatin in making nuclear matrix and supporting nuclear functions, therefore its occurrence in the nucleus requires an explanation. Constitutive (continued)

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Box 6.3 (continued) heterochromatin has a close physical link with the nuclear envelope and probably contributes to its stabilization. Cavalier-Smith (2005) suggests that facultative heterochromatin is a means for the cells to modulate nuclear size (and, indirectly, cellular size or cellular metabolic activity) without changing the genome size. This mechanism may be operative both in multicellular organisms, where cellular types of small sizes (for example the erythrocytes in birds) have abundant heterochromatin, but large cells (for example the neurons) have little heterochromatin, and in unicellular organisms with a life cycle involving different types of cells (for example Naegleria Fig. 6.16). If the eukaryotes ancestrally expanded their genome size in order to make larger cells, several cases are known in which evolution proceeded backwards (reviewed in Cavalier-Smith 2005). The Microsporidia, minute obligate intracellular parasites of animals, have virtually eliminated all non-coding DNA and have the smallest genome known in eukaryotes (from 19.5 Mb in Glugea atherinae to only 2.3 Mb and about 2000 genes in Encephalitozoon cuniculi), setting apart the rudimentary nucleus (nucleomorph) present in certain types of secondary chloroplast (Chap. 9). Encephalitozoon not only eliminated extragenic non-coding DNA but also almost all introns. Worth of interest is also the condition found in Apicomplexa, a group of alveolate protists including the Hematozoa and Gregarina. The Hematozoa (encompassing the malaria agent Plasmodium) are obligate intracellular parasites with very small cells that proliferate within the erythrocytes of their animal hosts; in line with the skeletal DNA hypothesis, they have very small genomes (only 25 Mb and about 5300 genes in Plasmodium). The Gregarina are extracellular parasites living in the intestine, celome cavities or gonads of invertebrates, especially annelids and arthropods; in contrast to their hematozoan relatives, the gregarines have enormous cells around 1 mm in length, with a nucleus as large as 90 μm in diameter. No information was available on gregarine genome size in 2005, when Cavalier-Smith presented his skeletal DNA hypothesis, nor is at the time of publication of this book, apart from scattered sequencing studies for phylogenetic purpose. CavalierSmith’s hypothesis predicts that gregarines have much larger genome sizes than their intracellular parasite relatives, mainly due to non-coding DNA. The ciliates (Ciliophora, Alveolata) have evolved a particularly elaborate solution to increase cell sizes. These protists have two types of nuclei, a small micronucleus and a large macronucleus. The micronucleus contains two copies of the whole genome (it is diploid) and is genetically inactive. The macronucleus arises from a micronucleus and is genetically active; during macronucleus development, the originally diploid genome duplicates several times and fragments into pieces each long no more than one or a few genes. During cell division, the micronucleus divides by mitosis, the macronucleus (continued)

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Box 6.3 (continued) instead uses a process named amitosis, which transmits the genetic material in an approximate way. Because of large-scale genome amplification, this has no damaging consequences for a number of cycles. During sexual reproduction, the macronucleus degenerates, whilst the micronucleus undergoes meiosis, caryogamy and mitosis, a new macronucleus developing from a micronucleus after syngamy. Thanks to nuclear dualism, the ciliate cell may attain large sizes, usually in the order of a hundred micrometres but in some species above one millimetre, whilst retaining a relatively small genome. A roughly isometric scaling of nuclear, cellular and genome sizes appear to be a universal character of eukaryotes. In a number of cases, however, genome sizes also correlate with other parameters such as cell division rate, respiratory or photosynthetic rate and, in complex multicellular organisms, with body size and developmental pattern. It is also important to note that the genomes so far investigated are small or relatively small, thus it is likely that exploration of larger genomes will produce critical novel insight.

6.11

Concluding Remarks

In contrast with their taxonomic diversity, the eukaryotes are extremely uniform in cellular, biochemical and molecular organization. Not only this indicates that their common ancestor (LECA) already possessed all the fundamental traits of the group, but also suggests that the original population was small and already well circumscribed by sexual reproduction (Chap. 7; Lane 2015). It is likely that multiple eukaryotic variants preceded LECA, yet apparently none of these survived to the present. The amazing biological success of LECA was most likely due to the mitochondrion or to a better-integrated and more efficient mitochondrion than in competing lineages. We can now conclude this complex story with a somewhat trivial question: are eukaryotes better than prokaryotes? Indeed, when considering genetic and metabolic versatility, prokaryotes have no equals. By ceaselessly experimenting novel genetic combinations, the prokaryotes managed to “invent” photosynthesis, aerobic respiration, nitrogen fixation, acetogenesis and methanogenesis, methanotrophy, the anammox pathway, numerous form of lytotrophy and much more (Martin and Russell 2003; Konhauser 2007). Eukaryotes, in contrast, adopted phagotrophy as the primary feeding mechanism and restrained sexual recombination within the relatively narrow space allowed by meiosis (Chap. 7). Consequently, the main way they could expand their metabolic repertoire was through endosymbiotic association with prokaryotes (Wernegreen 2012; Douglas 2014). If today all eukaryotes disappeared from the Earth, global productivity would reduce dramatically and large parts of the continents would become dry, inhospitable deserts. Yet, the

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Earth would most likely remain a planet teaming with life, and its geochemistry would not change radically. Conversely, if all prokaryotes suddenly disappeared, the geochemistry of the planet would be completely overturned and the eukaryotes almost certainly would not make it alone. In spite of their superior metabolic versatility, prokaryotes are exceedingly conservative in terms of morphologic and genomic complexity, to the point that bacteria living 3 billion years ago do not appear to be morphologically different from those existing today. We do not quite understand why prokaryotes have remained morphologically simple, despite being so diverse in their biochemistry and so versatile in their ability to extract a living from gases and rocks. Eukaryotes, in contrast, have a propensity to develop increasing complexity. Although usually retaining unicellular forms, all major eukaryote lineages independently evolved a multicellular organization, and five evolved complex multicellularity (Chap. 7). Eukaryotes have a superior ability in niche construction encompassing the inside of their cells, multicellular organs (e.g. the animal gut Chap. 10 and plant roots Chap. 11), and large-scale phenotypic extensions such as the soil, coral reefs and forest canopies up to multi-level oceanic and terrestrial ecosystems, for none of which there is a counterpart in the prokaryotic world.

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Chapter 7

Sexual Reproduction

Sex is not only unnecessary, but it ought to be a recipe for evolutionary disaster. . .By all rights, any group of animals that evolves sexual reproduction should be promptly outcompeted by nonsexual ones. . . .Why is sex a success, despite all its disadvantages? (Evolution: The Triumph of an Idea, Carl Zimmer)

Abstract Sex is a universal property of life encompassing any process that incorporates foreign DNA into functional genomes. Sex is essential for the conservation of biological information across time and, together, is a powerful mechanism of genetic innovation. Unlike prokaryotes, in which sex has no link with reproduction, eukaryotes perform sexual reproduction, an alternation of cellular fusion (syngamy) and meiosis better referred to as meiotic sex. Meiosis possibly evolved in early eukaryotes as a way to reversibly shift from haploidy to diploidy in response to environmental signals. Meiotic sex might then have emerged for maintenance of multi-chromosome, large-sized genomes. The evolution of mating types belonging to either of two sexes/genders prevented fusion of sister cells, thus fostering genetic recombination. Meiotic sex sets strong boundaries between species, at the same time promoting genetic isolation and the emergence of novel species. Species boundary in prokaryotes is conventional. Despite high cost, meiotic sex is almost universal in extant eukaryotes and was most likely present in the last common ancestor.

7.1

Introduction

The quotation at the head of this chapter shows how easily sexual reproduction and sex are treated as synonyms. Indeed, the confusion between the two terms is commonplace not only in everyday language, but also in scientific contexts. It is

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therefore appropriate to start this chapter with a short discussion of what sexual reproduction and sex really are. Sexual reproduction is a process unique to eukaryotes, consisting in the formation of a new cell, the zygote, by fusion (syngamy) of two specialized cells, the gametes. So defined, sexual reproduction is actually the opposite of a reproductive mechanism: from two cells, a single cell comes out, and usually the process requires the cooperation of two individuals. In genetic terms, each parent transmits only half of its genes to the progeny, whereas asexual reproduction permits a single parent to transmit the whole genotype (Glossary). This is the so-called “two-by-one” cost of sexual reproduction (Maynard-Smith and Szathmàry 1995). The need to facilitate gamete encounter and reduce gamete loss entails quite a few additional costs, including for example the need for flagella and chemo-sensorial mechanisms, the production of sexual attractors and inductors, the involvement of vectors (in flowering plants) or courtship (in many vertebrates) and, of course, sexually-transmitted parasites. In spite of the huge cost and potentially harmful implications, sexual reproduction routinely occurs in the majority of eukaryotes, often alternating with forms of asexual reproduction in which a new organism derives from somatic cells or from a single gamete, without syngamy. When present, asexual reproduction tends to predominate under favourable environmental conditions, whereas sexual reproduction intervenes in response to unfavourable conditions. An important property of sexual reproduction is that the zygote receives a complete chromosome set from each gamete. For the process to reiterate over time it is necessary that, after each syngamy and before the formation of new gametes, the chromosome number is halved through a special mechanism called meiosis. The life cycle of sexually-reproducing eukaryotes, therefore, encompasses a regular alternation of meiosis and syngamy and of a haploid and a diploid phase whose relative importance varies with the taxonomic group. The term sex refers to a diversity of mechanisms that introduce foreign DNA into cells. Less precisely but probably more effectively, we may define sex as the mixing of DNA from separate organisms. So defined, sex is a universal property of life and has no obligate link with reproduction. Thus, sexual reproduction is a (complex) form of sex, yet sex is not a synonym for sexual reproduction. The prokaryotes exhibit a diversity of sexual processes, usually described as transformation, transduction and conjugation (Redfield 2001). Because these processes do not involve cellular fusion or meiosis, we will collectively refer to them as “non-meiotic sex”, in contrast to meiotic sex in eukaryotes. Transformation is the direct uptake of DNA fragments from the environment, a process whose primary function is probably the use of DNA as food, essentially a source of phosphate and organic bases. Multiprotein complexes inserted in the cell membrane such as type-IV (Cascales and Christie 2003) and type-VI secretion systems (Silverman et al. 2012; Borgeaud et al. 2015) facilitate DNA uptake in bacterial cells. Transduction is an accidental cell-to-cell transfer of DNA mediated by viral vectors; viruses may occasionally incorporate DNA fragments from a host cell and

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transfer them to next host. Transduction is a rare event, yet it has great genetic relevance because of the large numbers involved (Griffiths et al. 2000). Molecular biologists use transduction to introduce foreign genes into a host cell’s genome. Conjugation is a more complex process controlled by genes carried by specialized accessory chromosomes (sex plasmids). These genes induce the development of special projections, the sex pili, that transfer a copy of the plasmid and occasionally also of a part of the main chromosome from a donor cell to a recipient cell (De La Cruz et al. 2010). DNA acquired by these mechanisms may be recognized as foreign DNA and degraded, or may be retained, expressed and transmitted to next generations. In the latter case, newly-acquired DNA is conserved as an independent plasmid or is inserted in the main chromosome, either as as an additional sequence (a process called illegitimate or non-homologous recombination) or in lieu of a pre-existing homologue sequence (legitimate or homologous recombination). The same enzymatic machinery responsible for DNA replication and repair, notably RecA and RecBCD enzymes (Redfield 2001; Hoff et al. 2018), controls these processes. For further details, we refer the reader to the excellent molecular biology textbook by Krebs et al. (2017). Although random and usually limited to short DNA sequences, sex is essential for the preservation of biological information. DNA is easily damaged by physical or chemical agents or simply in consequence of replication errors (Vijg 2014). The degeneration of biological information is an extremely serious biological issue: in the absence of repair mechanisms, the information would unavoidably be eroded over time, a phenomenon known as Muller’s ratchet from American geneticist and Nobel laureate Hermann Joseph Muller. Legitimate recombination permits the replacement of altered sequences with correct sequences from other cells. Thus, under tight control by natural selection, sex ensures the persistence of functional genomes through generations (Maynard Smith and Szathmàry 1995; Prasad Narra and Ochman 2006; Szöllősi et al. 2007; Takeuchi et al. 2014; Rocha 2016). A second important property of sex is in the convenience for genes to move from a genome to another, thus “experimenting” interaction with novel genes. From the “perspective” of genes, it does not matter that most of the copies dispersed in the environment get lost or enter wrong genomes; it suffices that at least one ends up in a genotype with above-average fitness, with which it will replicate and spread. A fitness-improving mutation is a most rare event, thus even more unlikely is the occurrence of multiple favourable mutations in the same genotype. Sex permits favourable mutations appeared in separate genotypes to associate in the same genotype and add up their effects. Widening the perspective, sex potentially enables genes to interact with all other genes in the surrounding environment. Indeed, the notion of selfish gene (Dawkins 1976) is founded on sex-mediated gene shuffling between genomes. A third effect of sex is the creation of new, chimeric genes by illegitimate recombination (Rocha 2003). A particularly effective mechanism for making new genes is “domain shuffling”, the duplication and random re-arrangement of

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sequences encoding for domains (Glossary) of separate proteins (Long et al. 2003). This is like trying to make new working software by putting together strings of preexisting software in random combinations and test the result until something usable gets out. Such an approach would probably work if one had an extremely large choice and extremely long times, exactly the conditions under which biological evolution works. Resuming, sex has two seemingly contrasting consequences: on one side, it ensures the persistence of biological information and functional genotypes across the time, on the other it is a powerful source of genetic innovation. The production of new genes or new gene combinations has an immediate positive impact in the ceaseless war against parasites in which all organisms are engaged (Lively 2010). A wide-ranging effect of sex is evolutionary flexibility, an enhanced ability to adapt to environmental changes and seize novel opportunities. This is so fundamental to survival that sex most likely played a crucial role in biological evolution since the very beginning of life history. It is important to correct the potential circularity of this reasoning: genomes evolve thanks to sex (and occasional favourable mutations), and it is convenient for genes to be part of genomes that practise sex. Nevertheless, sex was not invented by evolution with the aim to accelerate genome evolution; rather it appeared as a spontaneous by-product of the biochemical machinery for DNA duplication and repair, subsequently implemented by mechanisms for DNA uptake/transfer. Yet, it is extremely unlikely that life as we know it would have ever emerged from molecular chaos without sex.

7.2

Origin of Meiotic Sex

What said above about prokaryotic (non-meiotic) sex entirely applies to eukaryotic (meiotic) sex, yet with an important difference. Horizontal gene transfer is relatively rare in eukaryotes and sex occurs almost exclusively by cellular fusion and meiosis. Meiosis is an extremely effective mechanism of intraspecific genetic recombination, but it is equally efficient in preventing gene flow between different organisms/ populations. Thus, gene flow in eukaryotes almost exclusively occurs within groups of closely related individuals that we call species (Sect. 7.4). Meiotic sex is a universal character (apomorphy) of eukaryotes, there being very few groups that completely lack it. In several cases, closer scrutiny of eukaryotic organisms originally reported as asexual managed to reveal signs of sexual reproduction. In the other cases, the absence is almost certainly due to secondary loss. Indeed, a number of genes involved in meiosis are widespread in eukaryotes, including Giardia (Diplomonada, Excavata), a basal eukaryote in which sexual reproduction has never been observed (Ramesh et al. 2005). This suggests that LECA, the ancestor of present living eukaryotes, already had meiosis and sexual reproduction. Still, we are far from a full understanding of how and why such a complex mechanism emerged.

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Fig. 7.1 Active (a) and encysted cell (b) of the ciliate Blefarostomum. Dehydration or food shortage may induce encystation. When conditions return favourable, the cysts rapidly germinate, converting into active cells

It has been proposed that eukaryotes appeared in concomitance with the Great Oxygenation (2.45 GYA), and evolved sexual reproduction to recovery from genetic damages by reactive oxygen species (Gross and Bhattacharya 2010). This hypothesis is scarcely tenable, as current evidence does not support eukaryote origin much before 1.5 GYA (6.10). Cavalier-Smith (2002, 2010) separates the origin of meiosis from sexual reproduction. He suggests that eukaryotes evolved meiosis in parallel with mitosis, initially as a mechanism to correct errors in chromosome segregation and subsequently to shift from diploidy to haploidy in response to environmental signals. The haploid condition, characterized by a single chromosome set per cell (viz. a single copy of the whole genome), is more efficient in the use of resources because reproduction is faster and less expensive (Maynard Smith and Szathmàry 1995). The diploid condition is less efficient in the use of resources but more resilient to stress, as homologous sequences in one genome may compensate for faults in the other (Cavalier-Smith 2002). Other possible advantages of diploidy are discussed in Qiu et al. (2012) and Goodenough and Heitman (2014). A haploid cell can become numerically diploid if chromosome duplication is not followed by mitosis (endoreduplication), or if mitosis is not followed by cell division (endomitosis). Cavalier-Smith suggests that genome doubling by endoreduplication in early eukaryotes underpinned the evolution of encystation, the development of resting cells, or cysts, capable of surviving unfavourable conditions. Encystation is an effective survival strategy widespread in modern eukaryotes. Cysts typically have a thick cell wall containing degradation-resistant polymers such as chitin or sporopollenin, and may survive unfavourable conditions in a metabolically inactive condition (Fig. 7.1). A remarkable example is that of ciliate cysts trapped in Siberia permafrost for many thousand years, and still able to germinate (Shatilovich et al. 2015). As for all predators, the life of phagotrophic protists alternates abundance and famine, depending on the availability of prey, prevalently bacteria but often also

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other unicellular eukaryotes. The ability to form cysts, therefore, is probably ancestral in eukaryotes. Encystation might have been associated with chromosome doubling because diploidy is more permissive to the maintenance of a functional genome in resting cells. If so, meiosis turned out useful to re-establish haploidy at the moment of germination, in a reproductive cycle alternating active haploid cells and resting diploid cysts. Meiosis consists of two consecutive nuclear divisions (meiosis I and II) each similar to a mitosis, which produce four haploid nuclei and eventually four haploid daughter cells from one diploid mother cell (Ohkura 2015). In sexually reproducing eukaryotes, the diploid nucleus contains two genetically equivalent copies of each chromosome, known as homologous chromosomes (for sex chromosomes there are some further complexities that do not need being considered here). Before meiosis initiates, each chromosome duplicates, producing two identical copies (“sister chromosomes”) that remain connected by a shared centromere and by a set of meiotic cohesin proteins similar but not identical to cohesins involved in mitosis. During prophase I, the cell produces a second set of special proteins (SYCP1, SYCP2 e SYCP3) that form the synaptonemal complex, a sort of glue that binds together homologous chromosomes, aligning homologous sequences with great precision. At this stage, the chromosomes have already duplicated, thus the synaptonemal complex holds together chromosome tetrads consisting of two identical duplicates of each homologue. Chromosome pairing starts from their ends, or telomers, which at the onset of prophase I bind to a specific area of the nuclear envelope, producing a distinctive chromosome arrangement called “bouquet”. The synaptonemal complex dissolves at the end of prophase I. Unlike mitotic cohesins, which are hydrolysed at the onset of anaphase to permit sister chromosomes to migrate to either pole, meiotic cohesins persist throughout meiosis. Consequently, it is homologous chromosomes, not sister chromosomes, that segregate to the poles during meiosis I. Meiosis I thus produces two nuclei that are already functionally haploid, each containing two duplicates of each homologue; meiotic cohesin complexes are dismantled during meiosis II, and the four sets of chromosomes eventually segregate into four haploid nuclei. A second fundamental process occurring during meiosis, more precisely during prophase I, is the exchange of segments between paired homologous chromosomes. Aligned and bound together by the synaptonemal complex, homologous chromosomes are cut (usually in a single point) by an enzyme called Spo11 at exactly corresponding positions (or loci). This immediately activates repair enzymes that stick the stumps; with the same probability, the enzymes may stick together stumps from the same chromosome, thus re-establishing the original gene arrangements or stumps from homologues. Known as crossing-over, the latter event generates new gene arrangements (Subramanian and Hochwagen 2014). Because cutting and crossing is random, the crossing-over can produce a great number of novel gene combinations; for each couple of homologues the number of possible combinations is 2m, where m is the number of genes present on each chromosome (usually several hundreds to several thousands). Considering that recombination may also occur within genes, the potential variability is even larger, if the homologues are not

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identical. Differences among homologues are due to the existence of allelic variants of each gene, produced by mutation over time (gene polymorphism). Interestingly, the widespread occurrence of introns (Glossary) in eukaryote genes favours meiotic crossing-over between allele genes because the probability of crossing over between coding sequences (exons) separated by long introns greatly increases compared to the same coding sequences in the absence of introns. Meiotic recombination between alleles of the same gene is thought to be a major factor of protein evolution by combining mutations from different alleles and “trying out” novel exon combinations (Rogozin et al. 2012). A second, genetically relevant effect of crossing over is that random recombination continuously breaks up gene aggregations in chromosomes, thus making every single gene “visible” to natural selection. In the absence of crossing over, selection would be able to “see” the single chromosomes but not the genes that these convey. Consequentially, the most critical genes present in each chromosome would hide the accumulation of slightly harmful mutations in the other genes, causing a progressive degeneration of chromosomes. Even worse would be the consequences of the appearance of a single mutation that greatly increases the fitness. The mutant gene would spread and fix in the population but, without crossing over, the other genes that happen to be associated in the same chromosome would be fixed as well independently of their fitness. This would wipe out the alleles present in the population and not associated with the “winning” gene, thus causing a dramatic reduction in genetic diversity and evolutionary flexibility (Lane 2015). As already said, early eukaryotes might have used meiosis as a means to reestablish the haploid condition in cells that had become diploid by endoreduplication (Maynard Smith and Szathmàry 1995; Cavalier-Smith 2002; Goodenough and Heitman 2014). Ancestral meiosis probably employed a synaptonemal complex to bind duplicated chromosomes into tetrads, and made use of DNAases and DNA ligases to cut and join accidentally superimposed chromosome arms that would interfere with segregation. This initially had no genetic consequence, as the four chromosomes of each tetrad were identical. By fostering whole-genome recombination, however, meiosis was from the beginning a powerful mechanism for reconstituting functional genomes after exposition to stressing, potentially mutagenic conditions. Cellular fusion was the critical step that linked meiosis with sex. Whatever their origin, eukaryotes ancestrally evolved a flexible cellular envelope and mechanisms of membrane pinching-off and fusion for phagocytosis (Chap. 6). Cavalier-Smith suggests that these pre-adaptations led to the emergence of cellular fusion as a mechanism that associated chromosome doubling and cyst formation with sex under stressing conditions. A high level of chromosome homology is essential in meiosis to prevent random chromosome sorting. Therefore, the evolution of cellular fusion required mechanisms of cell recognition to ensure that merging cells were mutually compatible. In extant eukaryotes, integral membrane proteins functioning as superficial receptors mediate cellular recognition preceding syngamy (Stein et al. 2004). A system of cellular recognition might have evolved from membrane proteins responsible for

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phagocytic vesicular traffic. Likely candidates include SNAREs (Soluble NSF Attachment Protein REceptor), cadherins, integrins, C-type lectins, tyrosine kinases, all ubiquitous in eukaryotes. After perfecting meiosis and putting in place reliable systems of cellular recognition and fusion, eukaryotes “learnt” how to express these mechanisms in cells specialized for sexual reproduction, the gametes. In a world still entirely unicellular, negative selection towards fusion of gametes from the same mother cell elicited the separation of mating types. Zygotes produced by gametes deriving from different individuals have greater chances of possessing non-identical alleles; this condition, referred to as heterosis or hybrid vigor permits allele integration and is often associated with enhanced fitness (Goodenough and Heitman 2014). Cross-fertilization (fusion of gametes of different mating type) has additional costs, the first one being a reduction in the number of available partners (by 50% with two mating types). Eukaryotes have evolved a diversity of mechanisms to favour gamete encounter, such as pheromons, photoperiodism, courtship and pollination. There is a subtle, yet important difference between mating types and sexes/genders, the former uniquely expressing the ability (or inability) of gametes to merge and produce a zygote, the latter additionally implying differences in gamete morphology and reproductive strategy (Sect. 7.6). A further cost of sexual reproduction is the dissolution of well-adapted genotypes, a negative effect exacerbated by cross-fertilization and often mitigated in nature by regular reliance on asexual reproduction (Goodenough and Heitman 2014). This is probably the main reason why numerous eukaryotes, either uni- or multicellular, use sexual reproduction only in response to stress whilst they stick to asexual reproduction under favourable conditions (Fig. 7.2). Horizontal gene transfer is an effective means to maintain functional genomes across generations and permit them to adapt to novel environments, provided genome sizes remain relatively small as they are in prokaryotes. Mathematical analysis shows that the same is not true for significantly larger genomes as those typical of eukaryotes (Lane 2015). Thus, sexual reproduction might have emerged in eukaryotes as a mechanism of genome maintenance suitable to large genome sizes. Although costly, sexual reproduction has been rewarded by natural selection, as witnessed by its nearly universal diffusion among eukaryotes. Based on the relative position of syngamy and meiosis, three fundamental types of reproductive cycle are distinguished in eukaryotes: haplontic, diplontic and haplo-diplontic (Otto and Gerstein 2008; Qiu et al. 2012; Fig. 7.3). In the green unicellular alga Chlamydomonas, vegetative cells of two mating types, plus and minus, differentiate into gametes in response to environmental signals. The plus mating type expresses GSP1 and the minus mating type GSM1, two homeodomain genes (Glossary); the proteins expressed by these gene are cytoplasmic until gamete fusion, after which they associate together into a dimer, move into the nucleus, and activate diploid gene expression. A similar mechanism also occurs in ascomycetous and basidiomycetous fungi. This suggests that the ancestral mechanism controlling the transition from haploid to diploid gene expression in eukaryotes involved pairs of paralogous (Glossary) homeobox-containing genes (Bowman et al. 2016).

7.3 The Case of Parthenogenetic Rotifers

241

Fig. 7.2 In many eukaryotes the zygote functions as a resting cyst. Protected by a tough involucre, it arrests metabolic activity and survives unfavourable conditions in a dormant state. Upon the return of favourable conditions, the zygote germinates and usually divides by meiosis, producing four haploid cells (meiospores) that further divide by mitosis. (a) Zygospore of the zygomycetous fungus Rhizopus. (b) Encysted zygote (hypnozygote) of the green alga Micrasterias. (c) Immature zygotes of the green alga Zygnema

7.3

The Case of Parthenogenetic Rotifers

Sexual reproduction is the rule in eukaryotes, with relatively few documented instances in which this form of reproduction was lost and entirely replaced with asexual mechanisms. A most remarkable instance is that of bdelloid rotifers (Bdelloidea), minute animals living in freshwater habitats all over the world. Unlike other rotifers, bdelloid rotifers reproduce exclusively by parthenogenesis, viz. by division of unfertilized eggs (being formed without meiosis, these eggs are diploid just like somatic cells). A second remarkable trait of these animals is their ability to survive drought by activating desiccation-induced dormancy (anhydrobiosis) at any life stage (Ricci and Fontaneto 2009). As mentioned above, sex permits recovery from genetic damage and promotes swift genetic change, thus reducing vulnerability to pathogens and parasites. How do bdelloid rotifers manage without sexual reproduction? The trick seems to be rooted in desiccation

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Fig. 7.3 The three fundamental life cycles in sexually-reproducing eukaryotes

tolerance. In order to repair genetic damage after prolonged permanence in a dried condition, bdelloid rotifers evolved a particularly effective DNA repair system that apparently compensates for the lack of sexual reproduction (Hespeels et al. 2014). Anhydrobiosis helps these animals to get rid of pathogens and parasites, which generally do not survive extreme dehydration; in addition, the wind easily transports dehydrated rotifers over long distances because of their minute sizes, thus facilitating colonization of habitats free from infesting agents (Wilson and Sherman 2013). Completing the picture, these singular animals have a surprising ability to pick up foreign DNA and insert it in their genome, thus performing bacteria-like non-meiotic sex, a task favoured by their relatively small genome sizes (Boschetti et al. 2011). Thanks to this ensemble of adaptations, the abandonment of sexual reproduction in bdellois rotifers was a convenient choice. Despite the absence of sexual reproduction, in fact, bdelloid rotifers have evolved at a quick pace, producing over 450 species in about 30 MY from their appearance.

7.4

Sexual Reproduction Sets Strong Species Boundaries in Eukaryotes

Sexual reproduction strictly requires that syngamy involves cells similar enough for their genomes to work together harmoniously. Recognition mechanisms must therefore have evolved in parallel to sexual reproduction to prevent fusion of non-compatible cells and random genome mixing. Homologue-pairing by the

7.4 Sexual Reproduction Sets Strong Species Boundaries in Eukaryotes

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synaptonemal complex during meiosis has been a powerful control mechanism from the very beginning. The synaptonemal complex binds chromosomes by recognizing homologous sequences with great precision. If merged cells are too different, one or more chromosome pairs may not be recognized as homologues and meiosis is disrupted, with the consequence that the organism is not able to reproduce sexually. Meiosis block is important when other mechanisms do not work, for example mate recognition, gamete recognition, embryo developmental control. This is the case, for example, of interspecific hybrids; under certain conditions individuals belonging to different species may breed but, because of meiosis block, the hybrids are sterile. By definition, individual organisms belong to the same species if they are able to pass all recognition controls of sexual reproduction and produce fertile offspring. Organisms that fail to pass recognition control belong to separate species (Robert and Baylis 2003; Ridley 2004). Whilst enforcing genetic isolation among species, sexual reproduction may also promote speciation under certain conditions. A most cited example is population splitting by a geographical barrier, such as a mountain chain, a river or a deep valley, which interrupts the gene flow between two subpopulations. After separation, sexual recognition systems in the two groups progressively diverge due to the accumulation of mutations; after a sufficiently long time, the divergence is so deep as to prevent sexual recognition between individuals from the two groups. A different mechanism of speciation, frequent among plants but probably also common in the microbial world, is chromosome doubling in interspecific hybrids. This creates a supplementary copy of each chromosome, thus enabling the hybrid to perform meiosis correctly (Fig. 7.4). The fertile hybrid may give rise to a new species if its offspring is able to colonize new ecological niches, or to out-compete one or both parental species. The species definition presented above is based on the biological species concept (BSC) introduced by Ernst Mayr in 1942 and mainly applied to macroscopic eukaryotes, notably animals and land plants. In order to embrace the extant diversity, the BSC is currently formulated as follows: a species is a cohesive, monophyletic group irreversibly isolated from other species by reproductive and ecological barriers. A corollary to this definition is that different species in the same ecosystem necessarily occupy different ecological niches and, conversely, each ecological niche within an ecosystem can host only one species. The definition above provides a robust, albeit not entirely univocal framework for sexually reproducing eukaryotes. In fact, the real difficulty is not in defining the species, but rather in recognizing them. A classic example is that of ring species; a ring species is a connected series of neighbouring populations, each of which can interbreed with nearby populations, but with at least two “end” populations in the series that are too distantly related to interbreed, though there is a potential gene flow throughout the distribution range (Irwin et al. 2001). As we will see in Chap. 12, paleontologists face a similar problem: species change with time and fossils may record a gradual transition that is hard to translate into taxonomy.

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Fig. 7.4 Chromosome doubling in interspecific hybrids may recover fertility and produce new species

7.5

Species Boundary in Prokaryotes Is Conventional

Biological species are epiphenomena of sex, and exist only in sexual eukaryotes, but not in bacteria, which transfer genes laterally without sexual cell fusion. (Cavalier-Smith 2007)

The biological species concept is not applicable to prokaryotes, as these lack sexual reproduction and at the same time are prone to genetic promiscuity by horizontal gene transfer (HGT). Metagenomic analysis points to the existence of a vastly greater diversity in the prokaryotic world compared with the eukaryotic counterpart (Medini et al. 2005). Difficulty in prokaryote systematics is amplified by the paucity of morphological criteria, prokaryotic cells being usually small and little structured. On the other hand, because of a relatively high frequency of HGT, the use of metabolic or ecological traits for species demarcation in prokaryotes is often problematic (Shapiro and Polz 2015). The number of genes attributed to bacterial species is often much larger than the gene number in single strains or isolates (Dijksoorn et al. 2000). It is necessary, therefore, to distinguish between genome and pangenome, the first referring to the set of genes in a single cell or strain, the latter to the global gene repertoire present in all strains and isolates attributed to the same species (Medini et al. 2005; Tettelin 2008; Lapierre and Gogarten 2009; Mira et al. 2010). The pangenome usually encompasses a number of genes found in all strains examined (core or backbone genome) plus a vast pool of genes present in some but not all strains (accessory genome). The core genome is the part of the genome that characterizes the species as a whole, and consists of genes controlling essential functions such as gene duplication/expression and fundamental metabolic pathways; the accessory genome characterizes specific strains and consists of genes employed in facultative functions such as the use of specific metabolites or antibiotic resistance. The fact that bacterial pangenomes are usually much larger than the genotype of single cells greatly enhances the chance of gene recombination through sex. Because of the “purifying” effect of meiosis, there is no or very little difference between individual genomes and the pangenome in sexually-reproducing eukaryotic species (allelic variants are not considered in the definition of pangenome).

7.6 Gamete Differentiation and Genders

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In the last two decades, comparative analysis of homologous sequences, usually rRNA, has integrated and partially replaced the traditional metabolic approach in prokaryote systematics. By convention, strains or isolates are currently assigned to the same species if sequence divergence in their 16 S rRNAs is less than 1% (Cohan 2006; Cohan and Perry 2007). Apparently low, this is actually the average level of divergence detected in 18 S rRNA (the eukaryotic homologue of bacterial 16 S rRNA) of mammals belonging to different orders, for example a goat and a dog. It is not surprising, therefore, that bacterial species that fit this criterion often encompass vastly diverse strains. For example, numerous species of bacterial pathogens supported by 16 S rRNA affinity encompass “strains” that differ in the type of host, tissue localization, and transmission mode. As an attempt to overcome these difficulties, ecological criteria have been advocated for subdivision of “molecular species” into better-demarcated “ecological species” or “ecotypes”; yet, the likeness of horizontal transfer of ecologically relevant traits warrants a cautious approach (Cohan and Perry 2007). The occurrence of meiotic sex in eukaryotes and its absence in prokaryotes is not a mere difference in the reproduction mechanism; rather it reflects a deep divergence in the life strategy of the two types of cellular organization. Prokaryotic recombination leads to pangenomes, eukaryotic recombination through sexual reproduction leads to vertical inheritance (Ku et al. 2015).

7.6

Gamete Differentiation and Genders

The separation of gametes into mating types is universal in eukaryotes. In isogamous lineages, e.g. some algae (Chlamydomonas) and zygomycetous fungi, different mating types are morphologically indistinguishable. Eukaryotes that practice conjugation, viz. the exchanging of nuclei between cells (for example the ciliates and some charophycean algae), also are isogamous. More commonly, mating types have obvious morphological differences, a condition known as anisogamy. A special case of anisogamy is oogamy, characterized by two sharply different types of gametes: relatively large, immobile gametes (called oogametes, oospheres or egg cells), and relatively small gametes usually mobile by means of flagella (called sperms, spermatozoa, spermatic cells or anterozoids according to the taxonomic group and personal preference). In hermaphrodite eukaryotes, individual organisms produce two types of gametes; here too, syngamy is usually possible only between gametes from different individuals, thus implying the existence of more than two mating types. In unisexual eukaryotes, each individual produces gametes of only one mating type. When this condition combines with anisogamy, populations encompass individuals belonging to either of two sexes/genders, viz. male and female. Anisogamy (notably oogamy) implies an important difference in the reproductive strategy of the two genders: females produce a relatively small number of gametes but invest a greater amount of resources in each gamete, males produce small

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gametes in large numbers. Anisogamy probably evolved by disruptive selection, an evolutionary process in which extreme values for a trait are favoured over intermediate values. With opposing selective pressures to simultaneously maximize the number of gametes, their encounter rate and the mass of resulting zygotes, the fitness of both partners is maximized when one interacting gamete is small and mobile, and the other is large and provides most of the resources required for zygote development (Perrin 2012). Oogamy evolved many times independently, occurring in several lineages other than land plants and animals. In oogamous species, the male gamete usually contributes to zygote formation only with the nucleus, plus one or two centrioles in the lineages possessing a centrosome (Box 6.2), all the rest of the cytoplasm including mitochondria and plastids usually coming from the egg. By providing the zygote with a large number of organelles, oogamy reduces organelle segregation in early development; purportedly, this improves adult fitness by restricting variation between tissues in multicellular lineages (Radzvilavicius et al. 2016). Males have a higher potential reproductive rate and benefit more from multiple mating than do females because they invest less initially. Consequently, selection favoured investment in parental care in females and mate seeking and competitive behaviours in males. Because males can potentially fertilize several females, extra males do not increase the population growth rate, thus it would be more efficient if there were more females than males. Except than under very special circumstances (Gardner et al. 2012), however, nature never departs from a 1/1 ratio between the two genders. To understand why, let us make a mental experiment, imagining a population in which the female to male ratio is 3/1 and the average offspring number per female is 4. In such population, males would have a triple reproductive success relative to females, because each of them could averagely mate with three females; consequently, it would be more convenient for females to make male offspring. A mutant female able to make only male offspring would have an initial reproductive success of 12/6 (over an offspring of 4, the mutant female would make four males, each with a reproductive success of 3, the normal female would make 3 females and one male, for a total reproductive success of 6). Because of this, the mutation would spread across the population and the gender ratio would decrease, to stabilize near 1/1. The same reasoning works in reverse for a population with an initial male-biased sex ratio. In the absence of a special selection pressure, the 1/1 gender ratio is the only evolutionary stable condition, because it is not biased in favour of males or females. Any population that deviates from the 1/1 sex ratio will be shifted back to it by natural selection. Why sexually reproducing populations only have two genders? More than two genders of course would burden sexual reproduction with additional costs in terms of resources to allocate to the gametes and a reduction in reproductive fitness. Nevertheless, genetic and evolutionary benefits from sex would increase if there were three or more genders (with singamy involving fusion of three or more gametes, each contributed by one gender). The main reason why evolution never explored a multi-gender option is that the regeneration of haploidy from a triploid or poliploid cell would require a tri- or multipolar spindle and a segregation mechanism far more complex and

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error-prone than the existing bipolar mechanism. Interestingly, monoplastidic spore mother cells in bryophytes do employ a quadripolar microtubule system to ensure plastid transmission to each of the four spores produced by meiosis (as all land plants, bryophytes have a haplodiplontic life cycle). After plastid segregation at four equidistant poles, the quadripolar system converts into a bipolar spindle during meiosis I, followed by two new spindles perpendicular to each other during meiosis II (Brown and Lemmon 1997; Shimamura et al. 2003). Thus, we had better give over intriguing multi-gender scenarios to science-fiction: life on Earth can only afford two genders at most, at least as far as reproduction is concerned (De Loof 2018). In contrast to genders necessarily limited to two, multiple mating types have independently evolved in several eukaryotic lineages, from three in Dictyostelium, seven in Tetrahymena, 13 in Physarum polycephalum, and thousands in basidiomycetes, with singamy possible only between gametes of different mating types (Goodenough and Heitman 2014). A nearly universal trait of sexually-reproducing eukaryotes is uniparental transmission of mitochondria. In Chlamydomonas, mammals and land plants, possessing two mating types, this process involves either pre-syngamy exclusion or postsyngamy destruction of mitochondria from one mating type. The combination of meiotic sex and uniparental transmission exposes a uniform set of organelles to natural selection in the haploid condition at each generation. This ensures the elimination of inefficient or unregulated organelles that might otherwise infest and destroy the population, or even the species (Maynard Smith and Szathmàry 1995; Roze et al. 2005; Goodenough and Heitman 2014; Greiner et al. 2014; Lane 2015; also see Sect. 6.7). A similar bottleneck mechanism might be operative during development in complex multicellular organisms, with cells receiving defective mitochondria being eliminated by apoptosis (Johnston et al. 2015; Chap. 8). Uniparental transmission of plastids independently evolved in several lineages of photosynthetic eukaryotes (Greiner et al. 2014).

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Chapter 8

Multicellularity

Is then the evolutionary transition to multicellularity a difficult one or not? The blunt answer is: not at all, since multicellularity has arisen more than twenty times in evolution. However, there are only three lineages that produced complex organisms: plants, animals, and fungi. Three hits in 3.5 billion years are not that many. Szathmàry and Wolpert (2003)

Abstract Multicellularity evolved many times independently in both bacteria and eukaryotes, but only in the latter did it attain high levels of complexity. Multicellularity dramatically enhanced niche construction and ecosystem complexity. The key factor behind the success of multicellularity is increased fitness from labour division and cellular specialization. Multicellularity required the evolution of mechanisms of cellular communication and differentiation, with frequent instances of convergence across the taxonomic spectrum. Of 119 major eukaryotic clades currently recognized, 83 are exclusively unicellular and 36 encompass multicellular forms; among the latter, six clades evolved forms with a high level of cellular differentiation. Large-sized multicellular organisms evolved vascular systems for long-distance transport. The animals and land plants added internal extracellular compartments subject to homeostatic control. In closed-form multicellular organisms (e.g. the animals and volvocine algae), the body shape is determined during embryo development, after which stem cells control cellular turnover and isometric growth. Open-form organisms (e.g. land plants and fungi) retain totipotent cells that produce new organs throughout life duration. In weismannist organisms (essentially insects, vertebrates and volvocine algae), a germ line precociously separates from the somatic line. Complex multicellular organisms have three hierarchically interlinked levels of organization and three related levels of death, i.e. systemic, organ and cellular.

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Introduction

The unicellular condition gives life vast flexibility and efficiency: each cell directly interacts with the environment, takes up the necessary raw matter and discharges waste; cell division coincides with reproduction, generation times are short, individual accidental death only concerns one cell, and evolutionary adaptation can be exceedingly fast. Oddly, Lamarck thought of unicellular organisms (shortened to “unicells” in the following text) as evolutionarily recent forms that “had not yet had time to become multicellular”. Now we know that unicells have been in existence for billions of years before the appearance of complex multicellular organisms (multicells). Simple multicellular forms evolved many times independently in both prokaryotes and eukaryotes; complex multicellular forms evolved a few times uniquely in eukaryotes. Independent of taxonomy, multicellularity requires mechanisms of cellular communication and differentiation, which evolved in different lineages with a high level of convergence (Ruiz-Trillo et al. 2007; Niklas 2014; CavalierSmith (2017). For a multicellular form to evolve from a unicellular ancestor, it is necessary that benefits from being multicellular, ultimately expressed in terms of gene survival, outweigh advantages from unicellularity. Kin selection theory predicts that cooperation among cell lineages will be favoured when rb > c, where r is the genetic relatedness of recipient cell to actor cell, b is the fitness benefit to the recipient, and c the cost to the actor (Hamilton 1964a, b). In multicellular organisms developing from a single founder cell, also known as clonal multicells, cell relatedness is 1, because all cells share the same genome pending mutations. In some taxa a multicellular organization develops by aggregation of independent cells, thus cell relatedness is lower than 1. This type of organization, known as aggregative multicellularity, evolved at least seven times in eukaryotes as well as in some bacterial lineages such as the Myxobacteria. A major problem in aggregative multicellularity is evolutionary instability due to the emergence of competition between cells not sharing the same genome, which may disrupt cooperation and prevent functional integration (Grosberg and Strathmann 2007; Fisher et al. 2013; Brunet and King 2017; Sebé-Pedrós et al. 2017). This explains why most multicellular organisms are clonal multicells and why complex multicellularity (see below) uniquely evolved in clonal multicells. In fact, non-clonal multicellular aggregates such as those formed in cellular slime molds are only a transient stage in a mostly unicellular life cycle, with sexual reproduction as the only function (Brown et al. 2012; Wegener Parfrey and Lahr 2013; Kapsetaki et al. 2016; Nanjundiah 2016). Which benefits does a multicellular organization provide? Numerous, multifaceted factors play a part, with differences from case to case (Grosberg and Strathmann 2007). The key factor behind the success of multicellularity is most likely labour division and cellular specialization (West et al. 2015). In unicellular organisms, a single cell performs all vital functions; in multicellular organisms, specialized cellular types perform specific functions, thus increasing overall efficiency

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(Brunet and King 2017). Cellular specialization is not restricted to multicellular organisms, there being numerous protists that produce different cell morphologies in response to environmental signals, for example the amoeba Naegleria (Fig. 6.16) and Plasmodium (Cowman et al. 2016). In multicellular organisms, however, different cellular types cooperate in a highly integrated way. A second important factor that probably favours the transition towards multicellularity is reduction of predation by phagotrophic protists due to increased body size. In addition, multicellularity enhances competitiveness in benthic habitats (Kapsetaki et al. 2016). Uni- and multicellular forms tend to occupy different ecological levels; in most modern ecosystems, the decomposers are mainly unicells, the main primary producers can be either uni- or multicells, whereas primary and secondary consumers are mainly multicells. The most relevant effect of multicellularity, however, is enhanced niche construction (Chap. 1) and increased ecosystem complexity, of which coral reefs, kelp forests and land plant communities are some of the most dramatic examples. Multicellularity strictly depends on cell communication, which is essential for cellular assemblages to function as integrated systems. Rudimentary forms of cellular communication are present in unicellular organisms, both prokaryotic and eukaryotic. These are based on quorum sensing, a type of interaction that permits decentralized systems lacking an internal hierarchy to express a co-ordinate response to certain signals (Waters and Bassler 2005; Diggle et al. 2007). For example, Escherichia coli and other bacteria co-ordinately produce extracellular polymeric substances encasing the cells in a protective biofilm in response to cellular density exceeding a threshold level. Quorum sensing requires the single members of the population to be able to “quantify” the number of the other members with which they interact, and to express a standard response above a threshold level. Quorum sensing might have been the starting point for the evolution of the complex systems of cellular interaction underlying multicellular organization. The evolution of sponges from a choanoflagellate ancestor as reconstructed by Cavalier-Smith (2017) is an illuminating example of transition from uni- to multicellularity (Fig. 8.1).

8.2

Cellular Specialization and Levels of Organization in Multicellular Organisms

Although present both in prokaryotes and eukaryotes, clearly multicellularity is more widespread and attains the highest levels of complexity in the latter. Indeed multicellular eukaryotes, notably the animals and land plants, have profoundly modified the face of the world, whereas the same does not hold for multicellular prokaryotes. Several distinctive traits of the eukaryotic cell underpin the special propensity of eukaryotes to evolve multicellularity. These include (a) a cytoskeleton that senses

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Fig. 8.1 Speculative reconstruction of sponge evolution from a unicellular choanoflagellate ancestor. (1, 2) Individual choanoflagellates capture bacteria drawn by ciliary water currents to the outer surface of their collar complex. (3, 4) Binding of clonal choanoflagellates by branched stalks or collar microvilli produced sessile multicells. (5) Binding via cell bodies produced planktonic swimming balls. (6) Protosponges might have evolved from a ball of cells that attached to rock surface (cross-hatched) by means of non-flagellate cells (pinacocytes) and secreted extracellular material (mesohyl) to support flagellate choanocytes. (7) Competition for filtering larger water volumes led to larger, stronger, three-layered feeding laminas with mesenchymatic cells specialized in extracellular material secretion sandwiched between choanocyte epithelia. Internalization of choanocytes by the pinacocyte layer as observed during sponge larval metamorphosis (8) established the body plan of basal sponges (9), in which co-ordinated flagellar beating of coanocytes drives water flow through an inner cavity, thus increasing feeding efficiency. The genome of choanoflagellates contains numerous genes associated with multicellularity in animals, for example protein tyrosine kinases, C-type lectins and cadherins, but lack other genes involved in animal development such as certain classes of homeobox genes, many transcription factors and intercellular signalling genes (Ruiz-Truillo et al. 2007). (Adapted from Cavalier-Smith (2017), Creative Commons Attribution 4.0 International License)

and transmits spatial information (Box 6.1), enabling the cells to control their shape and perceive their position relative to other cells; (b) an endomembrane system that secretes extracellular matrices functioning as a glue and a supra-cellular skeleton (Sanderfoot 2007; Kloepper et al. 2008); (c) energy production by mitochondria (Lane 2014; Sect. 6.3). Morphogenesis in animals tightly depends on cytoskeleton-mediated cellular motility (Alberts et al. 2014). In contrast, in eukaryotes with walled cells the cytoskeleton controls morphogenesis by affecting cell wall architecture and cellular expansion (Burgert and Fratz 2009; Li et al. 2012). At the genomic level, eukaryotes’ ability to support large genomes creates a favourable environment for the evolution

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Table 8.1 Levels of organization in multicellular organisms Colony

Simple filament Branched filament Syphon, plasmodium, syncytium Pseudoparenchyma

Parenchyma

An ambiguous term employed for multicellular forms whose cells remain physiologically independent; colonies may develop by aggregation of free cells (e.g. myxomycetes and some green algae) or from a single parent cell (e.g. colonial choanoflagellates, colonial green algae, diatoms) Unbranched filaments elongating by diffuse cellular division (all the cells of the filament are able to divide) or by apical cell division. Cell division perpendicular to main filament axis Cell division in two planes permits lateral branching. The filaments may elongate by diffuse or apical division Multinucleate cells developing by repeated mitosis of parent cell not followed by cell division (syphons and plasmodia) or by fusion of originally separate cells (syncytia) Three-dimensional organization consisting of tightly aggregated branched filaments, each growing from an apical cell dividing in two planes Three-dimensional organization deriving from one or more parent cells (stem cells) dividing in three or more planes

of new genes by gene duplication and neo-functionalization (Andersson et al. 2015), thus accelerating the genetic change underpinning the transition to multicellularity. Pivotal to the emergence of multicellularity was the evolution of cell-to-cell adhesion structures that give the multicellular edifice the necessary compactness. In multicellular organisms with walled cells, the outermost cell wall layer ensures cellular adhesion. For example, in colonial volvocine algae, individual cells are embedded in a distinctive extracellular matrix of hydroxyproline-rich proteins (Domozych and Domozych 2014). In land plants and multicellular charophytes, cellular adhesion is mediated by a cell wall layer rich in acid polysaccharides (pectins) linked together by calcium ions; in brown algae, adhesion is mediated by a different type of acid polysaccharide and tannin-like compounds named florotannins; in fungi, cellular adhesion is mainly due to glycoproteins (Niklas and Newman 2013; Niklas 2014). The animals lack a rigid cell wall and cellular adhesion is mediated by an extracellular matrix rich in proteoglycans and glycoproteins and integral plasma membrane proteins including cadherins (for cell-to-cell adhesion) and integrins (for cell-to-matrix adhesion) (Wegener Parfrey and Lahr 2013; Alberts et al. 2014). With the exception of lineages lacking sexual reproduction, found for example among land plants and colonial invertebrates, all multicells retain unicellular phases (gametes, zygotes, spores), so cellular adhesion is temporally controlled and developmentally reversible. Defined on the basis of cellular adhesion, communication and co-operation, multicellularity evolved multiple times both in bacteria and eukaryotes, although with different levels of complexity (Table 8.1). In myxobacteria (a group of gram-negative bacteria), cells originally independent react to unfavourable conditions by aggregating into “fruiting bodies”, structures specialized in the production of resting spores. Actinomycetes (a group of gram-posititive

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Light

N2

PS I

C-compounds

NADH

ATP Nitrogenase

N-compounds Amino acids

Vegetative cell

NH4+

Heterocyst

Vegetative cell

Fig. 8.2 Some cyanobacteria such as Anabaena produce filaments comprised of three cellular types with distinct morphology and functions. Vegetative cells, the most abundant type, perform photosynthesis and other metabolic functions. The akinetes are large thick-walled cells capable of surviving unfavourable conditions, notably drought, and regenerating new filaments when environmental conditions return favourable. The heterocysts express nitrogenase, the enzyme complex that converts molecular nitrogen into ammonia used up to make organic nitrogen compounds. The heterocysts exchange materials with vegetative cells across micropores enzymatically drilled in the peptidoglycan wall. Unlike vegetative cells, the heterocysts only express photosystem 1; therefore, they can make ATP but cannot reduce carbon dioxide (Chap. 4). Inhibition of photosystem 2 expression is essential to avoid oxygen production within heterocysts, which would irreversibly inactivate the nitrogenase. All three cellular types contain the same genotype, but express it differently

bacteria) form branched filaments similar to fungal hyphae. Filamentous cyanobacteria such as Nostoc and Anabaena are able to differentiate several cellular types (Fig. 8.2; Kumar et al. 2010; Lehner et al. 2013). Colonial, filamentous and siphonal eukaryotes usually have a low level of cellular differentiation, often limited to the separation of vegetative and reproductive functions (Fig. 8.3). Cellular integration and mutual interdependence is minimal in colonial organisms such as Pediastrum and Hydrodictyon, in which every single cell performs fundamental functions such as photosynthetic assimilation and reproduction (Fig. 8.3; Umen 2014). Pseudoparenchymatous and parenchymatous eukaryotes show a higher degree of cellular specialization and integration, associated with specialized cell junctions and

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Fig. 8.3 Colonial organization in green algae (Chlorophyceae). (a) Planar colony of Pediastrum. (b and c) Mesh-like colonies of Hydrodictyon. (d) Spherical colonies of Volvox. In all cases, the cells are interconnected by hydroxyproline-rich glycoprotein matrix. In Pediastrum and Hydrodictyon new colonies develop endogenously within each cell of mature colonies. The founder cell divides repeatedly, producing a number of flagellate cells (up to 20,000 in Hydrodictyon); these cells are initially free within the parental cell wall, but after a while, they lose the flagella and aggregate into a new colony with the typical morphology of the species. Following the breakup of the parental cell wall, newly-formed colonies are liberated and grow to mature sizes with no further cell division. (c) A young (left) and a nearly mature colony (right) of Hydrodictyon. (d) In Volvox, new colonies ( f ) arise from large reproductive cells named gonidia, which differentiate in a predetermined number (eight in the instance shown) in the cortex of the parental colony ( p); the developmental pattern of Volvox colonies recalls gastrulation in animals (Chap. 10). Newly-formed colonies remain within the parental colony until this ages and breaks up. In response to environmental signals or sex-inducing pheromones, the gonidia are also able to produce gametes (either eggs or sperms), which generate new colonies sexually

elaborate three-dimensional body forms. A particularly high level of complexity occurs in animals and land plants (embryophytes), which have protective body coatings (mucilages, cuticles, exoskeletons) and an internal system of extracellular spaces finely controlled by homeostatic interactions (Knoll 2011). Of 119 major eukaryotic clades currently recognized, 83 are exclusively unicellular and 36 encompass multicellular forms (Fig. 8.4); among the latter, six clades have evolved multicellular complex forms (Fig. 8.5). Not only has multicellularity

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Fig. 8.4 Distribution of unicellular and multicellular forms in eukaryotes. LECA, Last Eukaryote Common Ancestor. Phylogenetic tree based on Burki et al. (2016) and Brown et al. (2018)

Fig. 8.5 A complex multicellular organization independenty evolved in six eukaryotic clades (in red). Defining properties of a complex multicellular organization include large sizes, a high level of cellular differentiation, long-distance transport systems and an internal extracellular environment under homeostatic control (the last essentially in animals and land plants)

evolved independently multiple times in eukaryotes, but each of these transitions also occurred at different times in the history of life (Sebé-Pedrós et al. 2017). Cell specialization emerges from asymmetric access to genomic information, which is interpreted in a cell-specific fashion through mechanisms of transcriptional gene regulation. The evolutionary transition from unicell to simple multicell does not appear to involve a significant increase in genome size nor in the number of genes. In contrast, complex multicells have larger total genomes than their unicell or simple multicell relatives (e.g. land plants vs charophycean algae, or animals vs choanoflagellates). Independent of the number of genes, a larger genome permits the cells to attain larger sizes (Box 6.3). Genome amplification in complex multicellular eukaryotes possibly underpins the development of large specialized cells such as the neurons in animals or vascular cells in land plants and brown algae (Cavalier-Smith 2005), although endoreduplication (multiple duplication of the

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Fig. 8.6 Land plant cells communicate through plasmodesmata, specialized junctions crossing the cell walls. (a) Electron microscope image of plasmodesmata connecting neighbouring cells. (b) Diagrammatic representation of a plasmodesma, with endoplasmic reticulum (ER) passing from cell to cell. ML middle lamella, CW1, CW2 cell walls of neighbouring cells, Pl plasma membrane

whole genome) is a documented alternative mechanism (Gerstein and Otto 2009). A dozen genes are necessary and in some cases sufficient for switching to multicellularity in four groups investigated: green algae, fungi, slime molds, and choanoflagellates. All known multicellularity genes encode extracellular matrix proteins and, in the case of clonal multicellularity, cytokinesis regulators, suggesting that the initial evolution of multicellularity in different branches of the eukaryote tree repeatedly converged on similar mechanisms (Brunet and King 2017). Complex multicellular organisms have macroscopic sizes. To overcome physiological restraints from molecular diffusion and permit far-away cells and tissues to communicate and exchange materials at a sufficient rate, they have independently evolved long-distance transport mechanisms. Remarkable examples are vascular tissues (xylem and phloem) in land plants, a phloem-like tissue in brown algae, vascular systems in animals, and microtubule-mediated transport systems in fungal hyphae and animal neurons (Knoll 2011). In land plants and multicellular charophycean algae (Chaps. 9 and 11), cells are interconnected by plasmodesmata, cytoplasmic bridges that cross the cell walls and are outlined by a continuous cell membrane (Fig. 8.6). Plasmodesmata permit cellto-cell diffusion of molecules with a size limit up to 70 kDa (Brunkard and Zambryski, 2016); the cells connected by plasmodesmata form a continuous

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Fig. 8.7 (a) Adjoining hyphal cells in ascomycetous fungi communicate across simple pores (arrow) associated with spheroidal structures named Borodin bodies (b). (b) Hyphal cells in basidiomycetous fungi communicate through a pore associated with cup-shaped membranous structures known as “parenthesomes” (arrows). (c) Cross cell walls In Florideophyceae (red algae) present large pores known as “pit connections”, which may be obstructed by a protein plug. The different types of structures associated with pores probably control the intercellular flow of materials

protoplasmic system known as “symplast”, which extends throughout the plant body (Fujita 2015). Plasmodesma-like structures independently evolved in members of the Chlorophyceae and Ulvophyceae, and in brown algae (Raven 1997; Terauchi et al. 2015). Different types of cellular junctions evolved in fungi and red algae (Fig. 8.7). Lacking cell walls, the animals evolved cell junctions specialized not only for cell communication but also for cellular adhesion (Alberts et al. 2014; Fig. 8.8). Minute pores about 20 nm in diameter cross the peptidoglycan cell wall between adjacent cells in the filamentous cyanobacterium Nostoc punctiforme; these pores are drilled by the enzyme AmiC2 amidase and permit the exchange of metabolites and transmission of signal information (Lehner et al. 2013). Some clades such as the green plants (Viridiplantae, Fig. 8.9) and red algae have explored the whole spectrum of morphological organization, encompassing unicellular, filamentous, syphonal, pseudoparenchymatous and parenchymatous forms (Lee 2008; Niklas and Newman 2013). The fungi are primarily filamentous organisms and evolved a pseudoparenchymatous organization twice, in the ascomycetes and basidiomycetes (Deacon 2006). Animals and land plants both have a

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Fig. 8.8 Adherens junctions, desmosomes and hemidesmosomes ensure cellular adhesion in animals. Gap junctions are open pores in the plasma membranes of adjoining cells, which permit cell-to-cell flow of materials with a size limit of about 1 KDa. Tight junctions stick together the plasma membranes of adjoining epithelial cells, preventing uncontrolled flow of water and solutes through intercellular spaces

parenchymatous organization (Figs. 8.9 and 8.10) but employ sharply different developmental patterns (see below). Instances are known of regressive evolution from a multicellular to unicellular organization, a prominent example being the yeasts, an informal assemblage of unicellular fungi independently evolved from filamentous multicellular ancestors at least three times (Lutzoni et al. 2004; Fig. 8.11). Another remarkable instance of “regressive” evolution is the Myxozoa, parasitic unicellular organisms now known to derive from early-branching Cnidaria with the loss of 70% of genes, only their multicellular spores retaining cnidocysts (Chang et al. 2015). Interestingly, the yeast Saccharomyces cerevisiae can easily produce multicellular aggregates of hundreds of clonal cells (Ratcliff et al. 2013). These clusters express elevated rates of programmed cell death (apoptosis, see below) which induce fragmentation into smaller clusters, thus circumventing growth constraints from nutrient flow limitations (Libby and Ratcliff 2014). A universal mechanism playing a major role in the development of complex multicellular organisms is programmed cellular death, or apoptosis, a process that eliminates cells no longer necessary. Apoptosis is essential for normal organ development in both animals and plants. For example, apoptosis is involved in the

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Fig. 8.9 Levels of organization in green plants (Chlorobionta or Viridiplantae). A parenchymatous organization independently appeared four times in this clade, in the Ulvophyceae, Trebouxiophyceae, Chlorophyceae, and Embryophyta (land plants). (From Niklas and Newman 2013, licence number 4467760366224)

development of leaves, flowers and fruits, as well as of water-conducting cells, protective hairs and other specialized cell types in plants; during metamorphosis in animals, whole organs are removed or re-modelled by apoptosis (McCarthy 2003).

8.3

Stem Cells and Germ Line

The life cycle of clonal multicellular organisms with sexual reproduction encompasses a unicellular phase, the zygote, functioning as the founder cell. Multicellular organisms lacking meiotic sex reproduce clonally by means of uni- or multicellular propagules. The early developmental phase of most multicellular eukaryotes consists of totipotent cells, so named because each has an equal ability to produce any of the cell types present in the adult form. With the exception of a few simple forms, the body cells of multicellular eukaryotes stop dividing after differentiation, further body growth and cellular turnover being ensured by stem cells, undifferentiated cells that maintain the potential for cell division throughout the life of the organism. Stem cell determination to produce specific cellular types involves epigenetic control of gene expression and is usually irreversible. As already mentioned in Chap. 1, the term epigenetics refers to changes in gene expression that are heritable through multiple cell division cycles but are not due to variations in primary DNA sequence changes. Major effectors of epigenetic control include regulators of DNA methylation and

8.3 Stem Cells and Germ Line

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Fig. 8.10 Levels of organization in the Amorphea. Higher fungi (Ascomycota and Basidiomycota) independently evolved a compact pseudoparenchymatous organization mainly used for the formation of sporocarps (or fruiting bodies) in sexual reproduction, whereas the vegetative body mainly consists of loosely branched filaments (mycelium). The animals have a parenchymatous organization, with the possible exception of most primitive sponges (Porifera) and Myxozoa. The choanoflagellates, considered to be (or to encompass) the sister group of animals, are unicellular or colonial. (From Niklas and Newman 2013, licence number 4467760366224)

Fig. 8.11 The yeasts are unicellular fungi evolved by reduction from a multicellular ancestor. Regression from multi- to unicellularity entailed a pronounced reduction in genome size and gene number. The image shows cells of Saccharomyces cerevisiae, an ascomycetous yeast that reproduces by gemmation. The cup-shaped structures visible on the surface of cells are scars left by past gemmation events; when the whole surface is covered with scars, the cell stops reproducing and dies. (Credit: http://gomumu.blogspot.com/2014/04/jamur-seni-foto-menajubkan-dari.html)

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histone modification as well as ATP-dependent chromatin remodelling enzymes. These interact with other regulators, such as DNA sequence-specific transcription factors and non-coding RNAs to “landscape” genome expression during development and differentiation (Khavari et al. 2010; Armstrong 2013). In consequence of mutation, cells can escape epigenetic control and engage in out-of-control proliferation, producing abnormal tissue masses that invade the organs; known as cancer, this condition is a dramatic example of the consequences of uncontrolled cellular competition. With reference to the growth pattern, multicellular organisms may have a closed or open form. In closed-form organisms, stem cells ensure the cellular turnover and isometric growth (a growth pattern that retains the general shape of the organism and the size ratio between different parts); the body shape of closed-form organisms is under tight genetic control and is established at an early stage of development. In open-form organisms, stem cells keep producing new organs for the entire duration of life; these organisms not only never stop growing but also lack a rigidly determined body shape. Typical closed-form organisms are the animals and colonial chlorophycean algae such as Hydrodyctyon and Volvox. Land plants are typical open-form organisms (Niklas 2000): although possessing a basic shape determined genetically (for example the typical shape of a fir tree or a cypress), single individuals have indefinite numbers of leaves, branches and roots. The shapes and sizes of open-form organisms are largely determined by environmental signals; an open growth pattern confers great morphological plasticity and is strongly adaptive in sessile organisms depending on resources dispersed in the environment, such as fungi (depending on organic and mineral nutrients) and plants (depending on light, carbon dioxide and mineral nutrients). In some multicellular organisms, germ cells (namely the cells that produce gametes) separate from somatic cells early in development, with the consequence that somatic cells perform all the functions necessary for life but are excluded from sexual reproduction. The organisms in which the germline separates precociously are named “weismannist organisms” from German biologist August Weismann, who introduced the distinction between soma and germ line and formalized the “germoplasma theory” according to which only mutations appeared in the germ line are transmitted to next generations. Weismannist organisms are not common: some animal clades (essentially the arthropods and vertebrates) and Volvox. In most multicellular organisms, including many invertebrates and land plants, there is no clear-cut separation between a soma and germ line, new germ cells continuously developing from stem cells in the mature organism. It is unclear which evolutionary forces stand behind an early separation of a germline. A recent model suggests that selection for mitochondrial quality drives either early or late germline separation, depending on high or low mutation rate of mitochondrial DNA, respectively (Radzvilavicius et al. 2016). In the first case, the germline is separated early during development and kept quiescent until sexual maturation, so that mitochondrial mutations occurred during somatic development are not transmitted to the gametes. In contrast, in organisms with moderate mutation rate, numerous rounds of somatic cell division preceding germ cell separation could permit mitochondrial selection by cellular competition, thus improving gamete quality. High mitochondrial mutation rates in arthropods and vertebrates is probably related to high metabolic activity (Lane 2015).

8.4 Concluding Remarks

8.4

265

Concluding Remarks

The death is the loss of homeostatic mechanisms that maintain living systems far away from thermodynamic equilibrium. This transition coincides with cellular death in unicellular organisms, but not necessarily so in multicellular organisms. Here, in fact, death may arise from alterations of supra-cellular organization preceding the loss of intracellular homeostasis. In other words, a multicellular organism may be dead but its constituent cells remain alive at least for a while. A remarkable example is HeLa cells, a cellular line derived from cervical cancer cells taken in 1951 from Henrietta Lacks, a patient who died the same year. Since then, HeLa cells have been kept in culture and multiplied, becoming an enormous boon to medical and biological research. Complex multicellular organisms have three hierarchically interlinked levels of organization, each depending on a specific set of homeostatic interactions. The first is the whole organism, whose existence in life depends on mechanisms controlling the interactions of organs and systems of organs. The second is the single organs, which remain functional for a while after the first level of organization is lost. The third is tissues and cells, which remain alive for some time after the loss of organ function and, as is the case for HeLa cells, may even be isolated and kept in culture under appropriate conditions. We can therefore conclude that complex multicellular organisms have three distinct levels of death – systemic, organ and cellular – following each other in this order. Legal determination of death in the developed world is made by medical professionals after checking irreversible cessation of heartbeat and breathing (cardiopulmonary death), or irreversible cessation of functions of the brain (brain death), both assumed to signal systemic death. In the immediate aftermath of systemic death, organs such as the heart, liver or kidneys can be explanted and integrated into a recipient systemic network. Organ ability to survive transplant depends on their homeostatic interactions with the rest of the organism. Despite being controlled by the nervous system, the heart has an autonomous pacemaker and can resume beating after transplant in the absence of a connection to the recipient nervous system. The functionality of other organs such as the kidneys, liver and lungs is mainly under the control of the endocrine system, which facilitates integration after transplant. Transplant of blood, skin, cornea and bone marrow is even easier, as these tissues are under feeble system and organ homeostatic control. In humans, tissues may be recovered from donors up to 24 h after the cessation of heartbeat. Particularly simple is organ transplantation in open-form organisms such as land plants, in which shoots, leaves and roots not only are easily grafted on recipient plants, but can also be induced to produce new plants autonomously. On the opposite side, brain transplant in animals is still a most remote possibility.

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References Alberts B et al (2014) Molecular biology of the cell. Garland Science, New York Andersson DI, Jerlström-Hultqvist J, Näsvall J (2015) Evolution of new functions de novo and from preexisting genes. Cold Spring Harb Perspect Biol 7. https://doi.org/10.1101/cshperspect. a017996 Armstrong L (2013) Epigenetics. Taylor and Francis, London Brown MW et al (2012) Aggregative multicellularity evolved independently in the eukaryotic supergroup Rhizaria. Curr Biol 22:1–5 Brown MW et al (2018) Phylogenomics places orphan protistan lineages in a novel eukaryotic super-group. Genome Biol Evol 10:427–433 Brunet T, King N (2017) The origin of animal multicellularity and cell differentiation. Dev Cell 43:124–140 Brunkard JO, Zambryski PC (2016) Plasmodesmata enable multicellularity: new insights into their evolution, biogenesis, and functions in development and immunity. Curr Opin Plant Biol 35:76–83 Burgert I, Peter Fratz P (2009) Plants control the properties and actuation of their organs through the orientation of cellulose fibrils in their cell walls. Integr Comp Biol 49:69–79 Burki F et al (2016) Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista. Proc R Soc B 283:20152802. https://doi.org/10.1098/rspb.2015.2802 Cavalier-Smith T (2005) Economy, speed and size matter: evolutionary forces driving nuclear genome miniaturization and expansion. Ann Bot 95:147–175 Cavalier-Smith T (2017) Origin of animal multicellularity: precursors, causes, consequences – the choanoflagellate/sponge transition, neurogenesis and the Cambrian explosion. Philos Trans R Soc B 372:20150476. https://doi.org/10.1098/rstb.2015.0476 Chang ES et al (2015) Genomic insights into the evolutionary origin of Myxozoa within Cnidaria. Proc Natl Acad Sci U S A 112:14 912–14 917 Cowman AF et al (2016) Malaria: biology and disease. Cell 167:610–624 Deacon J (2006) Fungal biology. Blackwell Publishing, Malden Diggle SP et al (2007) Cooperation and conflict in quorum-sensing bacterial populations. Nature 450:411–414 Domozych DS, Domozych CF (2014) Multicellularity in green algae: upsizing in a walled complex. Front Plant Sci 5:1–8 Fisher RM, Cornwallis CK, West SA (2013) Group formation, relatedness, and the evolution of multicellularity. Curr Biol 23:1120–1125 Fujita T (2015) Plasmodesmata: function and diversity in plant intercellular communication. J Plant Res 128:3–5 Gerstein AC, Otto SP (2009) Ploidy and the causes of genomic evolution. J Hered 100:571–581 Grosberg RK, Strathmann RR (2007) The evolution of multicellularity: a minor major transition? Annu Rev Ecol Evol Syst 38:621–654 Hamilton WD (1964a) The genetical evolution of social behaviour. Part I. J Theor Biol 7:1–16 Hamilton WD (1964b) The genetical evolution of social behaviour. Part II. J Theor Biol 7:17–52 Kapsetaki S, Fisher RM, West SA (2016) Predation and the formation of multicellular groups in algae. Evol Ecol Res 17:651–669 Khavari D, Sen G, Rinn J (2010) DNA methylation and epigenetic control of cellular differentiation. Cell Cycle 9:3880–3888 Kloepper TH, Kienle CN, Fasshauer D (2008) SNAREing the basis of multicellularity: consequences of protein family expansion during evolution. Mol Biol Evol 25:2055–2068 Knoll HA (2011) The multiple origins of complex multicellularity. Annu Rev Earth Planet Sci 39:217–239 Kumar K et al (2010) Cyanobacterial heterocysts. Cold Spring Harb Perspect Biol 2:a000315. https://doi.org/10.1101/cshperspect.a000315

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Chapter 9

The Chloroplast and Photosynthetic Eukaryotes

Major transitions in evolution do include adaptations of remarkable importance, but they also include reversals, redundancy and waste, improbable complexities, and often more chance than necessity. Keeling (2013)

Abstract The chloroplast primarily evolved in the unicellular ancestor of Archaeplastida (Plants) from a cyanobacterial endosymbiont around 900 MYA. A secondary chloroplast independently evolved in the Chlorarachniophytes (Rhizaria) and Euglenophytes (Excavata) from two different green algal endosymbionts, and in the “chromoalveolate” lineage from a red algal endosymbiont. It is debated whether the chromoalveolate chloroplast was transmitted vertically across the Cryptophytes, Haptophytes, Chromista and Alveolata as the chromoalveolate hypothesis maintains, or by serial symbiosis. Chloroplast evolution involved massive gene transfer from the cyanobacterial endosymbiont to the host nucleus; a minor part of the original genome remained in the chloroplast and was never completely lost. Protein translocation from the host cytosol to different sites of the chloroplast depends on multiple translocation mechanisms derived in part from the host, in part of cyanobacterial ancestry. The evolution of secondary chloroplasts involved the transfer of genes for chloroplast maintenance from the nucleus of the primary host to that of the secondary host; mechanisms derived from the secondary-host endomembrane system mediate protein translocation across extra envelope membranes of secondary chloroplasts. Chloroplast division involves a divisome complex in part of cyanobacterial origin, in part eukaryotic. Photosynthetic eukaryotes replaced ancestral glycogen with starch, accumulated either in the host cytosol or in chloroplast stroma, or with β-glucans in cytoplasmic vesicles. Multiple chloroplast transfer is a common occurrence in eukaryotes. A novel primary chloroplast independently evolved in the cercozoan amoeba Paulinella chromatophora. By transferring

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_9

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photosynthesis from the prokaryotic to eukaryotic world, chloroplast evolution dramatically increased global productivity, thus probably being a major driver of the Late-Proterozoic transition from a low- to high-oxygen planet.

9.1

Introduction

Eukaryotes are extraordinarily keen on establishing symbiotic associations with other organisms. Leaving apart the mitochondrion, there is virtually no eukaryote lacking any form of symbiosis, and surprisingly common are instances of multiple, matrioshka-like associations (Douglas 2014). Focusing on symbioses in which at least one of the symbionts is unicellular, we distinguish extracellular symbioses, in which the cells of the two partners remain separate, and intracellular symbioses (said endocytobioses or, more simply, endosymbioses), in which the unicellular symbiont is accommodated in a host intracellular compartment. Endosymbioses often derive from interaction of a heterotrophic host and a potential prey. In these cases, the intracellular symbiont, or endosymbiont, is engulfed by the host by phagocytosis and kept within a membrane-bound vesicle, or phagosome. Fungal and bacterial endosymbionts of plants penetrate host cells by enzymatically dissolving the cell wall, a mechanism recalling their likely origin from pathogenic ancestors; also in these cases, a membrane of host origin surrounds the endosymbionts much as in phagocytic endosymbioses. For the successful establishment of a symbiosis, the organisms involved must be mutually compatible. In the case of endosymbioses, the biochemical mechanism underpinning the killing and digestion of prospective preys or pathogens by the host must be circumvented. Likewise, damage to the host from excessive resource depletion or production of toxins by the endosymbiont has to be avoided (Dorrell and Howe 2012). Mutual compatibility, however, is only the start of the process; the establishment of a symbiosis requires a balanced genomic and metabolic integration of the partners, which is a long-term outcome of genetic change and natural selection. By establishing symbiotic associations, eukaryotes acquire novel metabolic functions and jointly the genetic machinery to control their expression. The evolution of the mitochondrion shows how powerful and pervasive symbiosis can be in eukaryotes. Prokaryotes, in contrast, tend to acquire novel metabolic properties by horizontal gene transfer rather than by cellular association. Nevertheless, there are several known instances of symbiotic associations of bacteria and archaea. Examples already mentioned (Chap. 6) are syntrophic associations of hydrogen-producing bacteria with sulphate-reducing or acetogenic bacteria, or with methanogenic archaea. Another example of prokaryotic symbiosis, whose physiological significance is still unknown, is the minute archaeon Nanoarchaeum equitans living attached to the cell surface of another archaeon, Ignicoccus hospitalis (Huber et al. 2012). The only known instance of bacterial endosymbiosis (i.e. of a bacterium living inside another bacterium) has been reported in a mealybug, Planococcus citri; this

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insect has a special organ, the bacteriome, whose cells contain a γ-proteobacterium, Tremblaya princeps, which in turn contains numerous cells of a β-proteobacterium, Moranella endobia. Despite its large sizes, Tremblaia has a very small genome (139 kb, with about 120 genes) and depends on its endosymbiont for fundamental metabolic functions (McCutcheon and von Dohlen 2011; Husnik et al. 2013). Although established in a very special environment (the inside of a eukaryotic cell), this unusual bacterial symbiosis has been summoned to support hypotheses of bacterial fusion or bacterial endocytosis in eukaryogenesis (Chap. 6). In contrast to prokaryotes, endosymbiosis is common in the whole spectrum of eukaryotic diversity, involves both prokaryotic and eukaryotic endosymbionts, and has great ecological relevance (Rosenberg and Zilber-Rosenberg 2016). This chapter deals with an ancient endosymbiosis that gave rise to the chloroplast, the photosynthetic organelle of eukaryotes. The evolution of the chloroplast and photosynthetic eukaryotes is a complex and fascinating story still to be completely elucidated. Phylogenomic analysis has given some cues about the organisms originally involved in the genesis of the chloroplast. The eukaryotic host was a freshwater phagotrophic bikont, the photosynthetic endosymbiont (or photobiont) a cyanobacterium close to present-living Nostocales or Stigonematales (Deusch et al. 2008; Dagan et al. 2013). During the evolution of the symbiosis, the endosymbiont genome strongly reduced, being in part lost, in part transferred to the host nucleus, only a small residue remaining in the organelle to form the chloroplast genome, or plastome (Barbrook et al. 2010). With the acquisition of the chloroplast, the eukaryote host abandoned the ancestral phagotrophic lifestyle, encased itself in a cellulosic cell wall and became an obligate autotroph. Its descendants generated the large clade named “plants” or, more formally, Archaeplastida (literally “ancient chloroplasts”). The chloroplast of plants is a primary chloroplast because it directly descends from an enslaved cyanobacterium; its envelope consists of an inner and outer membrane that are homologous with the inner and outer membrane of the cyanobacterial endosymbiont, respectively (cyanobacteria are gram-negative bacteria just like the bacteria that originated the mitochondrion). The phagosomal membrane, which originally must have enveloped the cyanobacterial symbiont, was lost. The peptidoglycan layer that in cyanobacteria lies between the inner and outer membrane, is no longer visible in the chloroplast of most plants, with the exception of Glaucophyta, a small basal group (Bhattacharya et al. 2007; Gould et al. 2008; Howe et al. 2008; Keeling 2010; Dorrell and Howe 2012). After the emergence of the primary chloroplast and the spread of plants, multiple secondary symbioses ensued in which phagotrophic eukaryotes engulfed unicellular plants and converted them into secondary chloroplasts. Secondary chloroplasts have additional bounding membranes besides the original two-membrane envelope, which are derived from the plasma membrane of the eukaryotic photobiont and/or the endomembrane system of the secondary host. Eukaryotic lineages with a secondary chloroplast are scattered among the Alveolata, Stramenopila, Rhizaria, Euglenida, Cryptista and Haptista side by side with non-photosynthetic lineages, the only major clade entirely lacking plastidiate forms being the Amorphea (Keeling 2013; Zimorski et al. 2014). In a number of instances, the chloroplast (either primary

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or secondary) lost the photosynthetic function and turned into a non-photosynthetic organelle. The general term plastid is used for the different types of chloroplast and their non-photosynthetic relatives. Molecular-clock analysis by Shih and Matzkeb (2013) dated the emergence of the primary chloroplast around 900 MYA, considerably later than inferred in earlier studies (see, for example, Douzery et al. 2004; Parfrey et al. 2011). Before the appearance of the chloroplast, eukaryotes mostly occupied the consumers’ trophic level; with the acquisition of the chloroplast (either primary or secondary), a number of eukaryotic lineages became primary producers, a trophic level until then restricted to prokaryotic life and largely dominated by cyanobacteria. The transfer of photosynthesis to the eukaryotic world met with great biological success; photosynthetic eukaryotes spread in freshwater habitats and the ocean, becoming the major primary producers almost worldwide, out-competing cyanobacteria in some of their original niches and colonizing novel niches in aquatic and terrestrial environments. Most likely, the rise of photosynthetic eukaryotes in the Neoproterozoic significantly contributed to the second oxygen rise and the disappearance of euxinic oceans (Sect. 5.6). Increased albedo caused by cloud condensation nuclei from photosynthetic eukaryotes (dimethylsulphoniopropionate converted into volatile dimethylsulphide by bacteria) may have contributed to the planetary cooling that plunged the Earth into the Cryogenian glaciations between 720 and 635 MYA (Feulner et al. 2015). Land colonization by plants at the beginning of Phanerozoic dramatically increased global primary productivity and brought oxygen atmospheric concentration to modern levels (Chap. 11). About 280 billion tons of carbon dioxide is annually converted into organic matter on the Earth today, the greater part by photosynthetic eukaryotes (Geider et al. 2001). Adaptation to novel light regimes involved changes in the set of photosynthetic pigments of cyanobacterial ancestry; notably, novel forms of chlorophyll replaced phycobiliproteins in some lineages. Nevertheless, chlorophyll a (essential for oxygenic photosynthesis) was retained in all photosynthetic eukaryotes, and the core of the photosynthetic machinery remained almost unchanged (Chap. 4). Besides photosynthesis, the chloroplast performs other major metabolic functions such as assimilation of ammonia into amino acids, assembly of iron-sulphur complexes, biosynthesis of aromatic amino acids, purine and pyrimidine bases, fatty acids, isoprenoids and phenolic compounds, dissipation of excess mitochondrial reducing potential (Dorrell and Howe 2012 and references therein).

9.2

Birth of the Primary Chloroplast

Phylogenomic analysis of the plastome traces the ancestry of both the primary and secondary chloroplasts to a single cyanobacterial endosymbiont (RodríguezEzpeleta et al. 2005; McFadden and van Dooren 2004). For a while, this was explained by assuming that the transformation of a free cyanobacterium into an organelle was an exceedingly difficult event with very few chances of occurring more than once in the history of eukaryotes. This perspective changed in 2005 with

9.2 Birth of the Primary Chloroplast

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Fig. 9.1 The emergent chloroplast of the testate amoeba Paulinella chromatophora evolved independently of the chloroplast in the other photosynthetic eukaryotes. This organelle, traditionally named “chromatophore”, has an envelope of two membranes and a middle peptidoglycan wall. Genomic analysis has shown that it arises from a cyanobacterium close to Prochloron or Synechococcus, and retains a genome of 1.02 Mb, intermediate between a “proper” chloroplast (about 200 Kb) and a free-living cyanobacterium (several Mb). The symbiont of Paulinella lacks many metabolic functions normally present in free-living cyanobacteria and is unable to survive outside of its host. Mature cells of Paulinella contain two chromatophores. During cell division, each daughter cell receives one of the chromatophores and then lets it divide once. (Credit: http:// www.plingfactory.de/Science/Atlas/KennkartenProtista/source/Paulinella_chromatophora.html)

the discovery of a novel primary chloroplast in Paulinella chromatophora, incidentally a species already known to science for over a century (Marin et al. 2005). First described in 1895 by Robert Lauterborn, Paulinella chromatophora (Fig. 9.1) is a filose amoeba (Cercozoa, Rhizaria) that inhabits shady sediments of freshwater bodies rich in organic compounds, poor in oxygen, with increased salinity and relatively low pH. Molecular phylogeny has revealed that the photosynthetic organelle of Paulinella (traditionally called chromatophore, i.e. “bearing colour”) arises from a cyanobacterium akin to Prochloron/Synechococcus, independently of the nostocalean/stigonematalean chloroplast lineage in the rest of eukaryotes. Paulinella’s symbiosis started about 60 MYA, much later than the primary chloroplast of plants, and is still progressing towards further host-photobiont integration (Bhattacharya et al. 2007). There are numerous amoebas related to Paulinella chromatophora, yet all living as bacterivorous phagotrophs and lacking chromatophores; in contrast to its heterotrophic relatives, P. chromatophora lacks digestive vacuoles and is probably entirely autotrophic (Nowack 2014). A second photosynthetic species of Paulinella, P. microporus, has been isolated in Japan. The two species share the same photobiont, therefore they most likely diverged from a common ancestor (Yoon et al. 2009).

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It is estimated that chloroplast maintenance requires over one thousand genes (Archibald 2015), only a minor fraction of which (100–250) is located in the organelle, the rest being part of the nuclear genome. Nuclear chloroplast genes include genes of cyanobacterial ancestry (about 50% of the total) and genes of eukaryotic origin, plus a number of genes probably obtained from proteobacteria and Chlamydia by horizontal gene transfer and secondarily deployed for chloroplast maintenance (Suzuki and Miyagishima 2009; Ball et al. 2013). The conversion of the photobiont into an organelle regularly transmitted across cell generations entailed the transfer of most essential genes from the photobiont to the host nucleus. Being shared with the mitochondrion and Paulinella chromatophore, as well as numerous other types of endosymbiosis, this is probably an obligate evolutionary path dictated by natural selection. The expression “gene transfer” does not mean that pieces of DNA are cut away from the endosymbiont genome and inserted in the nuclear genome. By depriving the endosymbiont of essential functions without any compensative mechanism, this would destroy the symbiosis from the very beginning. More likely, occasional lysis of one of multiple endosymbionts present in the same host cell disperses DNA sequences that have thus the chance of being incorporated into the host genome (Dorrell and Howe 2012; Fig. 9.2). Experiments on genetically transformed tobacco plants (Huang et al. 2003) have revealed that chloroplast-to-nucleus DNA transfer is more frequent than expected (about 0.06% of the progeny obtained in a single generation).

Fig. 9.2 Chloroplast evolution involved gene transfer from the cyanobacterial endosymbiont (green) to the host nucleus (blue). (a) Host cell containing several cyanobacteria. (b) One of these breaks down and disperses DNA fragments (red) in the host cytosol. (c) DNA-repairing enzymes insert DNA fragments into host chromosomes. (d) Proteins encoded by nuclear genes of cyanobacterial origin (magenta) are translocated from cytosol to cyanobacteria; the cyanobacterial genome reduces by losing genes successfully transferred to the host nucleus

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A part of the cyanobacterial genes transferred to the nucleus during chloroplast evolution were probably of no use; unbound from the constraints of natural selection, they freely mutated into pseudogenes and were eventually lost by deletion. The most important part of the cyanobacterial genome transferred to the nucleus were genes involved in chloroplast maintenance and photosynthesis; there is evidence that some cyanobacterial genes were deployed for functions not related to chloroplast maintenance. Estimates of nuclear genes of cyanobacterial origin range from about 6% in the green alga Chlamydomonas reinhardtii, to 11% in the glaucophyte Cyanophora paradoxa and as much as 18% in the angiosperm Arabidopsis thaliana (Burki et al. 2012), the increase reflecting extensive gene duplication and neo-functionalization. After the discovery of the emergent chloroplast in Paulinella, it seems possible or even likely that a chloroplast evolved several times in eukaryotes. Indeed, as observed by Howe et al. (2008), it is unlikely that the symbiont ultimately converted into a primary chloroplast was the first one that the host lineage had ever acquired; more likely, “tentative” associations with other photosynthetic organisms preceded the ultimately successful symbiosis. Some of these were probably lost very quickly, others persisted for a while and possibly left some of their genes in the host nuclear genome. The putative reservoir of nuclear sequences derived from previous photosynthetic symbionts might have facilitated the evolution of the primary chloroplast. According to this “shopping bag” model, the majority of the genes employed for chloroplast maintenance were derived from the final successful symbiont, but a part of them were probably obtained from older symbioses (Howe et al. 2008). Why then, barring the instance of Paulinella, did only one chloroplast lineage survive to the present? A likely answer is that this chloroplast was so efficient as to enable its hosts to occupy all ecological niches available and out-compete other types of chloroplast appeared earlier or later (Keeling et al. 2015), Paulinella being a remarkable exception possibly explained by its unique habitat. Fundamental steps of the process that converted a cyanobacterium into a chloroplast were: (a) the evolution of membrane-bound transporters mediating molecular exchange between the host and photobiont, (b) metabolic integration of the symbionts with the elimination of useless and redundant pathways, (c) synchronization of their reproductive cycle. As is typical of biological evolution, these “goals” were attained through numerous little steps, each granting a small gain in overall fitness, with natural selection rapidly eliminating “mistakes” and less effective competing solutions. The chloroplast envelope contains transporters mediating the translocation of simple molecules such as phosphate, ATP, sugars and sugar-phosphates to and from the cytosol. Free-living cyanobacteria encode all the transport systems required for the uptake of mineral ions from the outside, but lack genes encoding transporters for the export of organic solutes. The family of phosphate antiporters localized in the chloroplast envelope inner membrane, known as plastidial Phosphate Translocators, probably arise from an ancestral host-encoded carrier that exchanged phosphate ions from the host cytosol for phosphorylated organic compounds from the photobiont. Chloroplast evolution also involved ABC (ATP-Binding Cassette) transporters, carriers for amino acids, nucleotides and other small organic molecules and carriers for sulphate, potassium, magnesium and iron ions; most these are of eukaryotic origin, being derived from components of the host endomembrane

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Fig. 9.3 The primary chloroplast has six main topological sites: (1) outer and (2) inner membrane, (3) inter-membrane space, (4) stroma, (5) thylakoid membrane, (6) thylakoid lumen. The arrangement of the photosynthetic membranes in granal and stromal thylakoids indicates that the chloroplast shown in the figure belongs to the Streptophyta, a line of Archaeplastida encompassing charophyte algae and land plants

system (Weber et al. 2006; Deschamps et al. 2008a; Tyra et al. 2007; Karkar et al. 2015). In extant plants, proteins encoded by nuclear genes and destined to the chloroplast reach their target using tag peptides that function as a sort of postal address. By interacting with special translocons, these peptides ensure that each protein gets to the correct destination in the chloroplast. Tag peptides are encoded by DNA sequences inserted in genes immediately before the protein-encoding sequence, the same tag being used to label different proteins destined to the same location. The chloroplast is a complex organelle comprising at least six topologically distinct sites; each cytosolic protein destined to the chloroplast may be labelled with one to several tags (Fig. 9.3), which are sequentially recognized by different translocons and cleaved by specific peptidases after use (Jarvis 2008; Bodył et al. 2009a). Most of the proteins synthesized in the cytosol and destined to the chloroplast enter this organelle through the TOC-TIC machinery (Figs. 9.4 and 9.5), a chimeric system made of proteins in part of cyanobacterial ancestry, in part derived from the eukaryotic host. How such a complex system might have evolved is still a matter of speculation. Protein import may initially have been mediated by the ER-Golgi vesicular pathway, to be then gradually replaced by more efficient post-translational pathways. In line with this hypothesis, a vesicular mechanism of protein import operates in Paulinella chromatophores (Nowack 2014). A fundamental component of the TOC-TIC system

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Fig. 9.4 The greatest part (>90%) of over one thousand different proteins required for chloroplast maintenance is encoded by nuclear genes, synthesized on cytosolic ribosomes and posttranslationally translocated to their final target in the chloroplast following “instructions” from tag peptides and hydrophobic transmembrane domains. The chloroplast envelope hosts a complex translocation machinery encompassing the TOC (Translocon of the Outer envelope membrane of the Chloroplast) and TIC system (Translocon of the Inner envelope membrane of the Chloroplast) inserted in the outer and inner membrane, respectively. The TOC-TIC machinery mediates the insertion of inner-membrane proteins carrying a transit peptide followed by multiple transmembrane domains, and the import of stroma-directed proteins carrying a transit peptide. In performing these functions, the TOC-TIC system can recruit accessory proteins that vary with the type of protein translocated. The outer envelope membrane also contains free Toc75 molecules that mediate insertion of outer envelope proteins without the participation of other Toc components. Most proteins residing in the outer envelope membrane lack a transit peptide and insert autonomously using intrinsic transmembrane domains. The transport of proteins to the intermembrane space is poorly known. Some of the proteins entering the stroma are destined to the thylakoid membrane or the thylakoid lumen. The first comprise (a) proteins that spontaneously insert in the thylakoid membrane using transmembrane domains, and (b) proteins that use the signal recognition particle (SRB). The proteins directed to the thylakoid lumen reach their target through the Tat (Twin arginine translocase) or Sec (Secretory complex) pathway, each recognizing its own tag peptide. Specific peptidases (represented as scissors in the figure) cleave tag peptides after translocation; the stromal peptidase (Stromal Processing Peptidase) is homologous with the peptidase of the mitochondrial matrix (Mitochondrial Processing Peptidase). A number of alternative routes are known, including vesicular transport through the endomembrane system. The TOC-TIC complex is a molecular chimera made of proteins of cyanobacterial origin (Toc75, Tic20, Tic21) and proteins derived from the host (Toc34, Toc150, Toc64, Tic32, Tic55, Tic62). Thylakoid translocons SRP, Tat and Sec are entirely of cyanobacterial origin. (Adapted from Jarvis 2008, licence number 4457720764708)

is Toc75, a protein derived from Omp85. Omp85 is a channel localized in the outer membrane of cyanobacteria and other gram-negative bacteria, which binds proteins secreted by the cell in the periplasmic space (including other Omp85 proteins) and

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Fig. 9.5 (a) Sequential steps (1, 2, 3) of protein translocation from cytosol to chloroplast stroma. The process consumes a GTP and two ATP molecules per protein molecule translocated. (From Jarvis 2008, licence number 4457720764708). (b) Structure of the TOC-TIC complex in higher plants. Hsp70, Hsp90 and Hsp93 are chaperonins that maintain stroma-targeted proteins in an unfolded conformation until they enter the stroma, and help them fold correctly after the removal of the transit peptide by stromal processing peptidase (SPP). The ubiquity of the TOC-TIC complex in photosynthetic eukaryotes is evidence of chloroplast monophyly. (Adapted from Bodyl et al. 2009a, licence number 4457711398644)

inserts them into the outer membrane. There is evidence that Toc75 is able to translocate cytosolic proteins through the outer chloroplast membrane without the participation of other components of the TOC complex, suggesting that the outer membrane translocon initially consisted of Toc75 alone. The gene for Omp85 was inserted in the host genome and the protein started being synthesized in the cytosol, whilst its ancestral equivalent was still present in the outer membrane of the cyanobacterial symbiont. Omp85 recognized the cytosolic version as similar to itself and inserted it into the outer membrane of the cyanobacterial symbiont. Bodył et al. (2009a) suggest that, because the cytosolic version was on the outer side of the membrane, Omp85 inserted it with an inverted orientation, namely with the channel opening directed towards the cytosol, thus enabling it to function in protein translocation from the cytosol to the chloroplast. With time, the cytosolic version of Omp85 mutated, progressively becoming the protein we now call Toc75. By incorporating the Toc75 channel, the outer membrane of the nascent chloroplast became able to mediate protein import autonomously. Tagging of nucleus-encoded proteins to the emergent chloroplast permitted deletion of the corresponding genes (including Omp85) from the chloroplast genome without detrimental effect.

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Fig. 9.5 (continued)

In parallel, a number of proteins of eukaryotic origin associated with Toc75 to build the TOC complex. Consequently, the ER-Golgi vesicular pathway became redundant and the phagosomal membrane was lost without impairing protein import. Vesicular import to primary chloroplasts in extant plants appears to be restricted to few glycoproteins (Fig. 9.4). Gene transfer from the endosymbiont to the host nucleus was essential for functional integration, yet a second factor was probably at stake as well. Across generations, the endosymbionts tend to accumulate mutations that they, because of isolation within host cells, cannot recover by recombination with foreign DNA, as occurs in free-living bacteria (Chap. 7). Over time, the endosymbionts may

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degenerate to the point that the host is obliged to get rid of them. Transferred to the nucleus, endosymbiont genes have access to recombination through meiotic sex, which ensures the conservation of genetic information in sufficiently large populations (Chap. 7). Thus, the transfer of essential genes from the endosymbiont to the nucleus is intrinsically beneficial to the stability of the symbiosis (Keeling et al. 2015). The insertion of endosymbiont genes in the nuclear genome may also facilitate the re-establishment of a symbiosis in case the endosymbiont is lost, as is certainly the case in dinoflagellates (Sect. 9.6). Plastids and mitochondria usually contain multiple genome copies and organelle fusion is common, thus homologous recombination is likely to contribute to the maintenance of organelle genomes despite isolation from external DNA sources. As the mitochondrion, the chloroplast retained a part of the original genome, resisting evolutionary pressure for gene transfer to the nucleus. The CORR (COlocation for Redox Regulation) hypothesis proposes that genome retention is instrumental to ensure rapid redox regulation of gene expression in these organelles (Allen 2015). The CORR hypothesis is consistent with the absence of a genome in mitochondrion-derived anaerobic organelles (Sect. 6.7.1). It has also been suggested that some genes were retained in the organelles because they code for hydrophobic proteins whose import from the cytosol would be problematic, or for protein complexes whose synthesis and assembly are coupled and tightly regulated, a condition incompatible with gene transfer to the nucleus (Barbrook et al. 2006). The above hypotheses primarily address the need to keep photosynthesis genes in the chloroplast, and none of them applies to all lineages. Excluding all photosynthesis-related genes and all genes of the plastid transcription-translation apparatus, there are only a few protein-coding genes that remain in the plastid genome. For each of these, examples are known of successful plastid-to-nucleus transfer (or loss of the gene altogether), and none appears to encode a particularly hydrophobic protein or a protein that might require redox regulation from within the plastid. Consequently, one might expect that the loss of photosynthesis would allow the loss of the plastid genetic system in its entirety (Barbrook et al. 2006). Actually, unlike most instances of mitochondrion-derived organelles, there is no convincing report of plastids completely lacking a genome, suggesting that other factors besides those considered above dictate gene retention in plastids. A genome is present in plastids that have reversibly lost the photosynthetic function, such as amyloplasts and leucoplasts in land plants, which retain the ability to convert into functional chloroplasts. The same is true for colourless plastids in heterotrophic parasitic plants (e.g. Orobanche or Cuscuta), where the absence of photosynthetic pigments is due to a reversible block of their biosynthetic pathway. More surprising is genome retention in plastids that have irreversibly lost the photosynthetic machinery, for example the “apicoplast” in the Apicomplexa (Lim and McFadden 2010) and plastids in parasitic algae (Prototheca, Ewing et al. 2014). The only documented instances of complete plastid genome loss are organisms that appear to have completely lost the plastid, such as the apicomplexan Cryptosporidium parvum (Barbrook et al. 2006).

9.3 The Plants (Archaeplastida)

9.3

281

The Plants (Archaeplastida)

If the multicellular fossil Bangiomorpha pubescens is really a red alga, then the plants and the primary chloroplast should have appeared at least 1.2 GYA. More restrictive interpretations of palaeontological evidence (Sect. 6.10) and molecular clock analysis postpone their origin to about 900 MYA (Shih and Matzkeb 2013). With the acquisition of a photosynthetic endosymbiont, the unicellular progenitor of plants switched to an autotrophic lifestyle, encased itself in a cellulosic cell wall and gave up phagotrophy. Cellulose is a β-1-4 unbranched glucan occurring in several bacteria, including cyanobacteria, and in a number of eukaryotic lineages besides plants (dinoflagellates, Dictyostelia, brown algae and even Tunicates, a group of animals). The family of enzymes responsible for cellulose synthesis in plants most likely derives from CesA, a cyanobacterial gene inherited through the chloroplast (Nobles and Brown 2004). The cyanobacterial progenitor of the chloroplast probably possessed the nitrogenase machinery for nitrogen fixation but lost it during organelle evolution in spite of its potential value (Deush et al. 2008; Dagan et al. 2013). Because oxygen inactivates nitrogenase, nitrogen-fixing cyanobacteria have evolved special adaptations to reconcile nitrogen fixation with oxygenic photosynthesis, such as temporal separation by a biological circadian clock or spatial separation in different cellular types (Berman-Frank et al. 2003; Chap. 8). Nitrogen fixation requires such a low reduction potential as to be feasible only in the chloroplast, thus preventing the transfer of this metabolic pathway to any other compartment of the host cell. Chloroplast specialization as a photosynthetic organelle, therefore, precluded the maintenance of nitrogen fixation. The conversion of the ancestral cyanobacterial photobiont into a chloroplast also involved the loss of the respiratory cytochrome oxidase complex (Howe et al. 2008). From a common progenitor endowed with a chloroplast and a cellulosic cell wall, three lineages diverged: Glaucophyta, Rhodophyta and Viridiplantae (de Vries et al. 2014). The Glaucophyta are all freshwater organisms and the same is true for basal Rhodophyta and Viridiplantae. The order of divergence of the three clades is uncertain (Fig. 9.6), probably because of rapid evolution after separation from the common ancestor. A study using nucleotide ambiguity codes to eliminate synonymous substitutions allowed by codon-degeneracy suggests an early divergence of Glaucophyta, with the Rhodophyta and Vididiplantae as sister groups in a separate lineage (Li et al. 2014). The persistence of a discrete peptidoglycan layer in the chloroplast envelope of glaucophytes (Nowack 2014) is in line with a basal position of Glaucophyta; a peptidoglycan layer, however, was recently reported in the chloroplast of the moss Physcomitrella patens, although in this case it is not visible by electron microscopy after conventional chemical fixation. Indirect evidence suggests that the enzymatic complement for peptidoglycan synthesis was retained throughout the streptophytes (the archaeplastidal lineage encompassing land plants), with the exception of angiosperms (Hirano et al. 2016).

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Fig. 9.6 Origin of the primary chloroplast and Archaeplastida. The relative order of divergence of Glaucophyta, Rhodophyta and Viridiplantae is yet to be determined. The length of the horizontal branches is not to scale with divergence time

Each of the three plant lineages modified the set of photosynthetic pigments inherited from the cyanobacterium, because of adaptation to different habitats. The glaucophytes are all unicellular and display a combination of pigments (chlorophyll a, C-phycocyanin and allophycocyanin) that imparts them a bluegreen colour. Their chloroplasts contain unstacked thylakoids with phycobilisomes and dense structures similar to carboxysomes (Jackson et al. 2015). There are four formally recognized genera of glaucophytes, viz. Cyanophora, Cyanoptyche, Glaucocystis, Gloeochaete, encompassing 13 species. Besides a peptidoglycan layer in the chloroplast envelope, a distinguishing trait of glaucophytes is the occurrence of flattened membrane-bound vesicles tightly associated with the cell membrane, recalling the “alveoli” of Alveolata (Kugrens et al. 1999; Takahashi et al. 2015; Fig. 9.7). The Rhodophyta, commonly known as red algae (Fig. 9.8), is a wellcircumscribed group in traditional systematics, robustly confirmed as a clade by molecular systematics (Yoon et al. 2006). Distinctive red algal traits include: (a) Two novel phycobiliproteins, R-phycocyanin and phycoerythrin, besides allophycocyanin of cyanobacterial ancestry. (b) Total absence of flagella and centrioles. (c) A gene from a proteobacterium replacing the cyanobacterial gene for the large RubisCo subunit (RbcL) (Glaucophyta and Viridiplantae retain the cyanobacterial gene).

9.3 The Plants (Archaeplastida)

283

Fig. 9.7 The glaucophyte Cyanophora biloba contains a single chloroplast that divides immediately before cell division. (a) Light microscope image showing the dividing chloroplast. (b) Electron-microscope image of a chloroplast with a dense band of peripheral thylakoids (T) and a prominent carboxysome-like structure (Ca). (c) Detail of the same cell, showing peripheral thylakoids (T) with phycobilisomes and flattened vesicles close to the plasma membrane (arrows). (a From: http://fmp.conncoll.edu/Silicasecchidisk/LucidKeys3.5/Keys_v3.5/Carolina35_Key/Media/ Html/Cyanophora_Main.html. b, c Adapted from Kugrens et al. 1999, licence number 4457730151154)

(d) Floridean starch, a storage polysaccharide similar to amylopectin, synthesized in the cytosol. (e) Absence of glycosylphosphatidylinositol, a glycolipid used in the rest of eukaryotes to anchor proteins lacking hydrophobic domains to membrane bilayers. A further distinctive character of red algae is their relatively small genome; for example, the unicellular red alga Chondrus crispus has only 9600 genes vs ~14,000 in the green unicellular alga Chlamydomonas and the multicellular red alga Porphyra umbilicalis has ~13,000 genes (Brawley et al. 2017) vs about ~27,000 in the diminutive angiosperm Arabidopsis. Red algae encompass about 7000 species distributed in the subphyla Cyanidiophytina and Rhodophytina (Yoon et al. 2006). The Cyanidiophytina comprises unicellular species adapted to thermo-acidic environments (Cyanidium, Cyanidioschyzon, Galdieria) and lacking phycoerythrin (Brawley et al. 2017). The Rhodophytina encompasses six classes (Fig. 9.6) comprising unicellular, filamentous, pseudoparenchymatous and parenchymatous species. Most known red algal species belong to the class Florideophyceae in the Rhodophytina. Primarily freshwater organisms, red algae migrated to marine habitats several times independently and here they richly diversified. Most extant species are marine

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Fig. 9.8 Red algae (Rhodophyta, Archaeplastida). (a) Porphyridium cruentum (http:// lookfordiagnosis.com/mesh_info.php?term¼porphyridium&lang¼4). (b) Cyanidium caldarium (http://www.uniprot.org/taxonomy/2771). (c) Cyanidioschyzon merolae (http://schaechter. asmblog.org/schaechter/2007/09/sex-well-not-re.html). (d). Ptilota serrata (picture by Kjersti Sjøtun). (e) A detail of a red algal cell photographed by transmission electron microscopy. The chloroplast (Cl) contains numerous unstacked thylakoids covered with phycobilisomes. The white globules in the cytoplasm near the chloroplast (arrow) are starch deposits. N, nucleus

weeds, often found at greater depths compared to other seaweeds because of phycoerythrin ability to absorb blue light. With about 400,000 species, the Viridiplantae (green plants) is by far the largest group within the Archaeplastida and one of the greatest eukaryotic lineages. Major apomorphies of the group (Leliaert et al. 2011, 2012) include: (a) (b) (c) (d) (e)

The absence of phycobilisomes and phycobiliproteins. Chlorophyll b in addition to chlorophyll a. Thylakoid stacking (Fig. 9.9). Starch deposited within the chloroplast. The RbcS gene encoding for the small RubisCo subunit located in the nuclear genome (in chloroplast genome in Glaucophyta and Rhodophyta). (f) Ancestral biflagellate isokonty (i.e. flagellate cells primarily have two flagella of the same type). (g) Stellate structure in transverse section of the transition zone between the basal body and flagellar axoneme (Box 6.2).

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Fig. 9.9 Chloroplast ultrastructure in green plants (Viridiplantae). (a) The photosynthetic apparatus in the Chlorophytes consists of long bands of 3–5 thylakoids (T). (b) Stacked (G) and unstacked thylakoids (ST) are more clearly separated in the chloroplast of Streptophytes. In both cases thylakoid stacking reflects spatial separation of the two photosystems: closely appressed thylakoid membranes (known as partitions) are enriched in antenna complexes and photosystem 2 (Chap. 4), whereas thylakoid membranes in contact with the stroma are enriched in photosystem 1. The degree of thylakoid stacking changes in response to the intensity and spectral composition of incident light to balance the activities of the two photosystems (Pribil et al. 2014)

The Viridiplantae comprises two clades, the Chlorophyta (chlorophytes) and Streptophyta (Streptophytes), the first encompassing freshwater and marine species, the latter freshwater (Charophyta, charophytes) and terrestrial species (Embryophyta, or land plants), with few land plants secondarily adapted to freshwater and marine habitats. Primarily aquatic Viridiplantae are generally known as green algae. Unlike red algae, the green algal assemblage is not a clade because it excludes land plants (Fig. 9.10).

9.4

Secondary Chloroplasts

Secondary chloroplasts arise from eukaryotic endosymbionts, not from a cyanobacterium, and have additional enveloping membranes besides the two original membranes (Fig. 9.11). A secondary chloroplast is found in the Chlorarachniophyta (Cercozoa, Rhizaria), Euglenophyta (Euglenozoa), a part of the Stramenopila (brown algae, diatoms, crysophytes, xanthophytes and several minor groups), a part of the Alveolata (Apicomplexa, Chrompodellida, Dinophyta), and in Cryptophyta (Box 9.1) and Haptophyta, two lineages of uncertain taxonomic position (Gould et al. 2008, 2015; Keeling 2010, 2013; McFadden 2014; Stiller et al. 2014;

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Fig. 9.10 The Viridiplantae (green plants) is the largest and most diverse lineage of photosynthetic eukaryotes. Their green colour is due to a combination of chlorophyll a and chlorophyll b. (a–d) Chlorophyta: (a) Chlorella (Chlorophyceae); (b) Pediastrum (Chlorophyceae); (c) Acetabularia (Ulvophyceae); (d) Ulva (Ulvophyceae); (e–h) Streptophyta, Charophyta: (e) Cosmarium; (f) Spirogyra; (g) Coleochaete; (h) Nitella. (i–n) Streptophyta, Embryophyta: (i) Anthoceros (Anthocerotophyta); (l) Dryopteris (Monilophyta); (m) Cycas (Cycadophyta); (n) Lupinus (Angiospermae)

Bodył 2018). All these groups include or are related to heterotrophic taxa lacking a chloroplast and living as phagotrophs or parasites. Among these, the Apicomplexa (with the exception of plastid-less Cryptosporidium) have a colourless plastid known as the apicoplast, which witnesses the origin of the group from a photosynthetic ancestor. The apicoplast lacks photosynthetic pigments, yet it retains a genome and is essential for these microorganisms (Lim and McFadden 2010). The Chrompodellida, a recently discovered group of flagellate eukaryotes encompassing both heterotrophic (e.g. Colpodella and Voromonas) and photosynthetic forms (e.g. Chromera velia and Vitrella brassicaformis), has been resolved as the sister group of Apicomplexa (Oborník et al. 2011; Oborník and Lukeš 2013; Fig. 9.18). The chloroplast of chloroarachniophytes and the chloroplast of euglenophytes, the former with four enveloping membranes, the latter with three, independently evolved from two unicellular chlorophytes, probably both belonging to the Ulvophyceae-Chlorophyceae-Trebouxiophyceae (UCT) clade (Rogers et al. 2007). The compartment between the second and third envelope membrane (known as the periplastid space; Fig. 9.11) in chlorarachniophytes derives from the cytoplasmic

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Fig. 9.11 (a) Genesis of a secondary chloroplast from a eukaryotic photobiont. (b) Diagrammatic representation of membranes enveloping a secondary chloroplast. Membranes 1 and 2 are from the envelope of the original primary chloroplast. According to a widely accepted interpretation, membrane 3 (the periplastid membrane) derives from the plasma membrane of the photobiont, and membrane 4 (the epiplastid membrane) is a phagosomal membrane from the secondary host. An alternative interpretation holds that the phagosomal membrane and the photobiont plasma membrane were lost an an early stage of the secondary symbiosis and that the peri- and epiplastid membranes derive from a sheet of endoplasmic reticulum that enveloped the photobiont

Box 9.1 Are Plants a Clade? The two-membrane chloroplast envelope, a shared origin of Calvin cycle enzymes (Reyes-Prieto and Bhattacharya 2007) and of plastid solute transporters (Tyra et al. 2007; Colleoni et al. 2010), dozens of unique Chlamydia genes including two nuclear genes encoding for an isoamylase and a starch synthase (Huang and Gogarten 2007), and extensive within-group gene sharing (Chan et al. 2011) are interpreted with some confidence under the parsimonious scenario of Archaeplastida monophyly. In spite of these robust unifying characters, phylogenomic analysis has produced contrasting results. The first approaches robustly resolved the group as a clade, yet in recent years several independent studies have placed the Cryptophyta and related heterotrophic forms (collectively named Cryptista) in a sister position to, or even within the Archaeplastida (Kim and Graham 2008; Parfrey et al. 2010; Derelle et al. 2015; Burki et al. 2016; Brown et al. 2018; Fig. 6.19). This overcomplicates the phylogenetic scenario, because the Cryptophyta have a secondary chloroplast with an envelope of four-membranes and clear affinities with the (continued)

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Box 9.1 (continued) red algal chloroplast. Assuming that non-photosynthetic cryptists evolved secondarily by chloroplast loss, the association of Cryptista with the Archaeplastida might be an artefact due to unrecognized red algal genes transferred to the host nucleus through the secondary chloroplast. Alternatively, the Cryptista might be an ancestrally heterotrophic lineage related to the Archaeplastida, and the cryptophytes may have secondarily acquired a chloroplast from a red algal endosymbiont (Mackiewicz and Gagat 2014). Interestingly, both the Cryptophyta and Haptophyta have a variant of the chloroplast rpl36 gene acquired by HGT and replacing the original gene present in all other photosynthetic eukaryotes (Rice and Palmer 2006). The question is thus still open, requiring phylogenomic analysis of broader taxonomic and gene ranges, with focus on nuclear genes not of chloroplast ancestry.

compartment of the green algal photobiont and contains endoplasmic reticulum, eukaryotic ribosomes, and even a relict nucleus (known as the nucleomorph) retaining a small but still functional genome (Cavalier-Smith 2003; Fig. 9.12). During the establishment of the symbiosis, the nucleomorph retained only a small fraction of the genes originally present (284 genes in Bigellowiella natans), whereas the genes necessary for chloroplast maintenance were transferred to the host nucleus (Fig. 9.12). A similar situation as that described in chloroarachniophytes is present in cryptophytes, flagellate eukaryotes with a chloroplast bounded by four membranes and retaining a nucleomorph in the periplastid space (Curtis et al. 2012; Fig. 9.12). The cryptophyte chloroplast has a phycobiliprotein with a β subunit arising from phycoerytrin, and an RbcL gene of proteobacterial origin, both traits pointing to a red algal origin of the organelle, which is confirmed by phylogenomic analysis of the nucleomorph genome. Unlike red algae, cryptophyte phycobiliproteins do not form phycobilisomes but instead are located in the thylakoid lumen, and their α subunit is encoded by a nuclear gene (phycobiliproteins in red algae are entirely encoded by plastid genes). The chloroplast envelope consists of four membranes in Haptophyta, Stramenopila and photosynthetic Chrompodellida, only three membranes in Dinophyta, and in none of these lineages is present a nucleomorph. All except the Chrompodellida possess chlorophyll c, a form of chlorophyll also present in Cryptophyta but absent in the Archaeplastida and green algal-derived secondary chloroplasts (Table 9.1). The lack of a nucleomorph in most secondary chloroplasts reflects complete obliteration of the photobiont nucleus following the transfer of all relevant genes to the host nucleus. Gene transfer from the photobiont nucleus to the host nucleus

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Fig. 9.12 The chlorarachniophyte Bigellowiella natans (Cercozoa, Rhizaria) and the cryptophyte Guillardia theta have a secondary chloroplast derived from a green and red alga, respectively. In both cases, a relict nucleus named nucleomorph (NM) persists in the periplastid space. The cryptophytes and chlorarachniophytes, therefore, carry four genomes (nuclear, mitochondrial, nucleomorphic and plastidial) in their cells. The numbers refer to protein-encoding genes located in each compartment. The outermost plastid envelope membrane in cryptophytes is continuous with the host nuclear envelope and its surface is studded with ribosomes, which co-translationally insert nucleus-encoded, chloroplast-targeted proteins. C storage carbohydrate, MT mitochondrion, PY pyrenoid. (Redrawn from Curtis et al. 2012)

blurred the phylogenetic signal, with the consequence that phylogenetic analysis of lineages with a secondary chloroplast is particularly problematic. Unlike nuclear genes, very few genes were transferred from the photobiont chloroplast to the secondary-host nucleus (Dorrell and Howe 2012); this facilitates the reconstruction of phylogenetic interrelationships of secondary chloroplasts. The conversion of a eukaryotic symbiont into a chloroplast required novel systems mediating the translocation of proteins, phosphate, photosynthesis products and other metabolites across the additional membranes interposed between the chloroplast and the secondary-host cytoplasm. Secondary chloroplasts generally have four surrounding membranes (Fig. 9.11), of which the two innermost ones are homologous with the original envelope membranes and contain the TOC-TIC complex.

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Table 9.1 Photosynthetic pigments and chloroplast traits in different lineages of photosynthetic eukaryotes. Chlorophyll c, with the two variants c1 and c2, differs from the other forms of chloroplyll for the absence of the phytol chain and for a double bond between C17 and C18 in the tetrapyrrole ring. Among the Stramenopila, chlorophyll c is lacking in the Eustigmatophyceae and in a member of the Xantophyceae. Phycobiliproteins from the various groups have different spectral properties. Carotenoids reported in parentheses are specific to the group. For further information on taxonomic distribution of photosynthetic pigments, see Adl et al. (2012) and Silar (2016). For further details on storage polysaccharides, see Sect. 9.7

Taxonomic group Rhodophyta

Chloroplast envelope membranes 2

Nucleomorph Absent

Glaucophyta

2

Absent

Viridiplantae Chlorarachniophyta Euglenophyta

2 4 3

Absent Present Absent

Cryptophyta

4

Present

Haptophyta

4

Absent

Stramenopila (Heterokonta) Dinophyta (dinoflagellates) Chrompodellida

4

Absent

3

Absent

4

Absent

Apicomplexa

4

Absent

Photosynthetic pigments Chl a, phycobiliproteins (allophycocyanin, R-phycocyanin, phycoerythrin) in phycobilisomes, carotenoids Chl a, phycobiliproteins (C-phycocyanin and allophycocyanin) in phycobilisomes, carotenoids Chl a, Chl b, carotenoids Chl a, Chl b, carotenoids Chl a, Chl b, carotenoids (diadinoxanthin) Chl a, Chl c2, unique phycobiliproteins in thylakoid lumen, carotenoids (alloxanthin) Chl a, Chl c1, Chl c2, carotenoids (fucoxanthin) Chl a, Chl c1, Chl c2, carotenoids (fucoxanthin) Chl a, Chl c2, carotenoids (peridinin); variants from tertiary symbioses Chl a, carotenoids (Chromera velia: isofucoxanthin; Vitrella brassicaformis: vaucheriaxanthin) Absent

The epiplastid membrane in Cryptophyta, Stramenopila and Haptophyta is associated with ribosomes and is cytologically a part of the endomembrane system of the secondary host. In these algae, protein translocation across the epiplastid membrane follows the Sec pathway, whereas the Der complex (also called SELMA, from Symbiont ER-like Machinery) mediates protein translocation through the periplastid membrane (Bodył et al. 2009a; Bodył 2018; Fig. 9.13). The epiplastid membrane in chlorarachniophytes is not associated with ribosomes, thus protein translocation from the cytosol to periplastid space probably follows a vesicular pathway. The same applies to the secondary chloroplasts in the euglenophytes and dinoflagellates, both surrounded by three, not four membranes (see below).

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Fig. 9.13 The current knowledge of protein import in secondary chloroplasts is incomplete. The model shown is relative to secondary chloroplasts in Cryptophyta, Stramenopila and Haptophyta, where the epiplastid membrane is integrated in the host ER system and is associated with host ribosomes. Chloroplast proteins encoded by nuclear genes carry bipartite pre-sequences composed of a signal peptide (yellow) followed by a transit peptide (blue). The signal peptide emerges first from the ribosome and binds to a cytoplasmic protein known as the Signal Recognition Particle (SRP); this interaction temporarily stops protein synthesis until the SRP-ribosome complex binds to a Sec complex inserted in the epiplastid membrane (or other ER membranes). When this happens, the ribosome resumes protein synthesis and the elongating protein is translocated to the epiplastid space, where a peptidase cleaves the signal peptide. Once in the epiplastid space, proteins directed to the inside of the chloroplast can cross the periplastid membrane through two possible pathways. The first (left) involves the Der complex (also called SELMA, from Symbiont ER-like Machinery), a translocon located in the periplastid membrane and derived from components of the photobiont endomembrane system, followed by the TOC-TIC complex. In the second pathway (right), proteins cross the periplastid membrane and the outer envelope membrane using a pinocytotic pathway expressed by the photobiont, and then enter the chloroplast stroma using only the TIC translocon. In both cases, a second peptidase removes the transit peptide. Other tag peptides or transmembrane domains (not shown) mediate protein transfer to the thylakoid membrane or thylakoid lumen as in the primary chloroplast. (Adapted from Bodył et al. 2009a, licence number 4457711398644)

Gould et al. (2015) have challenged the widespread assumption that the epiplastid membrane of secondary chloroplasts derives from the host phagosomal membrane and the periplastid membrane from the plasma membrane of the eukaryotic photobiont. In their scenario, the host phagosomal membrane and symbiont plasma membrane were both lost, and the epi- and periplastid membrane derive from a sheet

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of endoplasmic reticulum that enclosed the secondary chloroplast at an early stage of evolution. This hypothesis finds support in the association of ribosomes with the epiplastid membrane in the Cryptophyta, Stramenopila and Haptophyta. The scenario becomes more complicated in the euglenophytes and dinophytes because the secondary chloroplast in these lineages has only three envelope membranes. To explain this trait, it has been suggested that either lineage acquired the chloroplast by myzocytosis (Keeling 2010; Bodył 2018); this is a type of phagocytosis in which a phagotrophic predator penetrates the prey cell with a straw-like feeding tube and sucks the cellular content or a part of it, leaving out the prey plasma membrane. If so, the outermost chloroplast membrane of Euglenophyta and Dinophyta would derive from the host phagosomal membrane.

9.5

Phylogeny of Eukaryotes with Secondary Chloroplasts

The first phylogenomic data in the 1990s revealed that the chloroplasts in Cryptophyta, Haptophyta, Stramenopila and Dinophyta (Alveolata) are related with the chloroplast in red algae. Based on the assumption that the evolution of a secondary chloroplast were an extremely complex and unlikely event, CavalierSmith (1999) hypothesized that a chlorophyll c-containing chloroplast (also referred to as the “brown chloroplast” in the following text) evolved only once and was then transmitted vertically. This model maintains that the Cryptophyta, Haptophyta, Stramenopila and Alveolata share a photosynthetic ancestor provided with a chlorophyll c-containing secondary chloroplast of red algal ancestry, thus forming a clade named “Chromoalveolata” (Cavalier-Smith 2003; Sanchez-Puerta and Delwiche 2008; Keeling 2009; Archibald 2015). Each of the above lineages includes or is phyletically related to non-photosynthetic taxa, for example the Oomycota in the Stramenopila, the Ciliophora (ciliates) and Apicomplexa in the Alveolata, the Centrohelida with the Haptophyta in the Haptista (Burki et al. 2016; Cavalier-Smith et al. 2015), and the Katablepharidae with the Cryptophyta in the Cryptista (Cavalier-Smith et al. 2015; Silar 2016). In order to account for this taxonomical distribution, the chromoalveolate model assumes that non-photosynthetic lineages arose secondarily due to the loss of the chloroplast and/or photosynthesis. Indeed, there are several well-documented instances of heterotrophic eukaryotes derived from photosynthetic progenitors; some of these, for example the Apicomplexa and some colourless euglenophytes, retain a non-photosynthetic plastid that clearly documents a photosynthetic ancestry. In others cases, for example in some dinoflagellates, the chloroplast was completely lost, yet genes of plastid origin were retained in the nuclear genome (Dorrell and Howe 2015). Although highly controversial since its formulation, the chromoalveolate hypothesis has received important support from phylogenomic analysis of plastid DNA demonstrating that chlorophyll c-containing chloroplasts are monophyletic and derive from a red alga nested in the Rhodophytina (Sanchez-Puerta et al. 2007; Janouškovec et al. 2010; Stiller et al. 2014; Fig. 9.14). Assuming that non-

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Fig. 9.14 Phylogenomic analysis of plastid DNA shows that (a) all chloroplasts derive from the same ancestral cyanobacterium; (b) brown chloroplasts form a monophyletic group that is sister to the chloroplast in a red algal lineage including Chondrus. (Redrawn from Stiller et al. 2014)

photosynthetic chromoalveolate lineages descend from a photosynthetic progenitor, the chromoalveolate model predicts that these lineages retain plastid genes in nuclear genomes. Apart from taxa obviously derived from photosynthetic ancestors such as the Apicomplexa, this prevision has received confirmation in ciliates (Reyes-Prieto et al. 2008). In contrast, no plastid genes were found in the nuclear genome of two oomycetes (Stiller et al. 2009). Phylogenomic analysis of plastid sequences strongly supports the Cryptophyta, Alveolata, Stramenopila and Haptophyta as a clade (known as the CASH clade), thus confirming brown chloroplast monophyly, yet no significant support for CASH monophyly was obtained from nuclear or mitochondrial sequences (Baurain et al. 2010; Archibald 2015). In line with this result, phylogenomic analysis of mitochondrial and nuclear sequences has shown that the Stramenopila and Alveolata are more closely related with the Rhizaria (with which they form the SAR clade; Chap. 6) than with the Haptophytes and Cryptophytes (Burki 2014; Burki et al. 2012, 2016). To accommodate this topology in the chromoalveolate model, it is necessary to assume that the Rhizaria derive from a photosynthetic ancestor. This is unparsimonious, considering that the Rhizaria is a non-photosynthetic lineage with the only exceptions of the Chloroarachniophytes and Paulinella, which acquired a chloroplast independently (Rogers et al. 2007; Nowack 2014). A second problem with the chromoalveolate model is that phylogenomic analysis based on mitochondrial and nuclear sequences consistently places non-photosynthetic stramenopiles (Oomycota, Bicosoecida, Labyrinthulida, Blastocystis) in a basal position relative to their photosynthetic relatives (brown algae, diatoms, Xantophyceae, Chrysophyceae, Eustigmatophyceae), the latter clustering together in a derived clade named Ochrophyta (Burki 2014; Silar 2016; Simpson et al. 2017). Within the Alveolata, the Ciliophora (ciliates) are basal to Dinophyta,

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Apicomplexa and Chrompodellida, the latter three forming a primarily photosynthetic clade named “Myzozoa” (Cavalier-Smith and Chao 2004; Reeb et al. 2009; Bachvaroff et al. 2011). Cryptophyte nuclear sequences were detected in the nuclear genome of the photosynthetic stramenopiles Phaeodactylum and Aureococcus but not in non-photosynthetic relatives Phytophthora and Blastocystis (Stiller et al. 2014), a condition at odd with the chromoalveolate model and better explained by serial symbiosis (see below). A third inconsistency of the chromoalveolate model is the well-known fact that chlorophyll c-containing photosynthetic eukaryotes use storage polysaccharides with varying biochemistry and cytology (Table 9.2). If the brown chloroplast had Table 9.2 Storage polysaccharides in eukaryotes. Glycogen and starch are α-(1–4) glucans; paramylon is a β-(1–3) glucan; laminarin, chrysolaminarin and mycolaminarin are β-(1–3) glucans with (1–6)-linked glucan side chains and varying substituents; mannan is a β-(1–4) polymer of mannose. Glycogen is the main storage polysaccharide in the Amorphea (Amebozoa + Opistokonta) and Metamonada, and is probably the ancestral storage polysaccharide of eukaryotes. Glycogen is also the main storage polysaccharide in bacteria Taxa Amoebozoa Opistokonta Chlorachniophytes

Storage polysaccharide Glycogen Glycogen Paramylon

Cercomonads Foraminifers Ciliates Dinoflagellates Apicomplexa Chrompodellida Glaucophytes Rhodophytes Green plants Oomycetes

Glycogen? Glycogen? Glycogen Starch Starch Starch Starch Starch Starch Mycolaminarin

Diatoms

Chrysolaminarin

Brown algae

Laminarin

Haptista

Haptophytes

Chrysolaminarin

Cryptista Excavates

Cryptophytes Euglenophytes

Starch Paramylon

Heterolobosea Kinetoplastids Metamonads

Glycogen? Mannan Glycogen

Amorphea Rhizaria

Alveolates

Plants

Stramenopiles

Cellular localization Cytosol Cytosol Cytoplasmic membrane-bound vesicles

Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Chloroplast stroma Cytoplasmic membrane-bound vesicles Cytoplasmic membrane-bound vesicles Cytoplasmic membrane-bound vesicles Cytoplasmic membrane-bound vesicles Periplastid space Cytoplasmic membrane-bound vesicles Unknown Cytosol

Data from Myklestad and Granum (2009), Stone (2009), and Ball et al. (2011)

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been inherited vertically from a common progenitor, the same type of storage polysaccharide should be found throughout the chromoalveolates (Sect. 9.8). Because of difficulty in reconciling the chromoalveolate scenario with molecular phylogenies and information from other sources, serial symbiosis hypotheses are currently receiving attention (Bodył et al. 2009b; Stiller et al. 2014; Bodył 2018). The serial scenario posits that a brown chloroplast evolved once from a red algal symbiont (in accordance with plastome phylogenomics pointing to chloroplast monophyly in chromoalveolates), and was then transmitted horizontally to other lineages through multiple endosymbiosis events. Figure 9.15 illustrates the chromoalveolate model and a model of serial horizontal transmission. In conclusion, current insight indicates that the brown chloroplast evolved only once from an enslaved red alga, yet it is to be ascertained whether this secondary chloroplast was transmitted vertically (by direct descent) or horizontally (by serial symbiosis) to extant chlorophyll c-containing lineages. Given the current taxonomic composition of the Haptista, Cryptista and SAR clades, it is not obvious whether an ancient endosymbiosis followed by multiple independent losses is more parsimonious than a scenario where a brown chloroplast evolved once and was then independently transmitted to several lineages. The serial symbiosis scenario does not

Fig. 9.15 (a) According to the chromoalveolate model, a phagotrophic eukaryote evolved a secondary chloroplast from a red alga and then diversified, giving rise to the various chlorophyll c-containing lineages and, with the loss of the chloroplast, to their non-photosynthetic relatives (e.g. the Ciliophora and Oomycota). (b) According to serial endosymbiosis models, a chloroplast derived by secondary symbiosis from a red alga was horizontally transferred to the progenitors of photosynthetic “chromoalveolate” lineages several times independently. The model presented proposes that a chlorophyll c-containing chloroplast first evolved in cryptophytes; a cryptophyte was then converted into a chloroplast in the progenitor of photosynthetic Stramenopila by tertiary endosymbiosis, and eventually a stramenopile was converted into the chloroplast of haptophytes by quaternary endosymbiosis. The ancestor of Myzozoa (dinoflagellates, Apicomplexa and Chrompodellida) might have horizontally acquired the chloroplast from a haptophyte or a stramenopile (not shown). (a Redrawn from Archibald and Keeling 2002; b Redrawn from Stiller et al. 2014)

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exclude the possibility that, in some lineages, heterotrophic forms are derived and photosynthetic ones primitive. This is most likely the case for Chrompodellida, a group of alveolates whose non-photosynthetic members retain numerous nuclear genes of plastid origin. The Apicomplexa, the sister group of Chrompodellida, lost photosynthesis but most of its members including the malaria agent Plasmodium retain a non-photosynthetic plastid. Importantly, the ciliates have numerous plastid genes in their nuclear genome (Reyes-Prieto et al. 2008; Slamovits and Keeling 2008), which may witness either a photosynthetic ancestry as assumed by the chromoalveolata model or simple horizontal gene transfer. The discovery that the nuclear genome of chromoalvelolate lineages contains plastid genes of green-algal ancestry adds further complexity to the topic; these genes include green-algal forms of phosphoribulokinase, chlorophyll a synthase, ferredoxin-NADPH reductase, chloroplast signal processing peptidase, the ammonium transporter AMT1, and five enzymes involved in carotenoid biosynthesis (Dorrell and Smith 2011). The green-algal phylogenomic signal found in chromoalveolates is suggestive of a cryptic symbiosis with a green alga (possibly a member of the Prasinophytes) predating the acquisition of a brown chloroplast. In other words, it is possible that the progenitor of chromoalveolates possessed a chloroplast of green algal origin, which was lost at some stage in evolution, but left signs of its past existence in the form of genes in nuclear genome that were subsequently deployed for the maintenance of a brown chloroplast of red algal ancestry. If necessary, this alone would justify the quotation from Keeling (2003) at the head of this chapter. In the light of what is currently known about chloroplast/nucleo-cytoplasm interactions, it is evident that the conversion of a photosynthetic eukaryote into a chloroplast is a more simple process than the evolution of a chloroplast from a cyanobacterium. The conversion of a photosynthetic eukaryote into a chloroplast, in fact, only requires the transfer of the necessary gene complement from the symbiont nucleus to the host nucleus, and protein targeting for translocation through additional membranes. The conversion of a cyanobacterium into a well-integrated organelle entails much longer and more complex evolutionary “work”. Next section will present several instances of eukaryotic endosymbionts at different stages of conversion into chloroplasts.

9.6

Chloroplast Thieves and Other Stories

Many eukaryotes are able to extract chloroplasts from algae and to retain them in their cells in a functional condition for a variable length of time, obtaining organic matter from photosynthesis. These chloroplasts are sometimes called kleptoplasts from Greek kleptein for stealing (Serôdio et al. 2014). A substantial difference between true chloroplasts and kleptoplasts is that the first are stable organelles that divide synchronously with the cells and are transmitted vertically, whereas the latter have a limited lifetime and must be replaced continuously. As predictable, this type

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297

of interaction encompasses a great diversity in the type of host, the source of chloroplasts and the degree of cellular integration. A classic example is found in ascoglossan molluscs of the genus Elysia, which extract chloroplasts from the xanthophycean alga Vaucheria litorea and host them in specialized cells of the intestinal epithelium. Here the chloroplasts remain functional for a time ranging from several hours to about 8 months, depending on the species, and supply the animal with products of photosynthesis (Yellowlees et al. 2008; Serôdio et al. 2014). The ability of some species such as Elysia chlorotica to maintain functional chloroplasts for a long time finds an explanation in their nuclear genome containing chloroplast genes obtained from Vaucheria by horizontal gene transfer (Pierce et al. 2009). Elysia chlorotica feeds on Vaucheria only occasionally, probably when it needs to replace an aged chloroplast pool; these animals become competent to host chloroplasts soon after larval metamorphosis, when they start feeding on Vaucheria. The chloroplasts not only supply the animal host with sugar but also provide organic nitrogen by assimilating ammonium, nitrite and urea (Teugels et al. 2008). Rather than in animals, however, the most impressive examples of chloroplast horizontal transfer are found in unicellular eukaryotes, where they range from relatively labile associations to highly integrated systems in which the photobiont has become a stable organelle. The freshwater ciliate Paramecium bursaria hosts hundreds of green-algal endosymbionts of the genus Chlorella (Fig. 9.16); these supply the host with photosynthetically-fixed carbon mainly in the form of maltose, whilst the host Fig. 9.16 The ciliate Paramecium bursaria hosts hundreds of Chlorella cells within individual vacuoles. Organic matter from the algal photobiont permits the ciliate to live autotrophically. Credit: http://shannenpscientist5. blogspot.it/2008/02/ paramecium-bursaria.html

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provides algal cells with nitrogen compounds and protection from other phagotrophs. The alga is vertically transmitted to daughter cells upon cell division. Treatment with the photosynthesis inhibitor DCMU or continuous darkness cause the loss of the symbiont; colour-less Paramecium cells can still grow heterotrophically by phagocytosis, yet they promptly reacquire the symbiont and re-establish a phototrophic lifestyle if returned to a normal light/dark cycle (Nowack and Melkonian 2010). A second remarkable example is Hatena arenicola, a member of the Katablepharidae, phagotrophic flagellates that molecular analysis places in the Cryptista. In natural populations, most H. arenicola cells harbour a photosynthetic endosymbiont identified as a member of the green algal genus Nephroselmis. The symbiont is bound by a single membrane, probably of phagosomal origin. When Hatena cells divide, only one daughter cell receives the symbiont, the other living heterotrophically until it engulfs a new Nephroselmis cell from the environment (Okamoto and Inouye 2006). Interestingly, colourless H. arenicola cells have a complex apical feeding apparatus that degenerates soon after the establishment of the symbiosis (Fig. 9.17). A third remarkable example of chloroplast theft is found in Myrionecta rubra (¼ Mesodinium rubrum), a ciliate harbouring cells of the cryptophyte Geminigera cryophila, each containing a single chloroplast. The host supplies the endosymbiont with essential nutrients such as ammonia, obtaining sugar in exchange. The endosymbiont divides within the host and is transmitted vertically across several cell generations. In some cases, the ciliate retains only parts of cryptophyte cells, notably the nucleus and chloroplast-containing cytoplasmic masses, all enclosed in a membrane of phagosomal origin (Fig. 9.19). The association is highly efficient, enabling

Fig. 9.17 (a) Diagrammatic representation of Hatena arenicola, a katablepharid flagellate hosting a green algal endosymbiont of the genus Nephroselmis (outlined by dotted red line). (b) When the host cell divides, only one daughter cell receives the symbiont. The symbiont-free cell reconstitutes the symbiosis by ingesting a Nephroselmis cell from the environment. Free-living Nephroselmis cells are about 10 μm in length; after being captured, their chloroplast enlarges to about tenfold its original size, whereas other cell components are degraded to various extents. (b, Redrawn from Nowack and Melkonian 2010)

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Fig. 9.18 Evolution of dinoflagellates and their plastids. Primarily provided with a chloroplast derived from a red algal endosymbiont and containing peridinin, dinoflagellates have lost photosynthesis several times independently, in some cases retaining a non-photosynthetic plastid, in others losing the chloroplast entirely but retaining plastid genes in the nuclear genome. Some dinoflagellate lineages have reacquired a chloroplast from various sources, establishing varying levels of cellular integration. Fucoxanthin-containing chloroplasts of haptophyte origin have three bounding membranes and do not retain any other cellular component of the original endosymbiont. Diatom-derived chloroplasts are probably polyphyletic and are surrounded by the four original membranes plus a fifth membrane of uncertain origin; diatom cellular structures including the nucleus and mitochondria persist in the space between the chloroplast and the fifth membrane. Lepidodinium chloroplasts descend from a green algal photobiont and are bounded by four membranes, of which the two innermost ones are the original envelope membranes, the third is the plasma membrane of the photobiont and the fourth is probably a phagosomal membrane. Greenalgal cellular remnants persist between the second and third membrane but do not include a nucleus or mitochondria. The phyletic tree shown in the figure resolves dinoflagellates as the sister group of the Apicomplexa-Chrompodellida clade. (Based on Dorrell and Howe 2015)

the ciliate to grow autotrophically and even produce massive blooms in coastal areas (Nowack and Melkonian 2010; Qiu et al. 2016). Even more advanced are symbiotic associations detected in some dinoflagellates. Dinoflagellates are a primarily photosynthetic alveolate lineage, but encompass forms that lost either the whole chloroplast or only photosynthesis, in the latter case retaining a non-photosynthetic plastid (Fig. 9.18). The chloroplast of photosynthetic dinoflagellates has a three-membrane envelope and contains a high concentration of peridinin (C39H50O7), a carotenoid with a peak absorbance in the bluegreen area (470–550 nm) of the electromagnetic spectrum. Besides “canonical” photosynthetic dinoflagellates, several species have replaced the original chloroplast with eukaryotic photosynthetic endosymbionts at various steps of cellular integration (Fig. 9.18). This is the case of Karenia and Karlodinium harbouring a haptophyte endosymbiont, Lepidodinium viride and Lepidodinium chlorophorum

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Fig. 9.19 Serial theft of chloroplasts. The cryptophyte Geminiphera cryophila, whose secondary chloroplast arises from a red alga, is exploited as a source of chloroplasts by the ciliate Myrionecta rubra. In order to ensure the maintenance of stolen chloroplasts (kleptoplasts), Myrionecta also harbours the nucleus of the cryptophyte. The dinoflagellate Dinophysis acuminata extracts the kleptoplasts from Myrionecta and maintains them in a functional state for some months. Numbers 1, 2, 3, 4 are the nucleus, chloroplast, nucleomorph and mitochondrion in a free Geminigera cell, respectively; 5 nucleus and 7 chloroplast and cytoplasmic remains of Geminigera within a Myrionecta cell; 6 nucleus and 8 mitochondrion of Myrionecta; 10 kleptoplast from Myrionecta in Dinophysis cell, plus 9 nucleus and 11 mitochondrion of Dinophysis. (Adapted from Wisecaver and Hackett 2010, Creative Commons Attribution License 2.0)

containing a green algal endosymbiont, and other species (Durinskia baltica, Kryptoperidinium foliaceum, Gymnodinium quadrilobatum, Peridiniopsis spp. and Peridinium quinquecorne) hosting diatom endosymbionts. In some diatomharbouring dinoflagellates, the original chloroplast was reduced to an eyespot apparatus, a sensorial structure that permits the microorganism to sense light direction and intensity (Park et al. 2014; Dorrell and Howe 2015). Numerous heterotrophic dinoflagellates are able to extract chloroplasts from photosynthetic eukaryotes. Amphidinium poecilochroum and Gymnodinium aeruginosum (¼ G. acidotum) obtain chloroplasts from cryptophyte cells (several in the former, only one in the latter) by myzocytosis (Sect. 9.3). These “kleptoplasts” retain the four original bounding membranes and lie within a fifth membrane of phagosomal origin; they have a limited lifetime and must be continuously replaced with fresh chloroplasts. A particularly interesting instance of serial chloroplast theft is Dinophysis, a dinoflagellate that extracts chloroplasts from Myrionecta by myzocytosis; as in a Russian nesting doll, Myrionecta chloroplasts derive from a cryptophyte, which in turn hosts a red alga-derived chloroplast (Kim and Archibald 2010; Fig. 9.19). The remarkable ability of non-photosynthetic dinoflagellates to obtain functional chloroplasts from a diversity of photosynthetic protists probably reflect the persistence in their nuclear genome of plastid genes inherited from their photosynthetic

9.7 Plastid Division

301

progenitors, which facilitate the integration of algal endosymbionts or temporal maintenance of kleptoplasts. Indeed, kleptoplastidy is so common among extant eukaryotes that it has been proposed as a possible mechanism for horizontal transmission of brown chloroplasts across chromoalveolate lineages (Bodył 2018).

9.7

Plastid Division

As for mitochondria, eukaryotic cells are unable to regenerate plastids the novo, although most of the genes necessary for making a plastid lie in the nuclear genome. It is therefore vital that plastids divide synchronously with the cell, in order to ensure vertical transmission of the organelles across cell generations. Chloroplasts divide using the plastid divisome, a complex molecular machinery located in part in the plastid stroma and in part in the cytosol. The stromal components of the plastid divisome are largely of cyanobacterial origin, whereas the cytosolic components are strictly eukaryotic (Chen et al. 2018). Independently of their cyanobacterial or eukaryotic ancestry, divisome proteins in land plants and most algae are all encoded by nuclear genes and synthesized on cytosolic ribosomes, with only a few algae retaining some that are plastid-encoded (Onuma et al. 2017). Divisome proteins synthesized in the cytosol are in part transferred to the plastid, where they assemble the stroma component, the rest remaining outside to build the cytosolic component. Plastid division requires not only the correct assembly of division components at the appropriate localization but also protein movement and contractile force for membrane constriction. Chloroplast and cell division are tightly linked in cells possessing a single plastid (many algae and, among land plants, some hornworts), whereas the two processes are not directly interlinked in multiplastidic cells. A major component of the chloroplast division machinery is FtsZ, a tubulin-like cytoskeletal protein derived from the cyanobacterial ancestor of chloroplasts and ubiquitous in bacteria (Rowlett and Margolin 2015). In both bacteria and chloroplasts, FtsZ assembles into a contractile “Z ring” that defines the division site. Most bacteria have only a single FtsZ gene; in contrast, the majority of photosynthetic eukaryotes encode two plastid-targeted forms of FtsZ, known as FtsZ1 and FtsZ2 in green plants or FtsZA and FtsZB in red algae. A third form, FtsZ3, has been found in a subset of photosynthetic eukaryotes, where its occurrence may be correlated with the retention of a chloroplast peptidoglycan wall (Yang et al. 2008; Basak and Møller 2013; Osteryoung and Pyke 2014; Chen et al. 2018). Whereas the basic mechanism of plastid division has been unravelled in some detail (Fig. 9.20), other aspects of the process remain poorly understood, for example how plastid and cell division are synchronized, how the cell controls plastid size, shape and number, how thylakoids divide, how other types of plastids besides chloroplasts divide. Plastid DNA is usually in the form of a single circular chromosome of about 120– 170 kb including two separate regions with the same sequence (except minor

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Fig. 9.20 The machinery responsible for plastid division, known as the plastid divisome, consists of a series of concentric structures shown in a three-dimensional perspective (a) and in section across its plane (b). The stroma contains the Z-ring, formed by two homologs of the bacterial protein FtsZ, known as FtsZ1 and FtsZ2 in green plants. ARC6, a homolog of the bacterial protein Ftn2, forms a ring inserted in the inner envelope membrane (IEM), which connects the Z-ring with a third ring located in the outer envelope membrane (OEM) and formed by the eukaryotic proteins PDV1 and PDV2. These bind to a cytosolic ring of ARC5, a protein belonging to the dynamin family. Dynamins are eukaryotic proteins that mediate membrane constriction and pinching off. The Z ring is confined to the mid-zone of the organelle by the chloroplast Min system, comprising ARC3, MCD1, MinD, MinE, which inhibits Z-ring assembly at non-division sites. The Z-ring mediates plastid constriction, whereas ARC5 mediates the final separation of daughter plastids. Mutants defective in a part of the machinery develop giant chloroplasts unable to divide. The model shown is still in part speculative and may require substantial integration in the near future

differences) but opposite orientations, known as the inverted repeats. These regions are highly conservative, their sequence being very similar in distantly-related taxa, and their presence in the plastid genome is thought to exert a stabilizing effect (Shaw et al. 2007). Inverted repeats also occur in cyanobacterial genomes, suggesting that they evolved before chloroplasts. Plastids usually contain several chromosome copies organized in separate nucleoids; this may account for the apparent absence of mechanisms controlling chromosome segregation during plastid division, contrasting with tight control in free-living bacteria (Toro and Shapiro 2010).

9.8

Storage Polysaccharides

Based on the type of storage polysaccharides, eukaryotes separate into two groups: those using α-polysaccharides and those using β-polysaccharides, the former deposited in the cytosol or in the plastid stromal compartment, the latter in membranebound cytoplasmic vesicles (Table 9.2). The main storage polysaccharide in bacteria, including cyanobacteria, is glycogen, a multibranched polymer made of α-(1–4) glucan chains with α-(1–6) bonds at branching points. Bacterial glycogen synthase produces α-(1–4) glucan chains using

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adenosine-diphosphate-glucose (ADPG) as the sugar donor. Glycogen is also the main storage polysaccharide in eukaryotic plastid-less lineages, notably the Amorphea and Metamonada (Excavata), and is probably the ancestral storage polysaccharide in the eukaryote domain (Ball et al. 2011). In contrast to bacteria, eukaryotic glycogen synthase uses uridine-diphosphate-glucose (UDPG) as a sugar donor. Bacterial and eukaryotic glycogen synthases also differ in the mechanism of action and have no recognizable sequence homologies. Besides the main enzyme, glycogen synthesis requires other enzymes for the production of sugar donors (ADPG or UDPG), the insertion of branches (α-1,4-glucan-6-glucosyltransferases, or branching enzymes) and final polymer processing (isoamylases, or de-branching enzymes). In photosynthetic eukaryotes, the storage of surplus photosynthate has taken a range of alternatives in both primary and secondary endosymbiotic partnerships. The main storage polysaccharide in Archaeplastida is starch, which consists of varying proportions of two types of polymer, amylose and amylopectin. Amylose is made of chains of α-(1–4)-glucose with very few or no branches, amylopectin instead presents a branch for every 24–30 glucose units (in glycogen there is a branch for every 8–12 glucose units). Amylose is denser than amylopectin, less prone to enzymatic hydrolysis and insoluble in water. The α-(1–6)-linked branches are regularly distributed in glycogen, whereas they are concentrated in certain areas of the polymer in amylopectin, where they tend to form double helical structures held together by hydrogen bonds. Differences in branch distribution affect the growth capacity of the two types of polymer. Because of the even distribution of lateral branches, glycogen growth is sterically hindered, i.e. there is not sufficient space for interaction of the chains with glycogen-building enzymes when the granule attains about 40 nm in diameter. Glycogen is thus present in cells in the form of amorphous granules of these limit size, with smaller granules representing intermediate states of glycogen synthesis or degradation. In contrast, amylopectin synthesis is not subject to steric hindrance, so the polymer forms granules of indeterminate size, consisting of alternating amorphous and crystalline layers (Ball et al. 2011). Starch synthesis in Archaeplastida involves a mosaic of enzymes derived in part from the cyanobacterium, in part from the eukaryote host. Especially important among these are isoamylases, a class of enzymes of cyanobacterial ancestry that remove a part of the branches introduced by branching enzymes, thus enabling the polymer to assume the compact semi crystalline conformation typical of starch; Chlamydomonas mutants defective in isoamylase activity produce glycogen instead of starch in their chloroplasts (Patron and Keeling 2005). Starch is a mixture of amylose and amylopectin in Viridiplantae, whereas it is generally made of amylopectin alone and is known as floridean starch in Rhodophytes (Patron and Keeling 2005). The Viridiplantae have several isoforms of starch synthase, all encoded by nuclear genes and targeted to plastids. These enzymes are bound to the starch granule during synthesis, thus they are referred to as granule-bound starch synthases; they use ADPG as the sugar donor and at least one is specialized in amylose synthesis. The Rhodophytes have two starch synthase isoforms, both encoded by nuclear genes, both cytosolic and both dependent on

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UDPG; one isoform binds to the starch granule whereas the other is soluble and is related to the glycogen synthase of primarily heterotrophic eukaryotes, e.g. the animals. Starch is synthesized in the chloroplast stroma in Viridiplantae, in the cytosol in Rhodophytes. The Glaucophytes have a similar starch pathway as in Rhodophytes. Plastidial starch is an exception rather than a rule among the diversity of starchstoring eukaryotes. Other lineages that use starch as a storage polysaccharadise besides the Archaeplastida are the Cryptophytes, Dinoflagellates and Apicomplexa. Starch localization in the periplastid space (Fig. 9.11) in Cryptophytes is consistent with homology of this compartment with the cytoplasm of the red algal ancestor of the cryptophyte chloroplast. In contrast, starch is deposited in the cytosol of the secondary host in Dinoflagellates and Apicomplexa, suggesting transfer of starch metabolism genes from the red algal nucleus to the host nucleus (Coppin et al. 2005; Deschamps et al. 2008c). As in red algae, starch synthase in these lineages has a marked preference for UDPG, but at least in some cases it is also able to use ADPG. Despite substrate differences, all granule-bound starch synthases in starch-storing lineages so far investigated have a clear cyanobacterial ancestry (Patron and Keeling 2005; Deschamps et al. 2008a, b). Because starch is absent in primarily heterotrophic eukaryotes (Ball et al. 2011), the occurrence of this polysaccharide in the Archaeplastida (and in a part of the chromoalvelolate assemblage) suggests an origin from integration of host and endosymbiont pathways at an early stage of evolution of the primary chloroplast. Based on evidence of starch synthesis in Group V nitrogen-fixing cyanobacteria, a lineage probably encompassing or akin to the cyanobacterial ancestor of the primary chloroplast, Deschamps et al. (2008a, b) suggest that the enzyme set necessary for starch synthesis was already in place in the original cyanobiont and that an initial step of host/endosymbiont integration was the transfer of starch synthesis to the host cytosol. Exactly how this occurred is not clear. Figure 9.21 presents a simplified scenario considering only a part of the enzymes involved. The insertion of a host carrier in the cyanobiont inner membrane permitted the host to obtain ADP-glucose from the cyanobiont; the transfer of genes for starch synthase and isoamylase from the cyanobiont to the host nucleus enabled the host to synthesize starch in the cytosol, replacing UDPG-dependent glycogen synthesis, thus attaining the condition found in red algae. It is not clear whether starch synthesis in red algae depends on a starch synthase of cyanobacterial or eukaryotic origin or a cooperation of both enzymes. The presence of amylose in Porphyridium and of a glycogen-like polysaccharide in Cyanidium (Shimonaga et al. 2007) suggests that the red algal lineage retained both enzymes. The starch pathway was rewired to the chloroplast in the green lineage by adding stroma transit peptides to nucleus-encoded enzymes. This change was possibly necessary to facilitate energy dissipation and offset increased risk of photochemical damage due to chlorophyll-rich antennas replacing phycobilisomes (Sect. 4.2) and adaptation to high light intensities (Deschamps et al. 2008b). In Euglenophyta and Chlorarachniophyta, membrane-bound cytoplasmic β-glucan (paramylon) replaced starch in the chloroplast (in both cases of greenalgal ancestry).

9.8 Storage Polysaccharides

305

Fig. 9.21 A simplified scenario for the evolution of starch in the Archaeplastida. (a) Cyanobiont/ heterotrophic host at an early stage of chloroplast evolution: the cyanobiont (Cy) produced starch using ADPG-dependent starch synthase (SS); the host produced glycogen in the cytosol, using UDPG-dependent glycogen synthase (GS). (b) Common ancestor of Archaeplastida: targeting of a sugar nucleotide translocator (SNT) of host origin to the inner chloroplast membrane permitted the host to obtain sugar-nucleotides in exchange for AMP and stopped starch synthesis in the nascent chloroplast (Ch); at this stage, the host was still producing glycogen in the cytosol. (c) Ancestor of Rhodophytes and Glaucophytes: chloroplast genes for isoamylase (ISO) and starch synthase (SS) were transferred to the host nucleus (arrows), permitting the host to produce starch in the cytosol using ADP-glucose from the chloroplast. (d) Ancestor of Viridiplantae: addition of stroma transit peptides to starch synthase (SS), isoamylase (ISA) and host branching enzyme (BE) (dotted arrows) relocated starch synthesis from the cytosol to the chloroplast. A possible explanation for this transition was the evolution of a novel type of light-harvesting complex replacing phycobilisomes (see main text). ADPG ADP-glucose, UDPG UDP-glucose, AGPase and UGPase ADP-glucose and UDP-glucose pyrophosphatase. (Based on Patron and Keeling 2005; Deschamps et al. 2008a, b; Ball et al. 2011)

Laminarin, the storage polysaccharide in Stramenopila, is a polymer of β-(1–3) glucan trimers bound to each other by β-(1–6) bonds and bearing a mannitol unit bound to the free C1 of a terminal glucose unit. Chrysolaminarin, present in some stramenopile lineages (e.g. Chrysophyta) and in the Haptophyta, has the same basic structure as laminarin but lacks the mannitol residue. Laminarin is found in both photosynthetic (e.g. brown algae and diatoms) and non-photosynthetic forms (e.g. the Oomycota, where it is known as “mycolaminarin”), thus raising the question whether this polysaccharide appeared in a common progenitor before the acquisition of the chloroplast (Michel et al. 2010).

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Chapter 10

The Animals

A living creature is always in the business of surviving in its own environment. It is never unfinished – or, in another sense, it is always unfinished. (Richard Dawkins 2004)

Abstract The transition from Proterozoic to Phanerozoic (542 MYA) has been traditionally associated with the appearance of animals in the fossil record; paleontological evidence currently antedates this event to at least 565 MYA. Novel phylogenetic inference challenges traditional phylogeny by substituting the ctenophores for the sponges at the base of the animal tree, implying that fundamental innovations such as the intestine and neurons either evolved independently in the ctenophores and cnidarian/bilateria, or were present in a common ancestor and were lost in the sponges. In line with a “reductive” scenario, old and novel evidence suggests that the absence of a canonical mesoderm in the Cnidaria is a derived character. The evolution of an intestine enabled the animals to switch from phagotrophy, a form of predation necessarily restricted to unicellular prey, to macrotrophy, predation of multicellular organisms. Thus, the intestine was a fundamental innovation that paved the way to the evolution of most other animal traits. The diffusion of animals in Cambrian oceans enhanced organic carbon sequestration at the ocean bottom due to the sinking of carcasses and faeces, thus probably contributing to coeval rise in oxygen concentration. The diffusion of filter-feeding animals reduced the bacterial component of phytoplankton and favoured larger-celled eukaryotic phytoplankton, causing a shift from the stratified, turbid and partly anoxic Proterozoic ocean to a clear-water Palaeozoic ocean dominated by eukaryotic algae. Likewise, predation by animals was a powerful driver of macroalgal evolution and deeply influenced the evolutionary trajectory of land plants.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_10

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10

The Animals

Introduction

The animals form the largest eukaryotic lineage, with nearly a million and a half species described and probably as many yet to be identified. Until the 1950s, the earliest macrofossils unambiguously interpreted as animals were from sedimentary rocks dated 542 MY, so this date was chosen to mark the transition from the Proterozoic (the eon of microscopic life) to Phanerozoic (the eon of life visible to the naked eye, Fig. 10.1). Fossil evidence documents the appearance of a great number of novel forms including representatives of most extant animal phyla in the relatively short time interval between 542 and 515 MYA, an event known as the “Cambrian explosion”. The richest sources of Cambrian animal fossils are the Chengjiang Fossil Site (China), and the Burgess Shale formation (British Columbia, Canada), with an age of about 530 and 508 MY, respectively (Gaidos et al. 2007) Starting in the 1950s, an increasing number of fossils displaying a differentiated multicellular construction and sizes ranging from millimetres to metres were discovered in sedimentary rocks from about 600 MYA to the onset of Cambrian. Most of these forms show no affinity to any known animal phylum, so they have been tentatively classified as a unique and extinct clade in the new kingdom Vendozoa, or Vendobionta, after the now-obsolete Vendian era. Particularly conspicuous among pre-Cambrian fossils is Dickinsonia costata, known from imprints and casts in sandstone beds dated to about 555 MYA (Fig. 10.2). Ranging from a few millimetres to nearly 1.4 m in length, and from less than one to a few mm in thickness, Dickinsonia lacks any convincing evidence for a mouth, anus or gut. This organism might have been able of slow movements and probably fed by absorption or phagocytosis on its bottom surface. In the 1960s, the ensemble of macroscopic forms predating the Cambrian started being called the “Ediacaran fauna”, after the Ediacaran Hills in South Australia where a part of the fossils had been detected. This denomination was more recently changed into Ediacaran biota (Xiao and Laflamme 2008). In 2004, the International Union of Geological Sciences formally named “Ediacaran” the last period of the Proterozoic. Some Ediacaran organisms display a suite of traits that has prompted interpretation as possible early forms of extant animal phyla, e.g. sponges (Poriphera), comb jellies (Ctenophora) or jellyfish (Cnidaria). According to even less restrictive interpretations, the Ediacaran biota would also include animals with a true bilateral symmetry (Peterson et al. 2008). In line with this position, molecular dating places animal origin in the interval 850-650 MYA (dos Reis et al. 2015). A more conservative reading of the fossil evidence suggests that the earliest animals emerged at least 565 MYA, thus predating the Cambrian explosion (Cunningham et al. 2017), a conclusion in substantial agreement with the timescale by Benton et al. (2015). The recognition of a pre-Cambrian animal evolutionary radiation helps explain the sudden appearance of a multitude of novel forms in the Cambrian, some of which of remarkable morphological complexity.

10.1

Introduction

Fig. 10.1 Chronological subdivision of the Phanerozoic eon

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Fig. 10.2 Dickinsonia costata, a macrofossil of uncertain interpretation dated to about 555 MYA. The scale (cm) on the top right corner gives dimensions. https://commons.wikimedia.org/wiki/File: DickinsoniaCostata.jpg

Problems inherent to molecular clock analysis have been mentioned in Box 1.1 (also see review by Wray 2015). It is appropriate to repeat that, because fossilization is a rare event depending on the concurrence of multiple factors, palaeontological data provide a minimum but not a maximum dating constraint. In fact, a fossil may demonstrate that an organism was existing at some date in the past, yet the absence of similar fossils of older age does not exclude that the same organism existed significantly before but never fossilized. On the other hand, some animal phyla that fossil evidence dates to the Cambrian, for example the Ctenophores, Tardigrades and Onicophores, have left no unequivocal fossils for the following 500 MY and would be considered long extinguished if living representatives were not still extant today. Traditional animal systematics, essentially based on morphology and embryology, portrays animal evolution as a stepwise accumulation of increasingly complex characters from more primitive to more advanced groups. Zoologists, however, have long recognized that evolution can also proceed in the opposite direction, generating simpler forms from more complex ones, or can follow parallel yet independent routes and produce false affinities. In fact, all living animal lineages have had the same amount of time to evolve after diverging from the last common ancestor, and all have gained and lost multiple traits. Sequence analysis and novel techniques of morphological investigation have introduced important changes in animal systematics. For example, the traditional association of Arthopods and Annelida in the phylum “Articulata” is now obsolete, and Platyhelminthes are recognized as a paraphyletic group. Modern research also sparked novel controversy, for example producing evidence that the Ctenophores, traditionally associated with the Cnidaria in the phylum “Celenterata”, are the sister group of all other extant animals,

10.2

Embryo Development and Body Simmetry

315

suggesting that fundamental animal innovations such as the gut, a nervous system and muscle cells independently evolved twice. In contrast, the Placozoa, a morphologically simple animal lineage once placed at the base of the animal phyletic tree, is now considered the likely sister group of the Cnidaria + Bilatera clade, i.e. “advanced” animals.

10.2

Embryo Development and Body Simmetry

Molecular phylogeny (King et al. 2008; Paps et al. 2013; Ruiz-Trillo et al. 2008; Shalchian-Tabrizi et al. 2008; Steenkamp et al. 2006) places the animals, or Metazoa, in the amorphean clade Opisthokonta along with fungi, choanoflagellates and a few minor groups (Fig. 10.3). The closest living relatives of animals are the choanoflagellates (Sebé-Pedrós et al. 2017), unicellular or colonial phagotrophic protists with a funnel-shaped collar of microvilli surrounding a single flagellum, which they use for the capture of prey (Box 6.2). The choanoflagellates are morphologically similar to the choanocytes, cells lining the body cavity of sponges and specialized in the capture of unicellular prey by phagocytosis. Distinctive characters (apomorphies) of the opisthokonts include male gametes with two basal bodies and a single posterior flagellum (Box 6.2), a unique sequence of 12 amino acids inserted in the protein EF1α (a component of protein synthesis machinery), chitin (a linear polymer of β-(1-4)-N-acetyl-glucosamine), and flat mitochondrial cristae. Within the opisthokonts, important apomorphies of animals are specialized cell junctions (Sect. 10.4), the dynamics of gametogenesis, structural details of male gametes, and specific traits of the mitochondrial genome. The Metazoa is the eukaryotic kingdom with the largest number of described species, over 1.5 million, divided into approximately 33 phyla. The real number of animal species is likely much larger, with estimates ranging from 8 to 163 million (Paps 2018). The animals also have the largest diversity of cell types and body plans among multicellular organisms. A conservative assessment of the animal pangenome identified 4670 protein-encoding gene families, of which 1286 are unique, namely not shared with any other group. Nearly three-quarters of the animal-specific gene families occur at the base of the phyletic tree, supporting the notion that animal evolution was a fast event (Srivastava et al. 2010). On the same line of enquiry, Paps and Holland (2018) analysed 62 genomes belonging to 13 animal phyla and 8 eukaryotic outgroups, identifying 1189 gene homology groups exclusive to the animal spectrum examined. Of these, 25 gene groups were found in almost all animal taxa examined and therefore were probably present in the last common ancestor of animals. These results point to a level of genomic innovation during metazoan evolution unparalleled in other lineages. Animal development involves the differentiation of basic morphological units known as germ layers. According to the evolutionary model proposed by Haeckel in the nineteenth century, the body of ancestral animals (the urmetazoa) was essentially a hollow sphere, the planula, outlined by a single cellular layer. The addition of

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Fig. 10.3 Phyletic position of the animals (Metazoa) within the eukaryotes. Molecular data strongly support monophyly of the animal lineage. Along with the Choanoflagellates (plus the Filasterea and Ichthyosporea, two minor groups), the animals form a clade named the Holozoa, which is sister to the Holomycota, a clade encompassing the Fungi plus some amoeboidal protozoa. Together, the Holozoa and Holomycota form the Opisthokonta, one of the two major clades in the Amorphea. The cladogram shows the order of divergence of clades but gives no information on their absolute ages, the length of horizontal branches being arbitrary. The root of eukaryotes (LECA) is tentatively placed between the Amorphea and the rest of eukaryotes, but other topologies are also considered (Sect. 6.9). For further details on animal phylogeny, see Shalchian-Tabrizi et al. (2008) and Paps et al. (2013)

a second layer gave rise to the gut, or intestine, a new body cavity communicating with the outside. Embryo development in the animals with a gut recapitulates this hypothetical succession. The zygote, representing the ancestral unicellular stage, divides to produce a mass of cells known as the morula; after a number of cell divisions, the morula forms a hollow sphere filled with liquid, the blastula; a second cell layer then develops by polar invagination of one hemisphere within the other in the process known as gastrulation. This forms a new cavity, the archenteron or primitive intestine, that communicates with the outside through the blastopore, whereas the pre-existing cavity, or blastocoele, is usually re-absorbed (Fig. 10.4). Gastrulation produces two germ layers, an ectoderm outside and an endoderm inside. During subsequent development, the ectoderm forms the epidermis and the nervous system, the endoderm gives rise to the epithelium lining the gut cavity and

10.2

Embryo Development and Body Simmetry

317

Fig. 10.4 Diagrammatic illustration of early embryo development in the animal assemblage traditionally known as the Eumetazoa (“true animals”), Gastraea or Gastraeozoa (“animals with an intestine”)

associated glands. In basal lineages such as the cnidarians, an ectoderm and endoderm are the only morphologically distinct germ layers developing during embryogenesis. The definitive body plan in these animals remains quite simple, essentially consisting of an epidermis derived from the ectoderm, an intestine derived from the endoderm, and an intervening mass of intercellular matrix and scattered cells (the mesoglea). In most other lineages, a third germ layer or mesoderm develops after gastrulation and produces connective and muscle tissues. The pattern of embryo development shown in Fig. 10.4 is found in the Cnidaria (diploblastic animals) and Bilateria (triploblastic animals), with important yet nonsubstantial variants in some lineages such as the Arthropoda and Vertebrata. The animals with gastrulation (therefore with a gut and extracellular digestion) and true germ layers (either two or three) are traditionally named Eumetazoa, viz. “true animals”. In sponges (Poriphera), the zygote produces a hollow sphere similar to a blastula; following a larval mobile stage, the development of the adult sac-like form involves cellular movements that have been suggested to be homologous with eumetazoan gastrulation (Leys and Eerkes-Medrano 2005; Fig. 10.5). Unlike eumetazoan germ

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Fig. 10.5 (a–c) Larval metamorphosis in some sponges recalls eumetazoan gastrulation, yet the cell layers formed during this process are not pre-determined; for example, cells from the prospective choanocyte layer (white) may detach and form epidermal cells (dotted). The same is observed during embryo-to-larva transition (not shown), suggesting that cellular rearrangements in sponge development are not homologous with gastrulation. In line with this conclusion, the sponges lack an intestine and extracellular digestion, and feed uniquely by phagocytosis mediated by choanocytes, uniciliate cells lining the body cavity in the mature form (c). (d) Transitional stage between the larval and adult stage in the sponge Sycon raphanus, showing gastrula-like invagination of the ciliate emisphere. (From Leys and Eerkes-Medrano (2005), license number: 4459500227354)

layers, the cell layers formed during embryo and larval development in the sponge Amphimedon queenslandica are not pre-determined to produce specific cellular types; nonetheless, the choanocyte-producing inner layer in this sponge specifically expresses a homolog of GATA, a gene participating in mesoderm specification in triploblastic animals. This suggests that eumetazoan germ layers and gastrulation evolved from pre-existing developmental programs used for the simple patterning of cells in the first multicellular animals (Nakanishi et al. 2014). We will encounter further examples in this chapter of morphologically simple lineages possessing genes that mediate the development of complex traits in more derived metazoan branches, suggesting that these genes initially evolved for simple functions and were later recruited in more complex patterning (Marshall and Valentine 2010). A morphological trait fundamental in animal systematics is body spatial symmetry (Fig. 10.6). In sponges, the body of adult individuals has no symmetry; among the cnidarians, jellyfish have a radial symmetry: their body can be divided into specular halves along at least two distinct planes. Most animals have a bilateral symmetry: their body can be divided into specular halves only along one plane, called the sagittal plane. Symmetry is often imperfect, with marginal differences between the parts, yet it gives important cues on animal phylogeny and evolution. The animals with bilateral symmetry form a clade strongly supported by molecular phylogeny, the Bilateria (Bilaterians). Besides a bilateral symmetry, bilaterians usually exhibit a dorsal-ventral polarity, the dorsal part lying above the intestine, the ventral part below. In contrast to descriptions in most zoology textbooks, the Ctenophores (Fig. 10.7) lack both a radial and bilateral symmetry, rather having a rotational symmetry of the second order: any plane passing through the oral-aboral axis divides the animal into two halves that are the same but rotated by 180
relative to each other (https://www.mathsisfun.com/geometry/symmetry-rotational.html). This type of symmetry is already visible during embryo development from the third cell division and represents a major departure from the body plan of other animals (Dunn et al. 2015).

10.3

Animal Systematics

319

Fig. 10.6 Radial (a) and bilateral symmetry (b) in the animal body plan

Fig. 10.7 (a) A ctenophore as usually presented in zoology textbooks. (b) A pelagic (mobile) ctenophore in the swimming position: unlike jellyfish, the mouth-containing oral pole is oriented forward relative to the direction of movement (arrow); in the rest position, instead, the mouth is up. Pelagic ctenophores swim by means of groups of multiciliate cells arranged in eight longitudinal bands called combs or ctenes. Unlike normal flagella, comb flagella have axonemes with a 9 + 3 structure. Ctenophores are efficient predators that trap the prey using a sticky secretion from “glue cells” (colloblasts) localized in the tentacles. The intestine of ctenophores has a single opening

10.3

Animal Systematics

Until the late 1980s, animal phylogeny was based on anatomy and descriptive embryology, and the tree generally agreed had essentially the topology shown in Fig. 10.8.

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Fig. 10.8 Animal tree as presented in textbooks in the 1980s. The Ctenophora were resolved as sister to the Cnidaria or, alternatively, to the Cnidaria + Bilateria. The Acoela were included within the flatworms (Platyhelminthes), the Arthropods were sister to the Annelids, and the position of the Lophophorates was undetermined. The Placozoa were placed at the base of the animal tree (not shown), or even regarded as possible surviving forms of the “Ediacaran Fauna”

Phylogenomic analysis has confirmed in part the traditional tree topology, yet it has also introduced major changes and novel controversy (Halanych 2016). Figure 10.9 shows a recently published tree (Giribet 2016) based on robust molecular evidence from independent groups (Dunn et al. 2014; Ryan et al. 2010, 2013; Moroz et al. 2014; Ryan and Chiodin 2015; Whelan et al. 2015, 2017), in which the Ctenophora is basal to all other animals and the Poriphera (sponges) is immediately above. All the other animals cluster in a clade named Parahoxozoa from Hox/ ParaHox genes (Box 10.1), putatively ubiquitous in the group but absent in the Poriphera and Ctenophora. The Parahoxozoa comprises the Placozoa and the Planulozoa, the latter being a large clade encompassing the Cnidaria and Bilateria and thus coinciding with traditional Eumetazoa. In a basal position within the Bilateria, this tree features the novel clade Xenacoelomorpha, comprising two lineages lacking an excretory system and traditionally treated as primitive Platyhelminthes. The rest of the Bilateria cluster in the clade Nephrozoa, so named from tube-like excretory organs (proto- or metanephridia) present in all members save instances of secondary loss. The Nephrozoa in turn encompasses the Deuterostomia and Protostomia, owing their name to the blastopore becoming the anus or the mouth, respectively (Box 10.2). The Deuterostomia comprises the Ambulacraria (Hemichordates and Echinodermata) and Chordata, the latter including the Vertebrata. With over a million species described, the Protostomia is much a larger clade encompassing the Spiralia, the Ecdisozoa and the Chaetognatha, the latter being a lineage of uncertain position. The Spiralia, so named from the spiral geometry of zygote cleavage, comprises the large clade of Lofotrocozoa (Mollusca and Annellida plus the “Lophophorates” of traditional systematics) and several minor groups whose interrelationships are yet to be determined. The Ecdisozoa includes animals that

10.3

Animal Systematics

321

Fig. 10.9 Hypothesis of animal phylogeny derived from multiple phylogenomic sources. Wellsupported clades are in green; relatively stable clades but with some conflicts between the data are in blue; putative clades yet to be thoroughly tested in phylogenomic analyses are in red. The divergence order of red clades is not given. The length of horizontal branches is arbitrary. (From Giribet (2016), license number 4459520879453)

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grow by ekdysis, i.e. moulting their protective cuticle; the most species-rich protostome phyla are the Arthropods, Molluscs and Nematodes. The key point of the phylogenetic scenario presented in Fig. 10.9 is the Ctenophora basal to the rest of animals, implying the dismissal of “Celenterata”, a traditional group encompassing the Cnidaria and Ctenophores (Fig. 10.8), and of the Eumetazoa clade. The placement of Placozoa in the Paraoxozoa receives support from the placozoan genome containing a gene (Trox-2) with clear sequence homologies with Hox/ParaHox genes (Box 10.1), and of genes homologous with those that control neuronal and muscle cell differentiation in Cnidaria and Bilateria (Jacob et al. 2004; Srivastava et al. 2008). This suggests that the morphological simplicity of placozoans, notably the lack of gastrulation, mesoderm, muscle cells and neurons, is a derived condition. The phylogeny presented in Fig. 10.9 implies that the Ctenophores evolved a mesoderm germ line, muscle cells and neurons independently of the other animals. In line with this inference, the genes that specify mesoderm and muscle cells in ctenophores are not homologous with their counterparts in the Cnidaria and Bilateria. Further support to the placement of Ctenophora at the base of animal tree comes from their lack of Hox genes (Box 10.1) and of a canonical microRNA machinery (Moroz et al. 2014). Phylogenomic research by other groups supports partially different conclusions. For example, Schierwater et al. (2009a) splits the animals into two major clades, the Triploblasta (¼ Bilateria) and Diploblasta, the latter encompassing the Cnidaria and Ctenophora clustered together in the clade Celenterata, plus the Poriphera and Placozoa (Fig. 10.10). This tree implies independent evolution of gastrulation and neurons in Diplo- and Triploblasts (Schierwater et al. 2009b) or, alternatively, their presence in the common ancestor of all animals and independent loss in the Placozoa and Poriphera. The genomes of the ctenophore Mnemiopsis leidyi and the sponge Amphimedon queenslandica include similar sets of neural components, suggesting that a rudimentary nervous system was present in the last common ancestor of animals and was secondarily lost in the sponges (Ryan et al. 2013) and the Placozoa (Ryan and Chiodin 2015). In line with this “reductive” scenario, old and novel evidence suggests that the absence of a canonical mesoderm in the Cnidaria is a derived, not a primitive character (Cavalier-Smith 2017). For example, molecular work in the sea anemone Nematostella (Cnidaria) is consistent with the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from a bifunctional endomesoderm of a diploblastic ancestor (Wijesenaa et al. 2017). Furthermore, there is evidence that a part of the genetic network underlying the development of the nervous system in bilaterians also occurs in cnidarians, suggesting that the last common ancestor of the two lineages already had a wellestablished nervous system (Kelava et al. 2015). Alternative trees to those presented here confirm the traditional placement of sponges as sister to all other animals, and resolve the Ctenophora as the sister clade to Placozoa + Cnidaria + Bilateria (Philip et al. 2009; Pick et al. 2010; Srivastava et al. 2010; Pisani et al. 2015; Feuda et al. 2017; Simion et al. 2017). The dispute on ctenophores-first versus sponges-first is far from being settled, supporters of each

323

Echinodermata

Bivalvia Annelida Hexapoda Arthropoda Crustacea Priapulida

Ecdysozoa

Gastropoda Mollusca

Lophotrochozoa

Chordata

Bilateria

Hemichordata

-

Deuterostomia

METAZOA

Triploblasta

Animal Systematics

Protostomia

10.3

Hydrozoa

Cnidaria

Cubozoa 27

Scyphozoa Ctenophora 53

Hexactinellida

Diploblasta

62

“Coelenterata”

Anthozoa

Porifera

98

Demospongiae Calcarea Placozoa Choanoflagellata

Fig. 10.10 Animal phylogeny by Schierwater et al. (2009a). The Diploblasta, encompassing Placozoa, Poriphera, Ctenophora and Cnidaria, is resolved as the sister clade to Triploblasta (Bilateria). This cladogram is no more parsimonious than Giribet’s model, because it implies that gastrulation, neurons and other shared traits evolved independently in Triploblasta and Ctenophora/ Cnidaria (Celenterata), or were present in the common ancestor of Diplo- and Triploblasta and were lost in the Poriphera and Placozoa. Creative Commons Attribution License

side interpreting the alternative conclusion as a tree reconstruction artefact. CavalierSmith (2017) shares the sponge-first model of animal phylogeny, based on the consideration that “among extant animals, only sponges could have evolved directly from a choanoflagellate without changing the ancestral feeding mode”. The similarity of sponge choanocytes to unicellular choanoflagellates was first remarked by James-Clark in 1886. Until recently, homology of the two cell types has gone largely unquestioned, gaining further support from molecular phylogeny

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resolving the choanoflagellates and animals as sister groups in a clade named “Choanozoa”, with the microvillar-flagellar complex as the main unifying apomorphy (Brunet and King 2017). Similarities between the microvillar-flagellar complex in choanoflagellates and choanocytes include a shared underlying ultrastructure and bilateral wing-like extensions, or “vanes”. Interestingly, a ring of microvilli associated with a single, usually rudimentary flagellum appears to be a deeply-wired trait in the animal clade (Brunet and King 2017). Homology of the collar-flagellar apparatus of choanoflagellates and sponge choanocytes has been called into question based on morphological, functional and developmental divergences (Maldonado 2004; Mah et al. 2014). As Brunet and King (2017) observe, the long time span elapsed after the separation of the two lineages (about 600 MY) may explain minor differences as adaptive or casual variations of an ancestral pattern, whereas similarities are more difficult to explain in terms of convergent evolution. Collar microvilli in choanoflagellates are directly transmitted to daughter cells by cell division along the apico-basal axis. Because of this fixed division orientation, cell proliferation in colonial choanoflagellates directly produces only linear chains or planar sheets; more complex shapes, such as a sphere, arise from cellular rearrangements that permit bending and, ultimately, “closure” of the sheet at the point where non-sister cells meet. This constraint seems to apply to any spherical colony formed by clonal division of motile flagellate cells, for example in volvocine green algae and possibly in calcareous sponges. In all other animals, cellular division during early embryo development can orientate along several axes, thus directly producing a spherical blastula (Brunet and King 2017). This ability may be associated with the presence of a centrosome (Box 6.2), which restricts expression of a full flagellar apparatus to specific cell types and developmental stages.

10.4

Cellular Junctions and Epithelia

The animal body tightly depends on specialized cellular junctions for structural and physiological cohesion. These belong to four major classes: anchoring, occluding, channel-forming and signal-relaying junctions (Alberts et al. 2014). The last include chemical synapses in neurons and immunological synapses in specialized immunocompetent cells, and will not be considered here. Anchoring junctions (Fig. 10.11) have essentially a structural function: they permit the cells to cling to each other and to the extracellular matrix (ECM), thus generating structures capable of resisting mechanical stress sometimes extremely intense. It is thanks to anchoring junctions that the cells of our skin form a robust protective coat and muscle cells transmit tension from contraction to tendons. The main components of anchoring junctions are integral membrane proteins of the cadherin and integrin families. Cadherins (so named from “calcium-dependent

10.4

Cellular Junctions and Epithelia

325

Fig. 10.11 Anchoring and occluding junctions in vertebrates. Desmosomes, hemidesmosomes and semi-adherens junctions are mediated by integrins, adherens junctions by cadherins. Septate junctions take the place of tight junctions in invertebrates. The occurrence of desmosomes in invertebrates is dubious

adhesion proteins”) carry multiple calcium-binding domains and interact with the actin cytoskeleton on the cytosolic side of the cell membrane to form cell-to cell adherens junctions. Integrins have calcium-magnesium-binding domains and mainly mediate cellular attachment to the extracellular matrix; they interact with intermediate filaments on the cytosolic side of the membrane to form the desmosomes and hemidesmosomes, or with the actin cytoskeleton to form semi-adherens junctions. Despite numerous reports of desmosomes in invertebrates, these junctions are defined by proteins (desmosomal cadherins and plakoglobins) found only in vertebrates (Leys and Riesgo 2011). Occluding junctions (Fig. 10.11) occur in epithelial tissues, where they function in preventing free diffusion of solutes across intercellular spaces. Occluding junctions in vertebrates are known as tight junctions, their counterpart in invertebrates being the septate junctions. The main proteins involved in tight junctions are claudins; homologs of claudins seem to participate in the formation of septate junctions in invertebrates. Vertebrate tight junctions form a sort of a belt, named the zonula occludens, encircling the apical end of each cell in the epithelium, below which are adherens junctions and other types of anchoring junctions; an opposite topical distribution of septate and anchoring junctions is observed in invertebrate epithelia (Fig. 10.13).

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Channel-forming junctions (Fig. 10.12) facilitate cell-to-cell diffusion of small molecules dissolved in the cytosol. They consist of two transmembrane complexes, or connessons, which cross the membranes of neighbouring cells and bind to each other to form an open pore; the connessons are made of connessins in vertebrates and innessins in invertebrates, two protein families lacking recognizable homologies. The apparent dichotomy between vertebrates and invertebrates as regards cellular junctions has no obvious explanation and is rather surprising because there is no clear-cut phylogenetic boundary between the two groups: indeed the invertebrate lineages Echinodermata, Hemichordata, Tunicata and Cephalocordata are more closely related to the vertebrates than to the rest of invertebrates (Sect. 10.4). Further molecular and ultrastructural investigation of critical invertebrate lineages will probably attenuate the differences, yet the issue appears to deserve more attention than so far received (Fahey and Degnan 2010; Leys and Riesgo 2011). Epithelial tissues typically possess all three types of cellular junctions described above, whereas other tissues in the animal body have anchoring junctions and often also channel-forming junctions, but lack occluding junctions. The epithelia consist of cells tightly linked in one to many layers, and function at the supra-cellular level of organization much as membranes do at the cellular level, i.e. in separating specific compartments and controlling the outward and inward flow of water and solutes. Fundamental functions of the animal body such as digestion, waste disposal, ion and water balance and gas exchanges depend on the activity of epithelial tissues. A distinctive, ubiquitous feature of epithelial tissues is the basement membrane (Fig. 10.11), a thin layer of underlying extracellular matrix containing a specific type of collagen (collagen IV). In contrast to what was common belief until a few years ago, it is now established that Poriphera have epithelia with a basement membrane (including collagen IV) and adherens junctions; structures similar to septate junctions have been reported in some calcareous sponges, whereas channel-forming junctions seem to be lacking in these animals (Leys et al. 2008; Leys and Riesgo 2011). At the genomic level, the Poriphera have been found to possess genes for integrins, cadherins and other proteins involved in anchoring junctions in other animals, but appear to lack genes for proteins involved in occluding junctions (e.g. claudin-like proteins), a result at odd with the alleged presence of septate junctions in calcareous sponges (Fahey and Degnan 2010). Trichoplax (Placozoa) has a dorsal and a ventral epithelium with adherens junctions but, oddly, with no visible basement membrane although the genome of this animal includes a gene for collagen IV besides genes for integrins and cadherins (Srivastava et al. 2008). Figure 10.13 illustrates the taxonomical distribution of the various types of cellular junctions in vertebrates and some invertebrate lineages. Following genome sequence determination in unicellular relatives of animals, gene notation work has revealed that these organisms likely possess homologs of fundamental components of the extracellular matrix (ECM) such as laminins, collagens and fibronectins, as well as mechanisms for cell-to-cell and cell-to-ECM adhesion. For example, the genome of choanoflagellates encodes for 20–30 predicted cadherin-like proteins and for C-type lectins that mediate cell-to-cell

10.4

Cellular Junctions and Epithelia

327

Fig. 10.12 Channelforming junctions create passageways linking the cytosol of neighbouring cells

Fig. 10.13 Distribution of different types of cellular junctions in animals. (Adapted from Tyler (2003), license number 4459520510498)

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adhesion in animals; the filasterean Capsaspora owczarzaki has a complete integrin complement for cell-to-ECM adhesion. Overall, this indicates that rudimentary adhesion mechanisms were already present in unicellular relatives of animals, thus providing a springboard for the unicellular-to-multicellular jump (Sebé-Pedrós et al. 2017). The evolution of multicellularity and morphological complexity in animals probably co-opted the machinery used in their extant unicellular relatives to regulate gene expression at a temporary level (different life stages), and repurposed it to simultaneously elicit differential gene expression in cell lineages (Paps 2018). Box 10.1: Hox/ParaHox Genes Hox genes encode for homeotic transcription factors, i.e. proteins containing a domain of about 60 aminoacids (homeodomain) that binds specific DNA sequences (promoters) and activate or inhibit the expression of downstream genes, thus acting as enhancers or silencers. Homeotic genes are present in all eukaryotes and modulate gene expression during the life cycle; in multicellular complex forms, homeotic genes also control tissue and organ development. A distinctive property of Hox genes in bilateral animals (Bilateria) is spatial collinearity, viz. the aggregation in clusters in an order that mirrors the way they operate along the anterior-posterior body axis, with genes at the 50 and the 30 end of the cluster being expressed in and patterning the posterior and anterior part of the body, respectively. Hox genes in Vertebrates and in amphioxus (Sect. 10.4) also show temporal collinearity: the genes controlling the anterior part of the body are expressed earlier than those controlling the posterior part. The relative genomic arrangement of Hox genes is conserved in phylogenetically distant taxa, a property generally referred to as syntheny (Garcia-Fernàndez 2006; Ikuta 2011). Collinearity and syntheny are a relatively common property of genes with coordinated expression patterns, probably reflecting spatial and temporal modulation of chromatin structure in the interphase nucleus (Box 6.3), but the mechanisms involved are still poorly known. After the discovery of Hox genes, other two families of homeotic genes were found in animals: ParaHox and NK genes. All together, these genes belong to the large class of animal homeotic genes named ANTP from the Antennapedia gene identified in the fruit fly (Drosophila melanogaster). Hox, ParaHox and NK genes have clear sequence homologies and are most likely paralogs derived from a single ancestral gene. Hox genes are expressed in all germ layers but predominantly in the ectoderm and ectoderm-deriving tissues; ParaHox genes are primarily expressed in endodermal derivatives and NK mostly in mesodermal derivatives. Homologs of bilaterian Hox/ParaHox/NK genes are present in the genome of cnidarians, and a gene with sequence homology with Hox/ParaHox genes was also found in Placozoa, whence the name Parahoxozoa given to the clade encompassing the Bilateria, Cnidaria and Placozoa in recent phylogenomic (continued)

10.4

Cellular Junctions and Epithelia

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Box 10.1 (continued) work (Fig. 10.9). No homolog of Hox/ParaHox/NK genes has yet been found in the Ctenophores, whereas the Poriphera have several NK genes but no Hox or ParaHox homolog. The occurrence of NK homologs in sponges suggests that at least one gene of the Hox/ParaHox/NK group was present in the common ancestor of Poriphera and Parahoxozoa. Hox/ParaHox/NK genes appear to derive from a protoHox founder gene in the common ancestor of Metazoa (Garcia-Fernàndez 2006). In this scenario, the protoHox gene produced an ancestral Hox-like cluster by multiple duplication and neo-functionalization. Further duplications generated a “protoHox supercluster” of four Hox, five ParaHox and four NK genes in the last common ancestor of Parahoxozoa (Placozoa + Cnidaria + Bilateria). In the Bilateria, the ProtoHox supercluster fragmented into three separates clusters (Hox, ParaHox and NK) that went through further events of duplication, dispersion and deletion. The Xenacoelomorpha have a cluster of three or four Hox genes (Heinol and Martindale 2008a; Moreno et al. 2009); most invertebrate Bilateria have two Hox clusters (for a total of 7–9 genes, suggesting a duplication event (or probably two independent duplications, in Protostomes and Deuterostomes); the Cephalocordates, in contrast, have a single Hox cluster. The Vertebrates went through two whole-genome duplications early in their evolutionary history (Sect. 10.4) and, with the possible exception of Agnates, have four Hox clusters (not all complete, some paralogs having been lost). ParaHox genes are associated in a cluster in Cephalocordates and Vertebrates (a condition considered primitive) and exhibit both spatial and temporal colinearity, whereas they are scattered in the genome in the rest of Bilateria; the opposite is observed for NK genes, which are scattered in Vertebrates but form a cluster in Protostomes (the latter being considered the primitive condition). Since the Hox, ParaHox and NK genes are associated with the expression and differentiation of the ecto-, meso- and endoderm germ layer, respectively, their occurrence in the genome of Cnidaria calls into question the diploblastic nature of these organisms. Perhaps even more importantly, the expression pattern of Hox genes in the sea anemon Nematostella vectensis suggests a hidden anterior-posterior polarity homologous with that in Bilateria. Interestingly, the sea anemones exhibit a bilateral, not radial symmetry in their inner anatomy. Is the origin of the three germ layers and of Bilateria located more basally in the animal tree? The detection of NK genes in sponges revives the traditional hypothesis that the Eumetazoa (Cnidaria and Bilateria) evolved from a paedomorphic (!Glossary) larva of an ancient sponge (Maldonado 2004). The Placozoa, an enigmatic group presenting a mixture of “primitive” and “advanced” characters (Box 10.2) may hold the key to this intriguing scenario.

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Box 10.2: Trichoplax, the Simplest Free-Living Animal In 1883, German zoologist Franz Eilhard Schulze published a short description of a novel animal discovered in an aquarium at Graz University hosting marine samples from the bay of Trieste, then belonging to the Austria-Hungary empire. The animal had the appearance of a flattened disc with a diameter of 1–3 mm, consisting of a ciliate epithelium and a loose internal network of “fiber” cells, and showed slow gliding movement on solid substrate. Schulze understood that this tiny animal had no obvious relationships with any known animal phylum, and named it Trichoplax adherens. After its discovery, this enigmatic animal went into oblivion for nearly a century, to be “discovered” again in 1969 by another German zoologist, Karl Gottlieb Grell, who created the novel phylum Placozoa to accommodate it. Since then, Trichoplax has been reported from numerous tropical and subtropical ocean sites worldwide; morphological/ecological diversity suggests the existence of other species besides that originally described by Schulze, although none has yet been formally described (Eitel et al. 2013). Microscopic analysis confirms the extreme morphological simplicity of Trichoplax (Fig. 10.14). The organism presents a clear dorsal-ventral polarity but lacks a longitudinal axis and any type of symmetry; moreover, it lacks an intestine, neurons, muscle cells and even an extracellular matrix and a basement membrane associated with the epithelia. Tricoplax moves on the bottom substrate by means of cilia from lower epidermal cells and can change its shape thanks to contractile properties of fiber cells. It feeds on unicellular preys by phagocytosis, but apparently may also digest small animals, such as nematodes or invertebrate larvae, using the space between the substrate and the lower epithelium as a temporary gastric cavity. Trichoplax reproduces by fission, with two (sometimes three) parts of the animal body moving away from each other until their connection breaks. Sexual reproduction has not been described, but putative oocyte formation is routinely observed in culture. Tricoplax has a genome of ~98 Mb, quite small for an animal (for comparison, the human genome is ~3200 Mb), and contains about 11.500 putative protein-coding genes (Srivastava et al. 2008). These include genes for transcription factors whose homologs in Cnidaria and Bilateria control blastopore development and the anterior-posterior body organization, or specify the endoderm germ layer, neurons and muscle cells, although none of these traits are present in Trichoplax. Two homeotic genes of the ANPT class (Trox-2, a likely Hox/ParaHox ortholog, and Not) are expressed in epithelial cells along the border of the animal. Although lacking any visible extracellular matrix and basement membrane, Trichoplax possesses genes for proteins that participate in building these structures in other animals, for example genes for collagen IV, α-, β-, γ-laminins, and fibronectin (Srivastava et al. 2008). (continued)

10.5

The Intestine and Animal Evolution

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Box 10.2 (continued) SS UE

B FC

Mc GC

LE

(A)

1 mm

(B)

Fig. 10.14 (a) Trichoplax adherens as seen under a scanning electron microscope. (b) Diagrammatic representation of inner histological organization. At least four cellular types are distinguishable: an upper (UE) and lower epithelium (LE), fiber cells (FC), and gland cells (GC). The upper epithelium contains prominent “shiny spheres” (SS) of lipid nature, possibly functioning as a deterrent against predators. (From Schierwater (2005), licence number 4459521388347)

The title given to this box demands a closing note. Molecular systematics has revealed that there are more simple animals than Trichoplax: the Myxozoa, a class of obligate parasites of animals including fishes, long considered protists and classified within the “Sporozoa” along with the Apicomplexa. Sequence analysis of 18S rRNA has revealed that the Myxozoa is a cnidarian lineage that adapted to a parasitic lifestyle going through a dramatic simplification of the original jellyfish body plan and concurrent extreme reduction of genome size and gene content (Chang et al. 2015).

10.5

The Intestine and Animal Evolution

The intestine is the animal body cavity in which ingested food is digested. The evolution of an intestine enabled the animals to switch from phagotrophy, a form of predation ubiquitous in eukaryotes and necessarily restricted to unicellular prey, to macrotrophy, predation of multicellular organisms. Considered under this perspective, the intestine is the fundamental innovation of animals. The nervous system, sensorial organs, muscles, in short everything that we associate to the animal lifestyle are ultimately just accessories and complements of the intestine. Indeed, we may correctly say that the animals are digestive bags. The absence of a true intestine in the sponges is the main reason why these organisms are traditionally classified in the Parazoa (“nearly animals”), a subkingdom separate from the rest of animals (Eumetazoa).

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An intestine is present in the Ctenophores, Cnidaria and Bilateria (save instances of secondary loss in forms specialized as parasites). Lacking an intestine, the Placozoa and sponges rely on phagocytosis as the only or principal feeding mechanism. As already mentioned, this taxonomic distribution implies deeply divergent evolutionary scenarios depending on the phylogenetic model adopted. The evolution of a pervious intestine or through-gut, with a second aperture developing after gastrulation, was a second major innovation in animal evolution. In a pervious intestine, food can move unidirectionally, which not only enhances the efficiency of digestion but also permits the animal to ingest food whenever there is the opportunity (as any dog owner knows). The single aperture of the intestine in the Ctenophores and Cnidaria derives from the blastopore (Fig. 10.4). In 1908, Karl Grobben divided bilateral animals between Protostomia and Deuterostomia, based on the different embryonal origin of the mouth (the inward aperture): from the blastopore in Protostomia, secondarily in the Deuterostomia (in which the blastopore instead becomes the anus). Because of this embryological divergence, it is thought that a pervious intestine evolved independently in the two groups (Heinol and Martindale 2009; Heinol and Martìn-Durán 2015), a conclusion now supported by placement of the Xenacoelomorpha (whose intestine has a single aperture) in a sister position to the rest of Bilateria (Fig. 10.9). Gene-expression studies have revealed that goosecoid and brachiury genes, two genes encoding transcription factors, are expressed in the mouth region both in Protostomia and Deuterostomia, as well as at the level of the single aperture in Convolutriloba longifissura, a member of the Xenacoelomorpha. This suggests that the mouth opening is homologous throughout the Bilateria despite its diverse embryological derivation (Martìn-Durán et al. 2016). Heinol and Martindale (2008a, b) argue that the intestine in the progenitor of Bilateria (dubbed the urbilater) had a single aperture that became the mouth in both Protostomes and Deuterostomes, whereas the anus evolved independently in either lineage (Fig. 10.15). This interpretation does not affect the validity of the two clades, supported by multiple lines of evidence (Telford 2006), but if confirmed, it would demand a change in the names assigned by Grobben. The intestinal lumen of animals hosts a diversity of microorganisms collectively referred to as the gut microbiome. The main benefit afforded by the gut microbiome to their hosts is digestion of recalcitrant material requiring enzymatic activities not present in the animal genomic inventory. An example is the ability of several animals including insects and vertebrates to feed on cellulose and other complex polysaccharides. There is evidence that the gut microbiome of mammals makes additional contributions to the host health by producing vitamins, assisting in the development of the immune system, increasing resistance to pathogenic bacteria, and attenuating inflammatory responses (Yeoman et al. 2011; Sommer and Bäckhed 2013; Davenport et al. 2017). The occurrence of a gut microbiome in relatively simple animals such as the earthworms suggests that metabolic cooperation of animals with microorganisms is an ancient trait that underpinned the emergence of extracellular digestion from the beginning. Of great importance for animal health also is the microbial community that inhabits the epidermis and the epithelia of body cavities such as the nephridia, oviducts, vagina, ureters and respiratory ducts. The

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Chordates

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Fig. 10.15 (a) According to traditional view, the last common ancestor of bilateral animals (dubbed urbilater) had a pervious intestine (gut), a central nervous system (NS) and a circulatory system (hearth). (b) A more recent model of the urbilater based on genomic analysis is morphologically simpler, with no circulatory system, a “diffuse” nervous system (NS) and an intestine (gut) with a single aperture homologous with the mouth in both protostomes and deuterostomes. (Redrawn from Hejnol and Martindale 2008a)

application of metagenomic analysis (Box 1.1) is fundamental for identifying microorganisms participating in animal microbiomes. The growing dataset shows that the gut microbiome is a complex and highly dynamic community that rapidly responds to changes in the diet (Amato 2013).

10.6

Chordates

The phylum Chordates (Chordata) deserves special attention in this chapter, not so much for its size, which is quite large (about 69.000 species described) yet not particularly impressive when compared to other groups such as the Arthropods (above one million species), but because it encompasses the Vertebrates, including humans. Appeared about 530 MYA, the chordates are one of the two extant lineages of deuterostomes, the other being the Ambulacria, with the Echinodermata and Hemichordata (Satoh et al. 2014; Fig. 10.9). The principal apomorphy of chordates, from which the group received the name, is the notochord, a rod of large cells enclosed in a robust connective sheath running dorsally along the anterior-posterior body axis. Due to turgor pressure, the notochord is a relatively stiff structure, yet flexible enough to permit ondulatory body movements from a bilateral musculature connected to it (Fig. 10.16).

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Fig. 10.16 The anatomy of the amphioxus Branchiostoma lanceolatum (Cephalochordates) illustrates the body plan of Chordates. 1 Anterior bulge in neural tube; 2 notochord; 3 neural tube; 4 post-anal tail; 5 anus; 6 Intestine; 7 circulatory system; 8 atriopore; 9 atrium; 10 pharyngeal slits; 11 pharynx (the intestine region immediately behind the mouth; its ventral wall contains the endostyle); 12 oral vestibule; 13 oral cirri (green); 14 mouth opening; 15 gonads (ovaries/testicles); 16 light sensors; 17 nerves; 18 abdominal fold; 19 hepatic caecum (an intestine outgrowth homologous with vertebrate liver). (Credit: Piotr Michał Jaworski; https://commons.wikimedia.org/wiki/File: BranchiostomaLanceolatum_PioM.svg)

A second important apomorphy of the chordate lineage is the tail, a muscular projection extending backward beyond the anus and containing an extension of the notochord. The combination of a notochord, a tail and a bilateral musculature produces a powerful propulsion system that has given a substantial contribution to the evolutionary success of chordates and especially of vertebrates. Further distinctive traits of chordates are: – A central nervous system consisting of a hollow tube dorsal to the notochord and the intestine; in the other bilateria with a comparable anatomy, the central nervous system consists of a single or double cord running ventrally. – A bilateral series of slits in the anterior part of the intestine (pharynx), ancestrally employed as a filter feeding apparatus and secondarily converted into respiratory organs (gills). – The endostile, a groove of ciliate cells in the ventral wall of the pharynx. In filterfeeding species the endostile produces mucus that is used to aggregate food particles; the endostile stores iodine and is probably a precursor of the thyroid gland in vertebrates. The Chordate clade encompasses three lineages with the rank of subphyla: Tunicates (or Urochordates), Cephalochordates, and Vertebrates (or Craniates). The Tunicates own their name to the tunic (or tunica), a smooth body coat of varying thickness and stiffness, made of proteinaceous material and cellulose (Fig. 10.17). The tunic is unique among invertebrate exoskeletons because it grows as the animal enlarges and does not need periodically shedding. The tunicates are mainly marine, hermaphrodite animals that feed on microplankton utilizing the perforate pharynx as a filtering organ. The group comprises the classes Ascidiacea, Appendicularia (or Larvacea) and Taliacea, for about 3000 species described. The Ascidiacea present a mobile larval phase provided with a notochord and superficially

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Fig. 10.17 (a) The tunicate Styela plicata (Ascidiacea). As typical of the group, these animals feed on suspended particulate matter by filtering seawater, which enters through the oral siphon (sb), circulates across the pharyngeal slits and is expelled through the atrial siphon (sa). Ciliary beating of pharyngeal cells maintains water circulation. (b) A detail of the tunic (Tu) photographed by light microscopy; this structure consists of material secreted by the epidermis (ep) with scattered cells, the vanadocytes, containing high concentrations of trivalent vanadium. Numerous muscle cells (mu) are visible under the epidermis. (c) A detail of the tunic photographed by transmission electron microscopy after treatment with a molecular probe specific for cellulose. Abundant deposition of gold particles (black dots) demonstrates the presence of cellulose microfibrils in the extracellular matrix. Also visible is a vanadocyte (Va). The enzyme that produces cellulose (cellulose synthase) in Tunicates is encoded by CesA, a gene of bacterial ancestry acquired by horizontal gene transfer (Sagane et al. 2010). (a) (From http://www.biologiamarina.org/ascidia-stiela/)

similar to a frog tadpole, and a vase-shaped adult phase that re-absorbs the larval tail and notochord, and attaches to rigid substrates, becoming a sessile filter feeder (Fig. 10.17). The Appendicularia (70 species) retain a body structure similar to the larval stage throughout life and live suspended in water as components of the zooplankton, using the tail for propulsion. They have modified the tunic into a mucous net (the “house”) that facilitates the capture of particulate food through the pharingeal apparatus. The Taliacea (73 species) are similar to ascidiacean adults but, unlike these, are free-floating (pelagic) organisms for their entire lifespan, swimming by propulsion from water expelled through the atrial siphon at the posterior end; their life cycle includes a colonial phase that sexually produces a solitary phase (the oozooid), which in turn regenerates the colonial stage by asexual reproduction. The Cephalochordates, better known as amphioxes or lancelets, comprise 32 recognized species assigned to three genera (Branchiostoma, Epigonichthys, Asymmetron), all marine. The amphioxes have an elongate, laterally flattened body recalling a small fish and live half-buried in sand, with their anterior end protruding outside. Like tunicates, the amphioxes are filter feeders living on plankton or organic sediments captured with the pharyngeal apparatus. The Vertebrates (about 66,000 species, of which more than half are fishes, the rest terrestrial forms) own their name to the vertebral column, also known as the backbone or spine, an articulated axial skeleton that replaces the notochord late in embryo development.

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Fig. 10.18 Phylogenetic tree of Chordates. In contrast with earlier analysis, more recent molecular phylogeny places the Cephalochordates in a basal position and resolves the Tunicates as the sister group to Vertebrates. Early in their evolutionary history, the Vertebrates underwent two wholegenome duplications, the first (black arrow) certainly preceding the divergence of Gnatostomes; the timing of the second doubling has yet to be determined with certainty (white arrows). The relative length of horizontal branches is arbitrary. (Redrawn from Bertrand and Escriva 2011)

The Vertebrates went through two successive genomic duplications at an early stage of their history (Fig. 10.18). This favoured the evolution of numerous novel genes from redundant sequences; as a result, vertebrates possess several paralogs for many genes that are present in single copy in the genomes of Tunicates and Cephalochordates. The evolutionary interrelationships of the chordate subphyla are contentious (Schubert et al. 2006; Satoh et al. 2014). Traditional zoology has long treated the Cephalochordates as the sister group to Vertebrates; in 1928 Walter Garland suggested that these two lineages derived by paedomorphosis (!Glossary) from the larval stage of a tunicate. Early molecular studies chiefly based on mitochondrial DNA and 18S rRNA supported this scenario, but subsequent work on nuclear sequences (Bertrand and Escriva 2011; Holland et al. 2008; Schubert et al. 2006) resolved the Tunicate as the sister group to Vertebrates and moved the Cephalochordates at the base of the Chordate tree (Fig. 10.18). Replacement of the nothocord by a robust axial skeleton of interconnected vertebrae, which can give support to a more powerful musculature, dramatically improved the propulsion apparatus of Vertebrates. A second important character of Vertebrates is their pronounced cephalization, with sensorial structures concentrated in the anterior part of the body, and underlying neural centres forming a brain protected by a new skeletal complex, the cranium (whence the alternative name Craniata given to the group). Important parts of the Vertebrate head derive from neural crest cells, embryonic cells unique to the group, suggesting that the head is an apomorphy of Vertebrates with no homolog in the other Chordates (Satoh et al. 2014).

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Fig. 10.19 An artistic reconstruction of Haikouichthys, a primitive agnate fish found as a fossil in the Chengjiang site in China. The arrow points to pharyngeal slits. (From: Giant Blue Anteater – Own work, Public Domain. https://commons.wikimedia.org/w/index.php?curid¼3157361)

The vertebrae and other parts of the internal skeleton of Vertebrates consist of a novel tissue made of cellular elements scattered in a dense extracellular matrix rich in collagen and proteoglycan and highly resistant to tension, torsion and compression forces. During the evolutionary history of vertebrates, more dense and rigid bone tissue containing hydroxyapatite (monohydrate calcium phosphate) replaced the relatively flexible and light cartilage present in earlier Vertebrates. Sodium chloride concentration in circulating fluids of the Vertebrate body, including marine forms, is lower than in seawater; in contrast, it is the same as in seawater in Cephalochordates and Tunicates. This suggests that the ancestral habitat of Vertebrates was in coastal lagoons and estuarine environments where seawater mixed with freshwater from rivers. This type of environment probably remained the main hatchery of vertebrate diversification for a long interval in Silurian/Devonian; with time, some lineages moved to the sea and became pelagic, others adapted to life in freshwater bodies, and one abandoned the ancestral aquatic habitat and became terrestrial (Long and Gordon 2004). The earliest vertebrates were fish-like animals lacking jaws and paired fins (the couples of pectoral and pelvic fins present in most modern fishes), named Agnates from Greek for “jawless”. The oldest fossils of Agnates, from the Chengjiang site in China, are dated to about 525 MYA (Fig. 10.19). Early agnates probably fed on organic detritus deposited on shallow bottoms using the pharynx as a filtering organ and a specialized musculature instead of ciliate cells to produce water circulation. The Agnates is probably a paraphyletic group, represented today by the Cyclostomes, primitive fishes so named as they keep their jawless mouth always open (Shimeld and Donoghue 2012). With about 60 species, the Cyclostomes encompasses the lampreys, living as ectoparasites on other fishes, and hagfishes (Mixini) living on dead/dying animals or in some cases preying on invertebrates. As in the other extant fishes, the pharynx of Cyclostomes has lost the filtering function and only works in gas and solute exchange. Evolving into gills, the pharyngeal slits became richly vascularized and developed a finely lamellate epithelium that enhanced the exchange of dissolved gas and ions between the blood and water. Most extant vertebrates are Gnatostomes, from Greek for “jawed mouth”, characterized by hinged jaws supporting the mouth rim. According to traditional comparative anatomy, the jaws evolved from a couple of gill arches, skeletal pieces that

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Fig. 10.20 (a) Ancestral jawless fishes had nine cartilaginous gill arches supporting eight gill slits. (b) In early jawed fishes (Placoderms), the first two pairs of gill arches were lost, and the third pair was modified into a hinged jaw. This left six gill arches to support the remaining five gill slits retained in use for gas exchange. (c) In modern jawed fish, the original fourth gill arch was converted into the hiomandibular bone to reinforce the connection of the jaws to the skull. (Adapted from: https://biology-forums.com/index.php?action¼gallery;sa¼view;id¼101)

reinforce the pharyngeal wall and keep the gill slits open (Fig. 10.20). This model, based on embryological data, recently received confirmation from genomic analysis revealing that the evolution of jaws in gnatostomes required a relatively minor change in the gene set that patterns the development of the pharyngeal skeleton in lamprey (Cerny et al. 2010). Further apomorphies of the Gnatostomes include bone tissue, teeth, pectoral and pelvic paired fins connected to the vertebral column, a third semicircular canal in the inner ear (there are only two canals in Agnates), myelinated nerves (absent in Agnates and invertebrates), and a more efficient sequence-recombination mechanism for antibody production (Colbert et al. 2001; Cooper and Alder 2006; Donoghue and Keating 2014).

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The oral skeleton may have primarily evolved as a support to the buccal pump, a muscular complex still used in modern fishes, which enabled the animals to inhale water through the mouth and force it to circulate through the gills, thus enhancing gas exchange. With time, this skeletal-muscular apparatus became stronger and was increasingly used also for catching food; with the concurrent evolution of teeth, this entailed a major shift in the ecological role of vertebrates from detritivores to predators. The earliest jawed fishes, known as the Placoderms, appeared in the Late Silurian (about 430 MYA), became widespread in Devonian oceans and extinguished at the beginning of Carboniferous. Most Placoderms were bottom-dwellers living in shallow marine and freshwater habitats. The last common ancestor of extant gnatostomes (Eugnatostomes) probably diverged from a Placoderm lineage at the end of Silurian and populated brackish habitats along marine coasts. The eugnatostome lineage early diverged into the Chondrichthyes (cartilagineous fishes) and Osteichthies (bone fishes). The Chondrichthyes replaced bone tissue with a special type of cartilage and moved to the sea, splitting into the Elasmobranchii (sharks and rays) and Holocephali (chimaeras). The Chondrichtyes accumulate urea (a waste from organic nitrogen metabolism) in the blood and other extracellular fluid to counterbalance higher ion concentration in seawater while keeping sodium chloride below seawater level. The Osteichthyes retained a skeleton made of bone tissue and diversified into rayfinned (Actinopterygii) and lobe-finned fishes (Sarcopterygii). The ray fins of Actinopterygii consist of a web of skin supported by bone rays. The lobe fins of Sarcopterygii contain muscles and a robust internal skeleton joined to the vertebral column by a single bone articulated in ways recalling the tetrapod limbs. The Actinopterygii moved to the sea and became pelagic, but retained hyposmotic internal fluids relative to seawater; because of this, they are obliged to compensate water loss by osmosis through the gills by drinking seawater and actively excreting excess salt through the gills (sodium and chloride ions) and kidneys (calcium, magnesium and sulphate ions). The Sarcopterygii remained brackish-water dwellers or moved to freshwater habitats, with only a few lineages becoming marine. At the Silurian/Devonian transition (around 420 MYA), they split into the Rhipidistia (“lungfish”) and the Actinistia (coelacanths), extensively diversified to form the largest group of bony fishes during the Devonian, and extinguished almost completely in the Mesozoic (except the tetrapod lineage and a few residual forms, see below). The Actinopterygii became the dominant fish group in the post-Paleozoic and modern world, currently encompassing at least 30,000 living species. Being a transition zone between river and maritime environments, estuaries and coastal lagoons are subject both to marine influences such as tides, waves and the influx of saline water, and to riverine influences such as flows of freshwater and sediment. The inflows of seawater and freshwater provide a continuous supply of nutrients in the water column and sediment, making estuaries and coastal lagoons highly productive natural habitats, but also producing wide fluctuations in

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environmental parameters such as salinity, temperature and oxygen concentration. Adapting to this changing environment, ancestral bony fishes evolved primitive lungs in the form of a couple of sacs connected with the intestine just behind the pharynx. These permitted the animals to breath air when oxygen concentration in water was too low. Primitive lungs were inherited both in Actinopterygii and Sarcopterygii. When the Actinopterygii moved to the sea, where they found deeper water and a more stable oxygen level, the lungs were modified into a swim bladder, a gas-filled organ that helps the Actinopterygii to control buoyancy without wasting energy in swimming. The Actinopterygii retained the swim bladder also when they moved back from the sea to freshwater habitats, and in some cases (e.g. Amia calva) reconverted the swim bladder into a lung-like organ to extract oxygen from air when its concentration in water is insufficient (Hedrick and Jones 1999). Unlike the Actinopterygii, the Sarcopterygii retained the ancestral lungs. These reduced to a single lobe filled with adipose tissue in Latimeria chalumnae e L. menadoensis, two coelacanth species survived to the present and living in the sea depth. In contrast, the lungs are important respiratory organs in lungfishes (Dipnoi), a sarcopterygian lineage encompassing today six freshwater species, four living in Africa (Protopterus aethiophicus, P. amphibius, P. annectens), one in Australia (Neoceratodus forsteri), and one in South-America (Lepidosiren paradoxa). Of these, only Neoceratodus is able to breath both with gills (extracting oxygen from water) and with lungs, the other five species using only the lungs. Terrestrial vertebrates, or Tetrapoda, diverged about 365 MYA from a rhipidistian lineage (Laurin et al. 2000). The transition to the terrestrial environment entailed major changes in anatomy (notably the conversion of paired fins into four limbs, whereby the name Tetrapoda), as well as in physiology and reproductive biology (Perry and Sander 2004; Triques and Christoffersen 2009). The Tetrapoda inherited the choanes from their rhipidistian ancestor; these are two openings in the roof of the mouth connecting the mouth cavity with the outside through the nostrils. Most bone fishes lack choanes; instead they have an anterior and posterior nostril on each side of the head, one close to the upper jaw, the other near the eye, connected to each other by a canal outlined by an epithelium rich in olfactory sensorial cells. In the swimming fish, water enters the anterior nostrils and gets out through the posterior nostrils, permitting olfactory cells to detect molecules in solution. In Tetrapoda, the olfactory canals opens in the mouth cavity through the choanes. These permit the animal to breathe whilst keeping the mouth closed, so they probably evolved in rhipidists as a means to inhale air through the external nostrils located dorsally on the head, as crocodiles do. It has long been debated whether the choanes derived from nostrils or evolved independently; analysis of genes controlling choane development supports homology with posterior nostrils (Janvier 2004). Interestingly, living dipnoi (the surviving representatives of rhipidists) also have choanes, but these are anatomically different from tetrapod choanes and probably evolved independently. A possible transitional form between rhipidists and early amphibians is Tiktaalik roseae, an extinct lobe-finned fish known from three partial skeletons found in Canada and dated to about 375 MYA (Daeschler et al. 2006). The tiktaalik presents

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a mixture of fish characters, for example scales, gills and fins, and tetrapod characters, notably toracic fins attached to massive shoulders and provided with mobile distal joints, and a robust ribcage probably able to support the weight of the animal’s body out of water (Daeschler et al. 2006). More advanced tetrapodomorph forms combining a fishlike tail and gills with amphibian skull and limbs are four species of Ichtyostega and Tinirau clackae, all appeared at the end of Devonian and extinguished in the Carboniferous (Pierce et al. 2012; Swartz 2012). In the Carboniferous, between about 360 and 300 MYA, the climate became warm and humid; forests of arboreal lycopods, horsetails and ferns covered large equatorial and subequatorial areas, providing novel habitats that stimulated the evolution of terrestrial invertebrates, mainly arthropods. The abundance of animal prey favoured an adaptive radiation of tetrapods, leading to the evolution of Amphibians. These occupied the apical position in the food chain of terrestrial ecosystems until about 300 MYA, when the forest cover decreased dramatically and amniotic tetrapods became dominant. The amphibians have relatively inefficient lungs and need to supplement pulmonary respiration with cutaneous respiration. For this reason, they lost the scale cover present in their fish ancestors and evolved a highly permeable skin rich of mucous glands whose secretion keeps the body surface constantly moist. Amphibians still lay eggs in water and have external fertilization. Development encompasses an aquatic larval stage, the tadpole, that metamorphosates into the adult when grown to a suitable size. Amniotic Tetrapods (or simply Amniotes) overcame the ancestral dependence on water for reproduction by evolving internal fertilization (the male discharges sperms directly in the female oviduct), cleidoic eggs (eggs enclosed within a protective shell and supplied with a water reserve) and embryonic annexes. The latter are structures produced by the fertilized egg along with the embryo proper and used to assist embryo development. Of fundamental importance is the amnion, a liquid-filled vesicle that provides an aquatic environment for the developing embryo within the shell. These adaptations permitted the amniotes to lay eggs on land, thus severing the ancestral link with aquatic habitats. Amniotic eggs are much larger than amphibian eggs due to more abundant reserve material (with the exception of viviparous mammals), and produce young similar to the adults, thus bypassing the larval phase. Further innovations improving adaptation of amniotes to land include improved respiratory, cardio-vascular and excretory systems; no longer relying on cutaneous respiration, the amniotes covered their body with a thick layer of keratin and various types of protective appendages (scales, hairs, feathers) (Strasser et al. 2014; Wu et al. 2004). The first amniotes, referred to as “basal amniotes”, resembled small lizards and probably evolved from an anthracosaurian amphibian around 320 MYA (Shedlock and Edwards 2009), then rapidly diverging into the Synapsida (including mammals) and Sauropsida (including living reptiles and the birds). See Fig. 1.5 for a phyletic tree of amniotes.

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The Impact of Animals on the Global Environment

The appearance of animals followed by at least 1.8 billion years the Great Oxygenation event and probably had links with two major events marking the end of Proterozoic: the rise of oxygen concentration from 0.8% or less to over 8% with concurrent oxygenation of deep ocean waters, and a series of major glaciations that terminated around 600 MYA (Erwin 2015). The increase in oxygen concentration was probably pivotal to the evolution of energetically demanding organisms like the animals, and Proterozoic glaciations may have favoured the emergence of the animals in the immediate aftermath by causing the extinction of pre-existing organisms and creating novel ecological opportunities (Reinhard et al. 2016). The other way round, there is evidence showing that the first animals had relatively low oxygen requirements (Mills et al. 2014, 2018) and that their global diffusion was by itself a factor underpinning the rise of oxygen concentration recorded at the Proterozoic/Palaeozoic transition (Butterfield 2009, 2011). In a world dominated by microbial organisms and unicellular predators, the sinking of organic matter to the ocean bottom was quite slow, most of this being probably oxidised by decomposers along the way. Animal carcasses and faeces sink much more rapidly than the remains of unicellular organisms, thus the diffusion of animals in Cambrian oceans probably enhanced organic carbon sequestration; as discussed in Chap. 4, this entailed the accumulation of equivalent amounts of oxygen in the ocean/atmosphere/continental crust system. Organic carbon sequestration in sediments in the form of animal pellets might have been so efficient as to cause the virtual disappearance of organic matter suspended in the ocean water, formerly much more abundant. A second major effect of the animals on ocean ecosystems probably stemmed from the diffusion of filter-feeding nutrition, which reduced the bacterial component of phytoplankton and favoured the evolution of larger-celled eukaryotic phytoplankton (Butterfield 2011; Mills and Canfield 2016). Fossil biomarker molecules point to a shift in marine planktonic population accompanying the early radiation of animals, from primarily bacterial during the Proterozoic to primarily algal during the Phanerozoic. This caused a shift from the stratified, turbid and partly anoxic Proterozoic ocean to a clear-water, well-mixed and generally oxic Palaeozoic ocean dominated by eukaryotic algae (Butterfield 2011). Predation by animals was a powerful driver of macroalgal evolution and deeply influenced evolutionary innovation in land plants. For example, if there were no terrestrial herbivores, land plants would have not evolved the terrific diversity of secondary metabolites later exploited as a source of drugs by humans. Without pollinators, the angiosperms would have never emerged, and terrestrial ecosystems would still have the relatively simple structure as in Palaeozoic/Mesozoic. Without large terrestrial grazers, there would have been no savanna/grasslands, and humans would probably have never set foot on the planet. Once again, this shows how evolution proceeds from interaction of unrelated events under the combined action of chance and necessity.

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Chapter 11

Land Plants

The best time to plant a tree was twenty years ago; the next best time is now. (Confucius 551–479 BC)

Abstract Land plants (Embryophyta) appear in the fossil record from about 470 MYA. Phylogenomic analysis favours the Zygnematophyceae (Charophytes) as their closest algal relative. Early land plants probably inherited somatic desiccation tolerance (poikilohydry) from their charophycean ancestor. Major innovations underpinning plant terrestrialization include sporophyte interpolation in an ancestrally haplobiontic cycle and symbiotic association with mycorrhizal fungi. Poikilohydry and a unisporangiate sporophyte permanently dependent on the gametophyte are ancestral traits retained in extant bryophytes. The evolution of a branched, autonomous sporophyte led to the emergence of polysporangiophytes in Mid Silurian. Homeohydry (the control of water loss) and xylem (a lignified water-conducting tissue) gave polysporangiophytes access to a multitude of novel habitats and niches, driving a dramatic increase in the biological diversity and complexity of terrestrial ecosystems. Roots and leaves evolved multiple times during the Devonian. Seed evolution in Late Devonian severed ancestral dependence on liquid water for sexual reproduction. With the assistance of their fungal associates, land plants are powerful geochemical agents. Their diffusion caused a dramatic decline in carbon dioxide concentration and an unprecedented rise of oxygen. By reducing carbon dioxide level, land plants cooled the planet, creating the conditions for the establishment of the current climatic regime. Land plant cover increases local rainfall and is essential for long-term maintenance of climatic conditions favourable to life on continental masses.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_11

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Introduction

The land has long exerted an irresistible attraction on photosynthetic life, which on numerous occasions abandoned the safety of ancestral aquatic environments to colonize terrestrial habitats. The main reason for this ancient appeal is in enhanced access to mineral nutrients and carbon dioxide (CD), both of which often limit growth in aquatic habitats (Voesenek et al. 2006). Chemical and physical weathering of superficial rocks, in part directly due to the activity of living organisms, produces an abundance of mineral nutrients on the land (Lenton and Watson 2011).1 Considering carbon dioxide, the advantage offered by the subaerial (viz. terrestrial) environment is not in higher concentration, the atmosphere being in a chemical equilibrium with surface water, rather in the fact that the diffusion rate of CD in a gaseous phase is about 10,000 times higher than in solution. Life is most likely ancestrally marine, yet only freshwater organisms have been able to become fully terrestrial. The reason is simple: the water circulating in the subaerial environment is essentially rainwater, which is almost devoid of dissolved salt; because of this, terrestrialization requires pre-adaptation to low-osmolarity conditions. Interestingly, the same provision applies to heterotrophic life including animals, which were originally born in a marine or brackish environment but moved to terrestrial habitats – several times independently – only with the intermediation of freshwater forms. Cyanobacteria were probably the first photosynthetic dwellers of subaerial habitats (Lenton and Daines 2016; Wellman and Strother 2015). Photosynthetic eukaryotes followed much later, probably starting with green microalgae like those inhabiting today the soil and wet rock surfaces, and then lichen-like organisms similar to extant Geosyphon (Sect. 5.4). Colonization of land by these pioneer organisms led to the development of thin soils that later favoured the diffusion of land plants. The main difficulty that life encounters in the subaerial environment is water loss: without special adaptations, living matter in contact with air not saturated with water vapour tends to dehydrate to levels incompatible with normal physiological functions. The animals were able to perform the transition from water to land several times and with relative ease, because they ancestrally possessed an internal environment subject to homeostatic control; in addition, the animals had sensorial and locomotor organs that enabled them to detect and reach water sources. In order to adapt to subaerial life, numerous organisms resorted to desiccation tolerance (DT, also known as anhydrobiosis or cryptobiosis): the physiological ability to survive cytoplasmic dehydration. Desiccation tolerance independently evolved in bacteria, algae, protozoans, fungi, even in some small animals, being a common adaptation in habitats subject to drying and wetting cycles such as ponds,

1 Rock weathering is a minor source of nutrients to life in aquatic environments because the establishment of chemical equilibria between reagents and products rapidly stops rock dissolution. Consequently, the main source of mineral nutrients to aquatic ecosystems, including the oceans, is nutrient leakage from emerged continental masses.

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Introduction

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marshes, small rivers, intertidal areas, and soil (Alpert 2005). Despite its wide taxonomic distribution, DT is not a simple trait; it entails the ability to retain cellular integrity at a level of hydration as low as that of dry paper for months or years and rapidly resume normal metabolic activity upon wetting. To understand how extraordinary this is, it is useful to remember that water not only is the essential solvent for most metabolic reactions, but also directly affects the functional architecture of fundamental cellular components such as membranes and proteins. At the beginning of their evolutionary history, land plants resorted to DT (Oliver et al. 2000). Bryophytes (liverworts, mosses and hornworts) are basal land plants still depending on this ecological strategy (Oliver et al. 2005). In response to evolutionary pressure from subaerial life, a later-diverged lineage of land plants deployed a completely different strategy. These plants covered their body with a protective cuticle impermeable to water and gases but transparent to light, added tiny apertures to control gas exchange with the atmosphere, an internal system of air-filled spaces to facilitate internal CD diffusion, and rooting structures to absorb water from the soil. These adaptations enabled the plant body to maintain a high water content independently of short-term fluctuations in water availability in the environment. Both ecological strategies met with amazing biological success: land plants colonized continental masses over a strikingly wide range of latitude, altitude and climatic conditions (Dìaz et al. 2016), on several occasions also returning to freshwater habitats and even expanding to marine habitats (Aires et al. 2011). With over 400,000 species formally described and probably a substantial number yet undiscovered, land plants are the second largest eukaryote group, outnumbered only by the animals. Primary production on landmasses is higher than in the ocean despite emerged land accounts for only one third of total planet surface. Because of high productivity, terrestrial ecosystems evolved complex food chains and immense biological diversity at all taxonomic levels. The astounding success of land plants owes much to symbiotic association with fungi that help them extract mineral nutrients from superficial rocks (van der Heijden et al. 2015). The oldest fossils ascribed to land plants are Ordovician spores dated to about 475 MYA, at least 65 MY after the first animals (Wellman et al. 2003). Fossils dated to about 420 MYA document the appearance of plants capable of controlling their water content; at the end of Devonian around 360 MYA, a land flora including the first trees covered vast continental areas. Land plants belong to a lineage of green plants named Streptophyta (from the Greek streptos ¼ twisted, and phyton ¼ plant). Besides land plants, the Streptophyta encompasses the Charophytes, a group of about 4000 species of freshwater and soildwelling algae ranging from simple unicellular and filamentous forms to pseudoparenchymatous complex macrophytes (Delwiche and Cooper 2015; Domozych et al. 2017; Leliaert et al. 2012). In contrast to early-diverged streptophytic lineages, whose cells divide by centripetal furrowing, cell division in more advanced charophytes (Zygnematophyceae, Coleochaetophyceae, Charophyceae) and in land plants involves centrifugal deposition of a cell plate under the control of a distinctive microtubule system known as the phragmoplast (Graham et al. 2000). It is universally agreed that land plants arose from a charophyte ancestor, yet it is still debated

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whether the origin is in the Zygnematophyceae, Coleochaetophyceae or Charophyceae. Earlier phylogenomic studies pointed to the Coleochaetophyceae or Charophyceae, a satisfying solution considering the relatively complex anatomy of these algae, yet more recent work favours the Zygnematophyceae (Civàň et al. 2014; Ruhfel et al. 2014; Wickett et al. 2014; reviewed in Harrison 2017). Unlike land plants, zygnematophycean algae lack flagella/kinetosomes and plasmodesmata (Chap. 8) and encompass only unicellular and simple filamentous forms, the former probably being phylogenetically derived (Delwiche and Cooper 2015; de Vries and Archibald 2018). Despite this, Zygnema turned out to be the closest lineage to land plants in plastid-genome coding capacity in a range of streptophyte algae (Wickett et al. 2014). Interestingly, structural divergences suggest that plasmodesmata evolved independently in land plants and Coleochaetophyceae/Charophyceae (Brunkard and Zambryski 2016), thus removing a potential obstacle to the acceptance of a zygnematophycean ancestry of land plants. A major innovation of land plants is a parenchymatous organization (Chap. 8) arising from stem cells dividing along three or more planes. With few exceptions (e.g. stoma guard cells and reproductive cells), the cells of land plant body are interconnected by plasmodesmata, thus forming a supra-cellular compartment known as the symplast; the cell walls and water-filled internal capillary spaces form an extracellular physiological compartment called apoplast. To enter the symplast, water and molecules in solution must cross a cell membrane but, once inside, they can freely move over supra-cellular distances by diffusion or mass flow (Glossary). Accession to the apoplast does not need crossing of a biological membrane.

11.2

An Early Step in Plant Terrestrialization Was a Change in the Ancestral Life Cycle

Land plants have a haplo-diplontic life cycle encompassing a haploid gametophyte that alternates with a diploid sporophyte (Fig. 11.1a). This type of life cycle is rather common among algae, for example Ulva (Chlorophyta) and laminarialean brown algae. A distinctive aspect of the reproductive cycle in land plants is that the sporophyte, at least at an early stage of its life cycle, is bound to the gametophyte and receives nutrients from this through the placenta, a specialized tissue located at the interface between the two generations. This condition is called matrotrophy, i.e. mother-mediated nutrition (Graham and Wilcox 2000); with reference to the juvenile phase of the sporophyte, land plants are formally referred to as embryophytes, i.e. plants with an embryo. The interpolation or antithetic theory proposed by Czech botanist Ladislav J. Čelakovský in 1874 and subsequently re-elaborated by Frederick Bower, holds that the algal ancestor of land plants had a haplontic life cycle (Fig. 11.1b) and that the sporophyte appeared de novo by interpolation of a number of mitotic cell divisions between syngamy and meiosis.

11.2

An Early Step in Plant Terrestrialization Was a Change in the Ancestral Life Cycle

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Fig. 11.1 Reproductive cycle in land plants (a) and charophytes (b). According to the antithetic theory, a sporophytic generation evolved de novo in the algal ancestor of land plants by interpolation of a number of mitotic divisions between syngamy and meiosis. Whereas a single zygote produces only four spores, a multicellular sporophyte can multiply this number by orders of magnitude, thus amplifying the outcome of sexual reproduction. Producing a larger number of spores is a favourable character on land because, unlike gametes, the spores tolerate desiccation and can freely diffuse in the air. The zygote of charophyte algae has a thick sporopollenin wall and survives drought in a dormancy condition; upon germination, it divides by meiosis into four haploid spores, usually flagellate, which produce an adult organism by mitosis (b). In land plants, the four spores produced by meiosis deposit a sporopollenin coat before release from the mother cell wall; this results in the formation of a distinctive Y-shaped structure on the inner (proximal) side of the spores, known as the “trilete mark”. Fossil spores with a trilete mark dated to about 475 MYA are the earliest paleontological evidence of land plants (Wellman et al. 2003). We may think of land plants as haplodiplontic streptophytes

The homologous theory proposed by Nathanael Pringsheim in 1878, instead, maintains that the algal ancestor of land plants had an alternation of generations, with morphologically similar and developmentally independent gametophyte and sporophyte, and that the sporophyte secondarily became dependent on the gametophyte because of terrestrialization (Blackwell 2003; Haig 2008). For over 40 years, the homologous theory enjoyed great consensus, despite obvious difficulty at explaining the divergence between the life cycle of bryophytes (in which the gametophyte is the dominant generation and the sporophyte is permanently dependent on the gametophyte) and the rest of embryophytes (in which the sporophyte is dominant and becomes autonomous after a short embryonic phase). Starting in the 1980s, strong evidence of a phyletic connection of embryophytes with charophytes, which have a haplontic life cycle as Bower predicted (Fig. 11.1b), led to a gradual dismissal of the homologous theory in favour of the antagonist model (Niklas and Kutschera 2010). According to the antithetic model, the sporophyte of early land plants was extremely simple, a mass of cells that at a point shifted from mitosis to meiosis, producing haploid spores. With time, the sporophyte elaborated a more complex body plan, developing a haustorium (or sporophyte foot) that enhanced nutrient uptake from the parental gametophyte, and an involucre of sterile cells that protected fertile cells within a sporangium. The sporophyte in some present-living bryophytes, for example the moss Tortula, does not go much further (Fig. 11.2a), and in most

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Fig. 11.2 (a) Scanning electron microscope image of the sporophyte in the early-diverged moss Tortula ruralis, consisting of a sporangium (sp) and a conical foot (p, indicated with a dotted line) embedded in the gametophyte tissue (ga). At maturity, the sporangium splits along four longitudinal lines to liberate the spores in the air. In this moss, the gametophyte produces a filament (or pseudopodium) that raises the sporangium to facilitate spore dispersal. (b) Sporophytes of the liverwort Monoclea forsteri, consisting of a sporangium (sp), a long seta (se) and a foot (not visible) embedded in the gametophyte tissue (ga); the sporangia split along a single line of dehiscence. In most mosses and liverworts, the sporophyte has a prominent seta (the lack of a seta in Tortula is probably due to reduction), but differences in development and inner structure raise doubts about homology in the two lineages. (Pictures kindly provided by Jeffrey G Duckett, Natural History Museum, London)

bryophytes the only addition to this basic body plan is the seta, an axial structure that helps the sporangium to emerge from gametophytic involucres and raises it in the air, facilitating spore dispersal (Fig. 11.2b). In polysporangiophytes, a later-diverged lineage of land plants, the sporophyte elaborated a more complex body plan, in part recruiting gametophytic structures, in part adopting original solutions, and emancipated from ancestral dependence on the gametophyte (Graham et al. 2000). Charophytes typically inhabit ponds, irrigating canals or the banks of meandering rivers, shallow habitats cyclically subject to flooding and drought. Under favourable conditions, charophytes reproduce asexually by zoospores, fragmentation, or simple mitosis in unicellular forms, resorting to sexual reproduction under stressing conditions such as overcrowding or high temperature due to water body shrinking. The algae usually survive seasonal drought in the form of desiccation-tolerant zygotes invested with a sporopollenin coat. Transported by the wind, animals or water itself, dormant zygotes may diffuse over long distances, thus functioning as stress-surviving and propagating structures. Upon wetting, the zygote swells, breaks the protective cell wall, and divides by meiosis into four haploid spores (only one of which survives in the Charophyceae); being the product of meiotic division, these are called meiospores. Each meiospore divides mitotically and produces a haploid organism that, in the appropriate conditions, complete the sexual reproductive cycle forming gametes by mitosis (Fig. 11.1b). In multicellular charophytes, including filamentous Zygnematophyceae, egg cell fertilization occurs within the parental organism and

11.2

An Early Step in Plant Terrestrialization Was a Change in the Ancestral Life Cycle

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the zygote is usually liberated only when the parental organism ages and disintegrates; in C. orbicularis the tissue surrounding the zygote develops a placenta-like structure recalling the embryophyte placenta (Graham and Wilcox 2003). Why did adaptation to subaerial life add a diploid multicellular phase to this cycle? To answer this question it is useful to examine the reproductive cycle of a typical bryophyte, which is presumably the closest extant proxy to the cycle of early land plants. After fertilization, the zygote is retained in the parental gametophyte and divides repeatedly by mitosis, producing a number N of spore mother cells (SMCs), each functionally equivalent to a charophytic zygote. Unlike this, however, SMCs do not form a sporopollenin wall nor become quiescent; instead they divide by meiosis, each producing four spores (the so-called spore tetrad). After completion of meiosis, individual spores deposit a sporopollenin-containing new wall, then dehydrate and suspend metabolic activity, thus turning into highly effective propagating structures. In more formal terms, the genetic program that controls sporopollenin deposition is expressed before meiosis in charophytic algae, after meiosis in land plants (Harrison 2017). The advantage of this simple change is evident when considering that a subaerial environment is much less favourable to syngamy than the aquatic medium. Sporophyte interpolation amplifies the reproductive output of each successful syngamy by N times, with N ranging from a few hundred to several thousand in bryophytes. When the sporophyte became more complex, N increased by orders of magnitude, each sporophyte being able to produce millions or billions of spores at each reproductive season. Sporopollenin is a substance of extraordinary resistance to biological and nonbiological agents. It forms the outermost wall layer of land plant spores, known as exine and often bearing distinctive superficial ornamentations (Fig. 11.3). From the chemical standpoint, sporopollenin is a highly cross-linked polymer containing fatty acids, phenolic and phenylpropanoid groups, plus traces of carotenoids, with a molecular structure not yet known in detail. Sporopollenin is thus empirically defined as the resistant, non-soluble residue left after treatment of biological materials with a hot mixture of acetic anhydride and concentrate sulphuric acid, which completely dissolves all other organic molecules present. Unsurprisingly, sporopollenin coats persisted in fossils for hundred million years, often preserving minute structural details useful for taxonomic identification. Spore exines evolved to be physically and chemically resilient, light shielding, elastic microcapsules; despite their toughness, spore exines present a myriad of nano-channels that permit water movement during cellular dehydration and rehydration (Mackenzie et al. 2015). Sporopollenin-like compounds also occur in chlorophytes, dinoflagellates and Dictyostelida, where they form protective coats of spores, cysts or other types of stress-tolerant cells. What is the function of sporopollenin? Here and there, the scientific literature reports vague comments about a protective role, sometimes also suggesting protection against desiccation, which is wrong because sporopollenin-coated cells are desiccation tolerant and sporopollenin coatings are permeable to water, due to their nanoporous structure. The most likely explanation for the widespread use of such an expensive material is protection against predators such as nematodes and myzocytic protists (Sect. 9.3).

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Fig. 11.3 Spores of Lycopodium clavatum as seen with a scanning electron microscope. The outermost layer of the sporal wall, known as the exine, is made of sporopollenin and presents species-specific ornamentations. The arrow points to the Y-shaped relief (known as the “trilete mark”) on the proximal side (i.e. the spore side oriented inward in the spore tetrad), which proves the meiotic origin of these spores. (From Taboada-Diego et al. (2014), Creative Commons Attribution 3.0 International License)

11.3

Early Land Plants

In line with the interpolation model and with the support of molecular phylogeny pointing to bryophytes as the earliest-diverged extant land plants, we may infer that the gametophyte was the dominant generation in early land plants. Unfortunately, no fossil remain has yet been discovered that gives a cue about how these ancient plants looked like. The hypothetical model shown in Fig. 11.4 suggests that the ancestral gametophyte consisted of parenchymatous bifurcating axes attached to the substrate by means of rhizoids (unicellular hairs ubiquitous in embryophytes and present in some charophyte algae, Jones and Dolan 2012). The sporophytes were permanently bound to gametophytes and lived just the time necessary for spore maturation. These plants might have lived in shallow water at the edges of ponds or marches, with sporophyte-bearing axes emerging to surface to permit spore dispersal in the air. We may even conjecture that partial desiccation of the water body acted as a signal inducing sexual reproduction and spore formation. This reproductive strategy is similar to that of living charophycean macrophytes, just with the addition of a sporophyte that amplified the reproductive outcome of each syngamy, and the use of air-borne spores instead of zygotes for survival and diffusion. Somatic desiccation tolerance, also referred to as poikilohydry (from Greek poikilos, variable, and hydor, water), is present in most extant bryophytes (both the gametophyte and sporophyte), with wide taxonomic differences in the level of tolerance (Oliver et al. 2000; Wood 2007). Poikilohydry is an essential prerequisite for the successful transition from water to land of a morphologically simple

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Early Land Plants

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Fig. 11.4 Hypothetical reconstruction of an Ordovician land plant (around 470 MYA). The gametophyte consisted of parenchymatous bifurcating axes living in shallow freshwater and attached to the bottom by rhizoids. Fertilization occurred in water and produced bryophyte-like sporophytes that at maturity emerged to surface and liberated the spores in the air. Amphibious plants like these probably expressed somatic desiccation tolerance

organism, thus it must have evolved very early in land plants if not present from the beginning. Little is known on the distribution of desiccation tolerance in charophytes. Many unicellular charophytes express the trait in all phases of the reproductive cycle (Graham et al. 2014; Holzinger and Karsten 2013; Rippin et al. 2017) and the same holds true for two Coleochaete species (Graham et al. 2012). Morphologically more complex forms such as Chara or Nitella, generally living in relatively stable and deep water bodies, express DT in the zygotes but their vegetative bodies do not survive desiccation. This limited evidence suggests that land plants inherited the gene set for desiccation tolerance from their charophytic ancestor. Poikilohydry independently evolved in fungi and even in animals such as rotifers, tardigrads, nematods, crustaceans and arthropods, accompanying adaptation to habitats subject to periodical drought (Alpert 2005). Poikilohydric organisms not only tolerate dehydration but are often also resistant to other types of stress associated with drought, notably high temperatures and high light irradiance. In fact, bryophytes and lichens are among the first organisms that colonize naked rocks and start soil formation. Poikilohydry, however, comes at a cost: poikilohydric organisms are active only in the hydrated state and stop metabolic activity as soon as cellular water content drops below a threshold level. Because of this, poikilohydric plants grow slowly, and even slower are lichens; in addition, poikilohydric plants tend to be small because desiccation tolerance does not fit well with hydraulics principles underlying water transport over long distances (Sect. 11.4). It is now clear that when we talk about land plants we do refer to two organisms, the gametophyte and sporophyte, which share the same genome (two homologous sets in the sporophyte) but are subject to diverging evolutionary pressures. Initially small and ephemeral, consisting of an unbranched axis with determinate growth and a single terminal sporangium, the sporophyte turned out to be by far better suited

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Fig. 11.5 Rhynia gwynne-vaughanii, a Devonian polysporangiate plant living over 400 MYA. (a) The sporophyte consisted of erect bifurcating axes bearing terminal sporangia, and prostrate axes attached to the soil by rhizoid tufts. (b) Transverse section of a sporophyte axis showing amazing preservation of cellular details; the epidermis had a distinct cuticle and stomata, the cortical parenchyma had air spaces, and internally there was a strand of vascular tissue (the black spot enclosed in dotted circle. (c) Reconstruction of the Rhynia gametophyte, originally classified as Remyophyton delicatum. Although much smaller that the sporophyte (cf. scale bars in a, c), the gametophyte had stomata and vascular tissue; sexes were separate, i.e. there were male and female gametophytes. (a Credit: Françoise Gantet. b From: https://s10.lite.msu.edu/res/msu/botonl/b_ online/library/steur/rhynie/gwynsten.html. c Redrawn from Kerp et al. 2004)

than the gametophyte to life under subaerial conditions, became autonomous and evolved a complex body plan. Concurrently, the initially dominant gametophyte went through structural simplification culminating in the reduction to a few cells in angiosperms (Graham et al. 2000; Kenrick 2017; Qiu et al. 2012). Particularly informative about the evolutionary history of early land plants is the fossil record present in the Rhynie Chert, a formation near the Rhynie village, Scotland. The Rhynie Chert contains extraordinarily well preserved remains not only of plants but also of animals and fungi from the Early Devonian (about 407 MYA), and since its discovery in 1910 by William Mackie it has been an inexhaustible source of material for generations of palaeontologists. Other sites of similar age occur in Wales (UK), Canada, China and Brazil. Fossils from these sites show that the land flora between 420 and 400 MYA included free-living vascularized sporophytes (Fig. 11.5). Ancient plants such as Cooksonia, Rhynia, Aglaophyton are the first known “polysporangiophytes”, plants with a free-living branched sporophyte producing several sporangia (Kenrick and Crane 1991, 1997; Willis and McElwain 2014). Polysporangiate sporophytes grow by the activity of an apical meristem that develops after an embryonic phase and remains active for the duration of life; thus the polysporangiate sporophyte has indeterminate growth and an open form (Chap. 8). In contrast, the unisporangiate sporophyte of bryophytes lacks a post-embryonic apical meristem and its body plan is entirely determined during embryo development, whereas the bryophyte gametophyte has a well-defined apical meristem. It is therefore reasonable to expect that the

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Early Land Plants

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apical meristem of the polysporangiate sporophyte evolved by deploying the gene toolkit that controls apical growth in the gametophyte. Quite surprisingly, genomic analysis has revealed that Class 1 KNOX genes are essential for the function and maintenance of the sporophyte apical meristem in polysporangiophytes but are not expressed in the gametophyte apical meristem of the model moss Physcomitrella (Sakakibara et al. 2008). In addition, Class 2 KNOX genes are essential to prevent expression of the gametophytic developmental pattern in the sporophyte of Physcomitrella (Sakakibara et al. 2013). This suggests that the sporophyte of polysporangiophytes evolved an apical meristem de novo, without using the preexisting gametophyte pattern. With the evolution of an apical meristem, the sporophyte became able to grow indeterminately and to branch by meristem duplication (Ligrone et al. 2012); arguably, this required that in parallel the sporophyte gained autonomy, because the gametophyte would have been unable to support an overgrown sporophyte. In fact, after a short initial phase during which it depends on the gametophyte, the sporophyte of extant polysporangiophytes establishes a contact with the substrate by means of rhizoids and becomes autonomous. It is appropriate to anticipate here that molecular phylogeny consistently resolves extant polysporangiophytes (also called polysporangiate plants or polysporangiates) as a clade (Box 1.1); in contrast, the phyletic status of bryophytes is contentious. Robust evidence supports paraphyly, with liverworts basal to the rest of land plants and the hornworts sister to polysporangiates (Liu et al. 2014; Qiu et al. 2006; Qiu 2008; Fig. 11.6). More recent phylogenomic work, however, advocates bryophyte monophyly or alternatively resolves the hornworts as the sister group to the rest of land plants (Cox et al. 2014; Wickett et al. 2014; Puttick et al. 2018; Sect. 11.5). Early Devonian polysporangiate sporophytes present three important traits: (i) a thick epidermal cuticle and stomata (Sect. 11.4), (ii) air spaces in subepidermal tissue, (iii) a central strand of vascular cells. Most likely, these sporophytes had evolved homeohydry, a novel ecological strategy that radically changed plant interactions with the subaerial environment (variants of the term such as homoiohydry and homiohydry are spelling mistakes consolidated in use). Homeohydric plants are able to control gas exchanges with the atmosphere; in the hydrated condition, they open the stomata to take up CD for photoassimilation; doing so, they lose water in the form of vapour and, if water uptake from soil does not cover water loss, incipient dehydration induces stomatal closure. This mechanism enables homeohydric plants to maintain a sufficient water content to support physiological activity independently of short-term fluctuations in environmental water availability (hours, days or months, depending on the species). Extant polysporangiate plants are homeohydric and usually do not express DT in vegetative tissues; therefore they die if the water content in their tissues drops below a critical level. All polysporangiate plants, however, retain the gene toolkit for DT and express it in the spores. Seed plants (Sect. 11.2) express DT also in the seeds, which in these plants substitute the spores as dispersing units. In particularly adverse habitats, a number of polysporangiate lineages secondarily evolved a form of poikilohydry by expressing DT in the vegetative body. These are sometimes called “resurrection plants” because in the dry state they look like they

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Fig. 11.6 (a) The stomata are smart valves located in the epidermis of non-woody aerial parts of the sporophyte. Their development is under the control of the one-cell-spacing rule, by which stomata are always separated by at least one normal epidermal cell. (b–c) Stomatal opening/closing is driven by osmotic force depending on the movement of ions (notably K+, represented as spots) and water in and out of guard cells. In modern homeohydric plants, the stomata respond to multiple factors including tissue hydration level, carbon dioxide (CD) concentration in air spaces, light intensity and wavelength, temperature and hormones. Light induces stomatal opening, the dark (or low light intensity) induces closing (with the exception of plants using CAM, a variant of the carbon-fixation photosynthetic pathway). Independent of light conditions, the stomata close if CD in air spaces exceeds a threshold level. Water stress by dehydration induces stomatal closing independent of light conditions and CD concentration. The stomatal apparatus is a minute computing system capable of modulating the aperture between a maximum and zero in response to the absolute and relative intensity of the signals perceived. This affords the plant a tight control of stomatal conductance, a fundamental physiological parameter expressed as millimoles of CD entering or water exiting the plant per square meter leaf surface per second. (Pictures By Ali Zifan – https://commons.wikimedia.org/w/index.php?curid¼50023738)

11.4

Stomata and Homeohydry

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were dead but return green and active in a few days after wetting. Secondary poikilohydry evolved several times independently in lycophytes, ferns and angiosperms, with different taxa putting in place partly divergent adaptive solutions (Rascio and La Rocca 2005; Farrant and Moore 2011). Unlike primary poikilohydric land plants (today represented by bryophytes), resurrection plants do not express DT constitutively but only in response to severe water stress. In addition, they do not usually tolerate fast rates of dehydration or frequent cycles of dehydration/rehydration, and undergo structural and physiological damage during dehydration; consequently, they resume growth only after a repairing phase of varying duration. What became of the gametophyte whilst the sporophyte proceeded in terrestrialization? The gametophyte of extant polysporangiophytes is small, morphologically simple and lacks stomata and vascular tissue. It generally develops during the wet season and is short-lived, persisting just the time necessary to produce one or a few sporophytes. In some tropical ferns the gametophyte is poikilohydric and long-lived, yet it remains dependent on liquid water for fertilization (Watkins et al. 2007). We will see that seed plants completely reversed the ancestral reproductive cycle by reducing the gametophyte to a few-celled organism developing within the sporophyte without ever establishing a contact with soil. Fossils from the Rhynie Chert and other sites in the world have revealed that some Devonian gametophytes had stomata and vascular tissue, although these gametophytes were much smaller than conspecific sporophytes (Kerp et al. 2004; Taylor et al. 2005; Fig. 11.5). This suggests that, in some early lineages, the gametophyte followed the sporophyte on the evolutionary pathway towards homeohydry, but subsequently the tendency prevailed towards anatomical and physiological simplification in favour of the spore-producing generation.

11.4

Stomata and Homeohydry

Cuticle, stomata and air spaces were fundamental innovations that enabled land plants to control gas exchange with the atmosphere and the rate of water loss (Raven 2002). The stomata consist of a couple of guard cells and an aperture that opens on a gasfilled chamber continuous with a system of air spaces permeating the parenchymatous body of the plant (Fig. 11.6). Although poikilohydric, moss and hornwort sporophytes do have stomata, air spaces and a cuticle, the latter being structurally simpler than in homeohydric plants but affording the sporophyte better insulation compared with the gametophyte (Ligrone et al. 2012; Duckett and Pressel 2017). The occurrence of stomata in bryophytes raises the question whether (a) stomata arose once in the putative concestor of a “stomatophyte” lineage including mosses, hornworts and polysporangiophytes, (b) evolved independently in separate lineages, or (c) evolved once in the last concestor of all extant embryophytes and were lost in some lineages (Rensing 2018).

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Stomatal development and patterning in the polysporangiate plant Arabidopsis is under the control of a genetic toolbox including the basic helix-loop-helix (bHLH) transcription factor genes SPEECHLESS, MUTE, FAMA, ICE/SCREAMs, as well as EPFs genes encoding for “mobile epidermal patterning” peptides. The discovery that orthologs of these genes are also present in mosses and hornworts and are required for stomatal development in Physcomitrella points to single stomatal ancestry (Chater et al. 2016, 2017). This conclusion fits well with phyletic trees placing the liverworts in a basal position and hornworts in a sister position to polysporangiophytes (Qiu et al. 2006; Qiu 2008; Fig. 11.9). Alternative topologies (Cox et al. 2014; Wickett et al. 2014; Puttick et al. 2018) imply multiple stomatal evolution (a less parsimonious solution contrasting with evidence from genomics), or stomatal loss in liverworts (a less parsimonious solution with no or very faint support in genomics, see Chater et al. 2017). Substomatal cavities in moss/hornwort sporophytes are liquid-filled prior to stomatal opening and become air-filled after opening. In contrast, air spaces in polysporangiophyte leaves are gas-filled before stomatal opening, a difference claimed to support independent stomatal origin in the two lineages (Duckett and Pressel 2017). Stomatal opening and closing cycles in advanced polysporangiophytes (seed plants) depend on both passive and active regulation. Passive stomatal movement is directly determined by variations in turgor pressure; active regulation depends on physiological responses to light, carbon dioxide concentration and abscisic acid (ABA) a plant hormone involved in plant response to water stress (Lawson 2009). The relative importance of passive and active regulatory pathways in bryophyte stomata is controversial. Early experiments (reviewed in Merced and Renzaglia 2017) demonstrated that moss stomata respond to light/dark signals and abscisic acid (ABA) in a similar way as polysporangiophytes. In line with these observations, Chater et al. (2011) reported a closing response of stomata to ABA, elevated CO2 concentration and darkness in expanding sporophytes of the mosses Physcomitrella and Funaria. In contrast, hornwort stomata are unresponsive to ABA and lack diurnal cycles, remaining permanently open in mature parts of the sporangium (Pressel et al. 2014; Renzaglia et al. 2017). The occurrence of stomata in moss and hornwort sporophytes is somewhat puzzling, as these are poikilohydric plants just like their parental gametophytes, although they are more resilient to water loss, probably because of more efficient waterproofing (Duckett and Pressel 2017). In several moss taxa, sporophyte growth after stomatal opening is negligible relative to pre-opening growth, suggesting that stomata have minor importance in sporophyte growth (Duckett and Pressel 2017). Likewise, mutation-induced loss of stomata in Physcomitrella sporophytes did not affect sporophyte growth but only resulted in delayed capsule dehiscence (Chater et al. 2016). These data support the view that the major function of bryophyte stomata is in assisting capsule dehydration and dehiscence in order to facilitate spore dispersal (Pressel et al. 2014). This inference, however, is at odds with the lack of stomata in polysporangiate sporangia including large-sized sporangia of eusporangiate ferns (Ligrone et al. 2012).

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Stomata and Homeohydry

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Fig. 11.7 Hypothetical transition from a bryophyte-grade unisporangiate sporophyte (a) to a freeliving polysporangiate sporophyte (b and c). Key innovations underpinning sporophyte emancipation were a persistent apical meristem (arrows) and the development of rhizoids; the first evolved de novo, the latter recruited a gene set already expressed in the gametophyte. Once free from gametophytic constraints, the sporophyte proceeded towards full homeohydry by evolving a better waterproofing cuticle and more responsive stomatal physiology. The model assumes that polysporangiophyte stomata are homologous with stomata in extant bryophytes, both having been inherited from a bryophyte-grade concestor. Alternative models assume independent evolution. The black spots represent stomata. (From Ligrone et al. 2012, licence number 4470101238513)

The distribution of stomata in mosses is quite erratic, with taxa possessing stomata and others lacking stomata, sometimes within the same order (Duckett and Pressel 2017); among hornworts, four genera over nine lack stomata (Merced and Renzaglia 2017). The erratic taxonomic distribution and inconsistent morphology/ physiology of bryophyte stomata most likely reflect a cost/benefit evolutionary trade off underpinning multiple loss or functional reduction. In a scenario assuming stomatal monophyly, the sporophyte of early polysporangiophytes inherited stomata, air spaces and a cuticle from a bryophytegrade ancestor (i.e. an ancestor with a bryophytic life cycle). This pre-adaptation may have facilitated the transition to homeohydry when the sporophyte became autonomous (Fig. 11.7). Besides a permanent apical meristem and branching, sporophyte emancipation required the expression of rhizoids, rooting structures that in bryophytes are uniquely gametophytic. The discovery that rhizoid expression is under the control of orthologous genes in the bryophyte gametophyte and polysporangiophyte sporophyte suggests that rhizoid expression in the sporophyte recruited a pre-existing gametophytic gene set (Jones and Dolan 2012; Tam et al. 2015; Honkanen et al. 2016; Proust et al. 2016). Homeohydry gave plants access to a completely new evolutionary landscape, thus engendering a dramatic increase in biological diversity and structural complexity of terrestrial ecosystems.

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Fig. 11.8 (a) Structure of the cuticle covering non-woody aerial parts of homeohydric plants. The waterproofing properties of the cuticle essentially depend on the layer referred to as the “cuticle proper”. (b) The cutin backbone consists of long-chain hydroxyacid monomers linked by ester bonds. (Redrawn from Yeats and Rose (2013))

An efficient waterproofing coat is as important as stomata in homeohydry. A layer of hydrophobic material known as the cuticle covers the leaves and young shoots of extant homeohydric plants. Although exceedingly thin, rarely exceeding 10 μm in thickness, the cuticle is a formidable barrier to the diffusion of gases and water. The cuticle is a chemically complex structure consisting of cutin and waxes, with the addition in some plants of a third component called cutan. Cutin is a branched polymer of inter-esterified hydroxyl fatty acids, with lesser amounts of glycerol, phenylpropanoids, and dicarboxylic acids. Waxes are heterogeneous ethers of longchain acids, alcohol and aldehydes (usually 20–40 C atoms). Cutan is a polymer of aliphatic chains of 22–34 C atoms linked together by ether bonds. Organic solvents dissolve waxes but not cutin or cutan (Bargel et al. 2006; Fernàndez et al. 2016). The cuticle consists of an external waxy layer (the epicuticular layer), a middle layer with a distinctive multilamellar structure (the cuticle proper) made of cutin associated with waxes, and an inner layer with a more homogeneous structure (the cuticular layer), made of cutin associated with cell wall polysaccharides (Fig. 11.8). Cutan, when present, is associated with cutin. In many plants, the epicuticular wax forms crystalline aggregates that confer the leaves and fruits a distinctive glaucous appearance. The cuticle proper almost entirely accounts for the waterproofing properties of the cuticle (Yeats and Rose 2013). Extraction of cuticular waxes with organic solvents increases cuticle permeability to water and dissolved solutes by 100- to 1000-fold, suggesting that the water barrier properties of the cuticle mainly depend on cuticular waxes, whereas the cutin polymer forms a supporting scaffold (Schreiber 2010).

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Stomata and Homeohydry

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Wax and cutin precursors are synthesized in the endoplasmic reticulum of epidermal cells and exported across the plasma membrane; they diffuse through the polysaccharide cell wall to the nascent cuticular layer and here they polymerize, possibly by self-assembly (Domìnguez et al. 2010). The mechanisms responsible for precursor transport across the cell membrane, diffusion through the cell walls and final polymerization are only partially known (Yeats and Rose 2013). Besides reducing the permeability of the epidermis to water and gases, the cuticle performs other important functions, notably plant protection from mechanical damage and potential pathogens such as viruses, bacteria or fungi. In many species, elaborate epicuticular crystals form a self-cleaning surface preventing dust and other debris from blocking sunlight on leaf surface; in some cases, the cuticle also acts as a screen to excessive ultraviolet radiation (Bargel et al. 2006; Yeats and Rose 2013). For obvious reasons, a cuticle as that present in aerial parts is lacking in the root. The root epidermis has a thin cover of material insoluble in organic solvents. A similar layer also coats the cell walls facing the air spaces both in the root and aerial parts of the plant (see below). This coating is much thinner than the cuticle (only about 10 nm), often shows a tripartite structure when examined under an electron microscope, and is similar to the first stage of cuticle development in aerial organs, called “procuticle” (Jeffree 2006). The coating obviously does not prevent water or ion movement, yet it may have a role in (a) protecting the plant from pathogens or saprophytes, (b) facilitating cell detachment during the genesis of air spaces, (c) establishing physical inter-organ boundaries during the development of leaves, flowers, inflorescences and lateral roots, thus preventing accidental fusion. A similar coating also invests the external surface of the plant body in bryophytes and charophytes such as Chara and Nitella, thus it is probably an ancient structure (Cook and Graham 1998; Jeffree 2006). Besides a cuticle and stomata, the third anatomical trait underpinning homeohydry is a system of gas-filled intercellular spaces, or simply air spaces. This is a major compartment of the plant body, accounting for about 5% of the total volume (Raven 1996) and functionally distinct from the symplast and apoplast (the latter being filled with water in the active plant). The air space system is an internal controlled environment that permits the plant to optimize CD uptake and water use. With the stomata open, CD uptake by photosynthetic cells facing the air spaces creates a steep diffusion gradient between the inside and outside; in contrast, under typical environmental conditions, the formation of a relatively moist layer in the close proximity of the plant (the so-called boundary layer) tends to reduce the water vapour diffusion gradient (Niklas 2000). The possibility to close the stomata when water uptake from soil is insufficient to balance water loss permits homeohydric plants to avoid dehydration, although at the cost of interrupting photosynthetic assimilation. Air spaces normally develop with a schizogenous mechanism, i.e. by partial separation of cell walls following specific chemical modifications. Whereas the biochemistry of cell wall separation underpinning abscission, for example the detachment of old leaves and mature fruits, is known in remarkable detail, current knowledge of air space development is surprisingly poor considering the importance of the topic

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(Ishizaki 2015). In pea root tissues, a ‘splitting layer’ structurally and chemically distinct from the pre-existing cell wall develops in the areas of prospective splitting, starting from the cell corners (Roland 1978). The motive force for cell detachment is cell expansion driven by turgor pressure. Air spaces develop in young leaves before stomatal opening; air space development in completely submerged plants kept in the dark excludes dependence on external sources of gas or photosynthetic production of oxygen. Raven (1996) suggests that the spaces may initially be filled by water vapour produced by cavitation as tension builds, followed by gas release from the intracellular water solution. The hydrophobic properties of the cuticle prevent water penetration through stomata and infiltration of air spaces upon wetting. Lysigenous air spaces develop from dead cells whose cytoplasmic contents, and in certain cases the cell walls as well, are enzymatically dissolved and the excess water reabsorbed by neighbouring living cells. Extensive air spaces of lysigenous origin develop in the stems, petioles and roots of plants such as bamboo and grasses. Vascular tissue is a fourth anatomical trait invariably associated with homeohydry in modern plants. Since the combination of a cuticle and stomata can reduce water loss to very low levels, homeohydry might have evolved in the absence of vascular tissue, provided the plant body remained small. Vascular tissue, however, was a necessary requisite for fully exploiting the evolutionary potential of homeohydry. Several mosses and liverworts have vascular systems based on capillarity. Water transport mediated by capillarity rapidly becomes inefficient over distances exceeding a few cm. Competition for light and mineral nutrients selected for larger sizes in polysporangiophytes, therefore demanding a vascular system not relying on simple capillarity. The evolutionary response was a miracle of hydraulics engineering, a novel tissue capable of transporting substantial amounts of water to over 100 m height using the difference in water potential between the soil and atmosphere as the only source of power (Sect. 11.6).

11.5

Land Plant Systematics

Although of common use, the denomination “land plants” is somewhat ambiguous. Indeed, poikilohydric soil-dwelling green algae like Klebsormidium or Chlorella are by all regards land plants, whereas true land plants such as Elodea canadensis or Posidonia oceanica are fully aquatic organisms. The denomination “embryophytes” is certainly less ambiguous, although a sporophyte associated with the gametophyte also occurs in the triphasic reproductive cycle of floridean red algae. Land plants are distinguished into unisporangiates and polysporangiates, the first with a dominant gametophyte, the latter with a dominant sporophyte. Because of this, when referring to a unisporangiate plant such as the moss Physcomitrella or the liverwort Marchantia with no other specification, one means the gametophyte, whereas for polysporangiate plants such as a fern or a baobab tree the default organism is the sporophyte.

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Land Plant Systematics

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Extant unisporangiates are the bryophytes. There is no macrofossil record of ancestral unisporangiate embryophytes, for example the source of trilete spores dated to about 475 MYA (Wellman et al. 2003). Modern bryophytes distribute among three lineages: the liverworts (Marchantiophyta, with about 10,000 species), mosses (Bryophyta, about 13,000 species) and hornworts (Anthocerotophyta, about 300 species probably less). Bryophytes are primarily poikilohydric plants, although the degree of desiccation tolerance varies widely among taxa (Oliver et al. 2005; Proctor et al. 2007), with lineages from wet habitats not surviving more than mild dehydration and expressing desiccation tolerance only in spores. Although it is widely maintained that the bryophyte grade of organization predated the polysporangiate grade, the fossil record of extant bryophyte lineages does not go beyond the Devonian (Tomescu et al. 2018). Tomescu et al. (2009) demonstrated that thalloid macrofossils from the Appalachian Basin, a sedimentary formation spanning from the Late Ordovician to Late Silurian (roughly 460–420 MYA), have the carbon isotope signature predicted for Ordovician and Silurian liverworts (Fletcher et al. 2006). Despite the absence of diagnostic morphological traits in the Appalachian fossils, this is consistent with the inference that a bryophyte-grade land flora predated the emergence of polysporangiophytes. The earliest polysporangiophyte fossils are from Late Silurian, about 420 MYA (Taylor, Taylor and Krings 2009; Kenrick et al. 2012). Phylogenomic analysis has resolved both the embryophytes as a whole and polysporangiophytes as monophyletic groups, whereas the bryophytes are paraphyletic in certain analyses (Liu et al. 2014; Qiu et al. 2006; Qiu 2008; Ruhfel et al. 2014; Fig. 11.9) or monophyletic in others (Cox et al. 2014; Wickett et al. 2014). Phylogenetic relationship among early land plants has turned out to be one of the most recalcitrant problems in phylogenetics. Unresolved controversy is probably due to a combination of factors including compositional heterogeneity of sequence data and the paucity of data for hornwort lineages, as well as biological effects such as horizontal gene transfer and a rapid adaptive radiation resulting in conflicts among gene trees (Puttick et al. 2018). As for phylogenomic research, molecular-clock analysis of the timing of embryophyte origin and diversification has produced contrasting scenarios. One sets embryophyte origin and the divergence of liverworts, mosses, hornworts and tracheophytes in a short interval in the Precambrian. An alternative analysis places the same events in a much longer interval spanning from 490 (Early Ordovician) to 420 MYA (Late Silurian), dating the origin of mosses to around 446 MYA (Late Ordovician) and the split of hornworts and polysporangiophytes to 432 MYA (reviewed in Magallòn and Hilu 2009). More recent work estimated a minimum age of about 450 MY for liverworts and 420 MY for mosses and hornworts (Clarke et al. 2011). Modern polysporangiate plants use lignin (Box 11.1) to reinforce cell walls in mechanical and vascular tissues. A lignified water-conducting tissue, called xylem, is a fundamental apomorphy of all extant polysporangiophytes, whence the unifying denomination tracheophytes attributed to these plants. Paleontological evidence,

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Fig. 11.9 Phyletic interrelationships of plants (Archaeplastida). Embryophytes and Charophytes form the Streptophyte clade. Competing candidates as the sister group to the Embryophytes are the Charophyceae, Coleochaetophyceae or Zygnematophyceae, the last being favoured by recent phylogenomic analysis. Whatever the solution, the Charophytes as presently defined is not a clade. Likewise, the bryophytes is a paraphyletic group in this cladogram, with the liverworts basal and the hornworts sister to the tracheophytes (Qiu et al. 2006). More recent work supports alternative topologies but does not resolve the issue (Puttick et al. 2018). Numbers are estimates of clade ages (MY). (From Bowman (2013), licence number 4460281508555)

however, documents Early Devonian polysporangiate plants (e.g. Aglaophyton and Horneophyton) apparently lacking lignified tissues (Kenrick and Crane 1991, 1997). These plants probably relied on turgor pressure for mechanical support, an option feasible in homeohydric plants but incompatible with poikylohydry (Bateman et al. 1998). Bryophytes do not synthesize lignin, but mosses and some large-sized leafy liverworts have mechanical tissues with robust cell walls that afford the plants the necessary stiffness to remain erect also in the dehydrated state; bryophytes with a thalloid body adhering to the substrate lack mechanical tissues (Goffinet and Buck 2013; Fig. 11.10). The basalmost clade within extant tracheophytes is the Lycophyta, with about 1300 species (Ambrose 2013), followed by ferns or Monilophyta, now including the

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Fig. 11.10 (a) Pallavicinia lyellii, a thalloid liverwort. (b) Dawsonia superba, a moss. (c) Anthoceros agrestis, a hornwort; the flat green body in (c) is the thalloid gametophyte, erect axes are sporophytes. (a) http://moss-notes.blogspot.it/2011/12/what-is-liverwort.html). (b) http://www. terrain.net.nz/friends-of-te-henui-group/local-mosses/giant-moss-dawson-superba.html). (c) http:// www.bryo.cz/index.php?p¼mechorosty_foto&gallery¼anthoceros_agrestis&id¼492

horsetails (Equisetopsida) to about 12,000 species (Schneider 2013). Traditional systematics clumps the Lycophyta and Monilophyta under the denomination pteridophytes, meaning fern-like plants (Fig. 11.11). The phylogenetic tree in Fig. 11.9 clearly shows that the pteridophytes is not a clade; the term, however, is still in use for tracheophytes with a free-living gametophyte, distinguishing them from seed plants. For understanding the origin and biological significance of the seed, it is useful to make a short digression. In bryophytes and most pteridophytes, the spores have all the same size and shape. This condition is known as homospory and the plants that display it are said “homosporous”. Homospory does not affect sex determination; in most homosporous species, each spore produces a bisexual gametophyte that makes both male and female gametes; in others, the spores are genetically pre-determined to produce either a male or a female gametophyte despite being morphologically indistinguishable. Some pteridophyte lineages (the Selaginellales in the Lycopodiophytes, the Azollales/Marsileales lineage in the Monilophytes) and the seed plants independently evolved heterospory, a change in spore morphology that mirrors gamete dimorphism in oogamous species (Chap. 7; Doyle 2013). Heterosporous plants produce two different types of spores: the microspores and megaspores (or macrospores). The microspores are relatively small and develop in large numbers in microsporangia. The megaspores have larger sizes and develop in megasporangia (a single tetrad of four megaspores in Selaginellales or only one functional megaspore in the other cases). In heterosporous pteridophytes, the micro- and megaspores are released in the environment, where they produce diminutive free-living gametophytes (known as the micro- and megagametophyte, respectively), which lack photosynthetic tissue and remain almost entirely confined within the sporal wall. These gametophytes rely on

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Fig. 11.11 Major pteridophyte lineages. (a, b) Lycophyta. (a) Lycopodium annotinum, with erect cones bearing multiple sporangia. (b) Selaginella kraussiana. (c–e) Monilophyta. (c) Polystichum polyblepharum (a fern). (d) High magnification of the lower (abaxial) side of a fern frond, showing a dense array of sori, each containing 20–30 tiny sporangia. (e) Equisetum arvense (a horsetail). (a) http://www.actaplantarum.org/semi_dispersione/dettaglio_specie.php?id¼14&n¼1 (picture by Aldo De Bastiani). (b) http://luirig.altervista.org/schedenam/fnam.php?taxon¼Selaginella+kraussiana. (c) http://www.vivaces.be/polystichum-polyblepharum/. (d) http://m4.i.pbase.com/u10/rak_929/ large/41482294.DSC_0671.jpg. (e) http://www.herbar.org/liecive-rastliny/rastlina/praslicka-rolna/

nutrients stored in the spores by the parental sporophyte; particularly abundant are the reserves stored in megaspores, to support initial growth of the young sporophyte. Seed plants further implemented this reproductive strategy (Doyle 2013). Their microspores germinate before they are released from the microsporangia; the immature gametophyte still enclosed in the sporal wall, or pollen grain (Fig. 11.12), can complete development only on the megaspore-producing part of a compatible sporophyte. The megasporangium of seed plants is named ovule, a somewhat unfortunate denomination that caused generations of students to mistake this diploid multicellular organ for a female gamete. A single megaspore develops in each ovule. Unlike pollen grains, the megaspore of seed plants is not released in the environment but germinates within the ovule, producing a megagametophyte, consisting of a maximum of about 1000 cells in early-diverged seed plants (gymnosperms) to only

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Land Plant Systematics

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Fig. 11.12 Pollen grain of Pinus (Gymnosperms, Coniferae). The immature microgametophyte visible within the sporal wall consists of a tube cell, a generative cell (that will produce two sperms) and two prothallial cells that die precociously. The sporal wall forms two “wings” that facilitate pollination by the wind. (From: http://imgarcade.com/pine-pollen-grain.html)

seven cells or less in angiosperms. At maturity, the megagametophyte produces the egg cells, up to ten in gymnosperms, only one in angiosperms. Pollen grains need to reach an ovule to complete development and produce male gametes. Seed plants depend on the wind (anemophily), in some instances water (hydrophily) or, most frequently in angiosperms, on animals (zoophily) for pollen transport (pollination). Once reached the ovule (or the stigma, a specialized structure evolved in the angiosperm flower to provide the microgametophytes access to the ovules), the microgametophyte completes development with the production of the pollen tube, a filament that liberates two sperms in the proximity of the egg cell(s). After fertilization, the zygote immediately starts dividing to produce a multicellular embryo. When the body plan of the future sporophyte has been defined, the embryo interrupts development, activates DT, dehydrates and enters dormancy. At the same time, the cells of the ovule tegument(s) impregnate with lignin their cell walls and die, forming a robust envelope (the testa) that protects the embryo during dormancy. The final product of this complex process is the seed, an innovation that definitely severed the ancestral dependence of land plants on liquid water for sexual reproduction (Fig. 11.13). In seed plants, the parental sporophyte takes charge of embryo nutrition. This however does not suppress matrotrophy, as the embryo is still born and develops within a megagametophyte that mediates nutrient translocation from the parental sporophyte. Reserve material stored in the seed (either in the embryo or in an accessory tissue known as the endosperm) permits the young sporophyte to develop much faster than in seedless tracheophytes, thus dramatically increasing its fitness. Seed plants are formally named Spermatophytes (from Greek sperma, seed). They encompass the gymnosperms (from Greek gymnos ¼ naked and sperma ¼ seed, lit. “plants with naked seeds”) and angiosperms (from Greek angeion ¼ vase, lit. plants with seeds enclosed in a receptacle); in gymnosperms the seed has no

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Fig. 11.13 Megasporangium-to-seed transition in Coniferae. The mature seed is a chimeral structure made of parts belonging to three different organisms: (i) the testa from the parental sporophyte, (ii) the endosperm from the parental megagametophyte, and (iii) the embryo, viz. the new sporophyte

envelope other than the testa derived from the ovule, in angiosperms the seed in enclosed in a fruit, a novel structure that contributes to seed dispersion. Phylogenomic analysis supports extant spermatophytes, as well as gymnosperms and angiosperms, as monophyleytic groups (Figs. 11.14 and 11.15), but gymnosperm/angiosperm interrelationships are yet unresolved. The earliest spermatophytes documented by fossils are fern-like plants called pteridosperms, appeared in the Late Devonian, about 360 MYA. The pteridosperms attained maximum expansion in the Carboniferous/Permian (360–250 MYA) and extinguished at the end of Cretaceous (66 MYA), except few forms that survived up to the Eocene (about 50 MYA). Three of the four present-living gymnosperm lineages, i.e. the conifers, cycads and ginkgophytes, were already present in the Late Carboniferous. By the upper Permian (about 260 MYA), over 60% of the global flora was composed of gymnosperms and the arborescent lycopods and sphenopsids that had dominated the Carboniferous flora were in decline (Willis and McElwain 2014). Molecular-clock analysis of gymnosperms substantially agrees with the fossil record (Renner 2009). The earliest undisputed angiosperm pollens are from the Early Cretaceous, about 130 MYA (Taylor, Taylor and Krings 2009); molecular-clock analysis, however, points to an earlier origin of angiosperms (Clarke et al. 2011). Because of the abundance of stored reserves, seeds are an excellent source of starch, protein and fat to animals; a multitude of insect, bird and mammal species feed on seeds and co-evolved with spermatophytes. After the transition to agriculture, humans became heavily dependent on the seeds of a few angiosperm species, notably wheat, rice and maize (Chap. 12).

11.6

The Xylem: A Lignified Water-Conducting System

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Fig. 11.14 Timetree of gymnosperms. The numbers indicate the divergence succession. C Carboniferous, CZ Cenozoic, J Jurassic, Ng Neogene, P Permian, Pg Paleogene, Tr Triassic. (Redrawn from Renner (2009)) Fig. 11.15 Phylogenetic tree of angiosperms. The acronym ANA is from the basal clades Amborellales, Nymphaeales, Austrobayleiales. The length of horizontal branches is arbitrary. Cladogram based on Wickett et al. (2014)

11.6

The Xylem: A Lignified Water-Conducting System

The xylem is a vascular tissue specialized in long-distance transport of water and mineral nutrients in tracheophytes. The xylem is a complex tissue made of vascular cells, or vessels, and associated parenchyma cells. The vessels terminate development with apoptosis, or programmed cell death (Chap. 8) and lack a cytoplasmic content at maturity, the only part of these cells that persists after death being their

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purposely-modified cell walls. During differentiation, the young vessels grow up to several mm in length essentially by accumulating water in vacuoles. The original cell wall, or primary wall, yields to turgor pressure under the action of relaxing enzymes and acid secretion. Once attained final sizes, developing vessels deposit a secondary cell wall inside the primary wall; because the secondary wall is scarcely extensible, its formation terminates cell expansion. The secondary wall of xylem vessels is discontinuous, presenting numerous interruptions, or pits, where only the primary cell wall is present. Pit size, shape and distribution pattern are under the control of a cortical microtubular cytoskeleton (Roberts et al. 2004) and vary widely not only among different taxa but also with vessel location in the plant body (Carlquist 2012). The last step before apoptosis is lignification, which consists in the substitution of lignin for the water permeating the organic matrix of the cell wall. Lignification mainly affects the secondary cell wall, the primary wall remaining mostly nonlignified (Fig. 11.16d). Unlike normal cellulosic walls, lignified walls are resistant to compression and torsion forces besides tension (Box 11.1); this is an essential property that permits xylem vessels to develop a negative pressure in their inside (see below). Because of the hydrophobic character of lignin, the lignified secondary wall is impermeable to water and solutes; thus the pits are the only way for water to enter or exit the vessel lumen and to move from vessel to vessel (Fig. 11.16c). During final vessel differentiation, the primary wall area occluding the pits, known as the pit membrane, is chemically modified in order to reduce resistance to water flow (Lucas et al. 2013). Basal tracheophytes and most gymnosperms have tracheids, relatively thin and long unicellular vessels with superimposed extremities; the angiosperms, barring a few basal lineages that retain tracheids, have multicellular vessels made of files of cells whose end walls are partially or completely removed, thus reducing intra-vessel resistance to water flow (Lucas et al. 2013; Fig. 11.16a, b). The xylem forms longitudinal strands running in the plant body from the root tips to the leaves. The parenchyma cells associated with the vessels are essential for xylem functioning. They isolate the vessels from air spaces, thus protecting them from cavitation (see below). In addition, xylem parenchyma cells modulate vessel conductance (see below) by modifying the porosity of pit membranes in response to drought stress (Nardini et al. 2011b), re-activate embolized vessels by filling them with water (Nardini et al. 2011a; Brodersen and McElrone 2013), and seal no longer functional vessels to prevent fungal spreading. Despite apparent simplicity, the xylem is a vascular system of amazing efficiency, capable of transporting substantial volumes of water at relatively fast rates (up to several metres per hour) and to considerable height (over 100 m) with no energy expenditure besides the cost of construction and maintenance. For better appreciating the innovative properties of xylem, let us briefly examine the vascular tissues in poikilohydric plants. Mosses and a minority of liverworts have vessels specialized for long-distance water transport. Like xylem vessels, these arise from cells that modify their cell walls either by perforating them (liverworts) or reducing them to thin, diaphanous sheets (mosses), and conclude development with apoptosis (Ligrone et al. 2000).

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The Xylem: A Lignified Water-Conducting System

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Fig. 11.16 (a) Xylem is the lignified water-conducting tissue of tracheophytes. The vascular units in the xylem of pteridophytes and gymnosperms are elongated unicellular elements with superimposed extremities, known as tracheids. Tracheid-to tracheid water flow occurs through pit pairs in adjoining cell walls. (b) The vascular units in angiosperm xylem are multicellular vessels made of aligned cells (vessel elements) whose end walls are partially or completely removed during development, thus reducing resistance to water flow. A multicellular vessel may consist of few to several hundred vessel elements, with a maximum reported length of 70–80 cm. As for tracheids, water flow between adjoining multicellular vessels occurs via inter-vessel pit pairs. Pits on side walls are the only way for inward and outward water movement in both uni- and multicellular vessels. (c) High magnification of a pit membrane showing a myriad of nanopores. (d) A detail of the stem xylem in Lycopodium after treatment with an antibody against lignin. The dark staining of secondary cell wall (SW) indicates heavy lignification; the primary wall including pit membranes (PW) shows no staining. (c) From Jansen et al. 2009, licence number 4461350836007. (d) From Ligrone et al. 2008, licence number 4460340542009

A fundamental difference from xylem vessels is that bryophyte vessels are not lignified; this permits them to collapse and expand according to the hydration state of the plant (Fig. 11.17). Water transport in this sort of vessel essentially depends on capillarity, the physical process that causes water to rise against gravity in thin glass tubes or in blotting paper. Capillary rise depends on water interaction with the hydrophilic wall of the conduit, the height of rise being inversely proportional to the conduit radius (Fig. 11.18). Capillary spaces in plant cell walls are small enough for water to rise to a height of several hundred meters, more than enough even for giant trees such as Eucalyptus regnans or Sequoiadendron giganteum. The maximum height attainable by water, however, is not the principal issue in plant water relations; even more important is hydraulic conductance, the amount of water transported by the vascular system (or any other type of conduit) per unit time (m3 s1). Hydraulic conductance (C) varies

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Fig. 11.17 Water-conducting cells in the moss Polytrichastrum in the hydrated (a) and dehydrated condition (b). (From Ligrone et al. (2012), licence number 4460340740844)

Fig. 11.18 Water in glass tubes tends to rise against gravity to a height inversely proportional to the tube radius. Expressing the radius (r) in micrometres (1 μm ¼ 106 m), the height of rise (h) in metres is given by the relation: h ¼ 14.9/r For example, the capillary rise of water in a glass conduit with a radius of 10 μm is about 1.5 m. The same relation is a good approximation for capillary rise in plants

with the radius (r) and length (L ) of the conduit, and with the pressure difference (ΔP) between its extremities, according to the relation known as the HagenPoiseuille law: C¼π r4 ΔP=8η L

ð11:1Þ

where η is the viscosity of water (or generally of the liquid flowing in the conduit). Note that, in a capillary conduit, ΔP is the suction force exerted by the water meniscus.

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The Xylem: A Lignified Water-Conducting System

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According to Eq. 11.1, the conductance is directly proportional to the fourth power of the radius of the conduit, meaning that an increase/diminution of the radius by a factor 2 causes the conductance to rise/decrease by a factor 16. As an example, a cylindrical conduit with a radius of 20 μm has the same conductance as 16 conduits each with a radius of 10 μm. A reduction of the conduit radius, therefore, increases the maximum height attainable by capillary rise but dramatically reduces water flow. The resistance R to water flow is the reverse of conductance (R ¼ 8η L/π r4ΔP). A biological consequence of these opposite tendencies is that capillarity is a convenient mechanism for water transport in plants of small sizes that tolerate somatic desiccation, an unavoidable outcome when water loss by transpiration exceeds conductance. Bryophytes extensively rely on capillary transport not only across internal vascular tissues but also within specialized surface structures (Proctor 2000). Homeohydric plants have much larger average sizes than bryophytes, thus capillarity is normally unable to ensure an adequate water supply to aerial parts. Xylem was probably not essential for the emergence of homeohydry, but it certainly was for the evolution of homeohydric plants taller than a few cm. Competition for light, water and mineral nutrients strongly selected for larger sizes, thus also driving the evolution of more efficient water-conducting systems. Capillarity has but a marginal role in xylem transport. The motive force of water flow in xylem vessels is the difference in the energy of water molecules in the soil and atmosphere (Sperry 2010; Lucas et al. 2013; Venturas et al. 2017). Water energy is an important physiological parameter known as the water potential (Ψw) and expressed in pressure units (megapascal, MPa; 0.1 MPa ¼ 1 bar ¼ 0.987 atm). The water potential increases with pressure and temperature, and decreases in consequence of water interaction with dissolved molecules or hydrophilic surfaces. Pure water under a pressure of 1 atm (¼ 0.1013 MPa) and at a temperature of 25  C is conventionally assigned Ψw ¼ 0. The water present as vapour in the atmosphere has a potential varying from zero to extremely negative values, depending on relative humidity at constant pressure and temperature (Fig. 11.19). Water spontaneously moves across gradients from higher to lower potential, by diffusion at the cellular level (Fig. 11.20) or by mass flow in supracellular systems such as the plant xylem or vascular systems in animals. Now, let us imagine a plant that, early in the morning, opens the stomata in response to sunlight. Being in contact with unsaturated atmosphere (for example, with a relative humidity of 85%), our plant starts losing water by transpiration; very quickly, the water potential in leaf air spaces drops from a value close to zero to a negative value (Fig. 11.21). Reacting to this change, water from adjoining parenchyma cells diffuses as vapour to air spaces; the intracellular water potential decreases because of increased solute concentration. Consequently, water diffuses from nearby xylem vessels, where the water potential is still relatively high, to parenchyma cells. Being lignified, xylem vessels cannot shrink, thus water exit creates a negative pressure in their inside which draws water from adjoining vessels. The tension produced propagates across the plant body and eventually creates a negative water potential in the root xylem; this elicits a water flow from the soil to the

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Fig. 11.19 Atmospheric water potential varies with relative humidity, viz. the amount of water present in the atmosphere as vapour, expressed as percent of the saturation amount. The atmospheric water potential is zero in saturated atmosphere (100% relative humidity), but it drops to an extremely low value ( 14.2 MPa ¼ 140.17 atm under standard conditions) at a relative humidity as high as 90%. The relative humidity of the air in temperate areas is normally much below 90%

Fig. 11.20 The osmotic potential is the component of the water potential depending on water interaction wih solutes. The osmotic potential is always negative. (a) A plant cell in contact with a hypertonic solution (viz. a solution with a lower water potential than intracellular water) loses water by diffusion across the cell membrane. The net flow of water ceases when the water potential within the cell is the same as outside. (b) A plant cell in contact with a hypotonic solution or pure water absorbs water. The net flow of water ceases when increased pressure exerted by the expanding cell wall on the intracellular solution (known as the turgor pressure) counterbalances the residual difference in osmotic water potential. The model shown assumes that the cell membrane is a semipermeable membrane, i.e. is permeable to water but not to solutes. This is generally a good approximation for most biological membranes, due to the presence of water channels (aquaporins)

root that covers water loss through the aerial part. The plant water content, therefore, remains stable as long as the water potential in the soil is higher than in the root. When the soil water potential becomes too low, the plant is no longer able to absorb water and is obliged to close the stomata to prevent desiccation (Venturas et al. 2017; Fig. 11.21). According to the cohesion-tension theory, the water filling the vessels may be thought of as forming continuous filaments that are pulled upwards by the suction force exerted by unsaturated atmosphere. These little columns do not break because water molecules bind to each other by hydrogen bonds (on average three bonds per

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Fig. 11.21 Homeohydric plants transport water through the xylem exploiting the difference between the water potential in the soil and atmosphere. In a transpiring plant, water spontaneously flows across this potential gradient from the roots to the leaves. A negative value of Ψw in xylem vessels is referred to as “xylem tension”, a rise in xylem tension meaning that xylem Ψw becomes more negative. The values of Ψw reported in the figure are typical of trees from temperate areas

molecule in liquid water). Molecular cohesion by hydrogen bonds can withstand very high tensions (around 25 MPa) on condition that the continuity of the water column is not interrupted. The xylem typically operates under tensions between –0.8 and –3 MPa, but in some cases values as low as 13 MPa have been recorded. Under these conditions, xylem vessels are at risk of cavitation, or gaseous embolism, i.e. the formation of a gas bubble in their inside that interrupts water flow. Gas may penetrate the vessels from air spaces in the adjoining parenchyma or may form spontaneously in the vessel lumen under high tension. Whatever its origin, a gas bubble expands explosively under negative pressure, breaking the water column and inactivating the vessel (Lucas et al. 2013; Venturas et al. 2017). The principal function of pit membranes is prevention of vessel-to-vessel gas spreading, which would completely inactivate the vascular system, with disastrous consequences for the plant. Embolism is a serious problem for plants, especially in conditions of low relative humidity and high temperature, which stimulate transpiration and raise tension in xylem vessels. The value of xylem tension that induces a 50% reduction of xylem hydraulic conductance by cavitation (P50) varies widely among species, reflecting the range of tension adaptively experienced in the habitat of origin. Factors affecting vessel sensitivity to cavitation are poorly known. A high porosity of pit membranes increases hydraulic conductance but may also enhance the risk of cavitation and vice versa; likewise, an increase in vessel diameter greatly enhances hydraulic conductance but also increases vulnerability to cavitation, suggesting a trade-off between efficiency and sensitivity (Cochard et al. 2010).

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Xylem parenchyma cells can re-activate embolized vessels during the night by filling them with water under positive pressure (Nardini et al. 2011a; Brodersen and McElrone 2013). The plant maintains the stomata open as long as water uptake from soil balances water loss by transpiration; when the water balance becomes negative, the stomata promptly close and transpiration stops almost completely. Vessel inactivation by cavitation may oblige the plant to reduce stomatal conductance, thus negatively affecting photosynthetic activity. Plants that tolerate low water potentials in the photosynthetic tissue maintain the stomata open for a longer time than more sensitive plants, and consequently their xylem vessels can develop particularly high tensions. Whilst enhancing plant fitness in dry environments, this exposes xylem to the risk of vessel implosion due to cell wall collapse. Plants may prevent implosion by reducing the diameter of vessels or reinforcing their cell walls; the first solution negatively affects hydraulic conductance, the second is costly as implies greater investment in cellulose and lignin biosynthesis. The evolutionary solution is once again the result of a cost/benefit trade off: each species produces vessels strong enough to stand the average stress in their natural habitat but not more. In addition, because xylem vessels tend to cavitate at lower tensions than those necessary for implosion, some species deploy reversible vessel cavitation to prevent irreversible vessel implosion (Brodribb 2009). Earlier or later, however, vessels go out of function with time. In perennial plants, the vascular cambium, a secondary meristem developing in mature areas of the stem and root, cyclically produces new vessels that replace non-functional vessels. In annuals, the life cycle covers the favourable season and terminates with the death of the plant, thus making replacement of aged vessels unnecessary. Box 11.1: Lignin, Humus and Coal Lignin is a complex biopolymer essentially consisting of aromatic alcohols called monolignols, i.e. p-hydroxycinnamic (or p-cumaryl) alcohol and its derivatives conipheryl and sinapyl alcohol (Fig. 11.22). When incorporated in lignin, p-cumaryl, coniferyl and sinapyl units generate p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) residues, respectively (Wang et al. 2013; Weng and Chapple 2010). Monolignols derive from phenylalanine via a series of reactions catalysed by enzymes of the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL), and exported across the cell membrane by ATP-driven transporters. Polymerization occurs in the cell wall by the action of class III peroxydases and laccases exported to the apoplast; these enzymes oxidize the monolignols using hydrogen peroxide or oxygen, respectively, and convert them into radicals that spontaneously polymerize (Wang 2013). This mechanism permits uniform insertion of lignin in a pre-existing cell wall matrix and produces a chemically heterogeneous polymer because monolignol radicals polymerize randomly. After polymerization, lignin is (continued)

11.6

The Xylem: A Lignified Water-Conducting System

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Box 11.1 (continued) HO

HO O

HO OH

O O

O

O

O

O

HO HO OH

O

OH

O

OH

OH

O O

O O

O HO HO

O

OH

O

HO

O

O

polymerization

O O

p-cumaryl alcohol OH

OH

O O

OH

OH O

OH

O OH

O

coniferyl alcohol

OH

O

OH O

OH

OH

O

O HO

O

O

HO

HO

HO

O

O O

O

O OH

sinapyl alcohol

O O

O

O HO

O

LIGNIN

Fig. 11.22 Lignin precursors and partial molecular structure. Lignin is synthesized in the cell wall by polymerization of precursors exported from the cytoplasm

enzymatically bound to hemicellulose by covalent bonds; crucially, because of water removal by lignin, the hydrogen bonds linking hemicellulose to cellulose become permanent. Lignification imparts cell walls high resistance to bending, torsion and compression forces, which adds up to resistance to tensile forces typical of cellulosic cell walls (Fig. 11.23). Fossil evidence suggests that lignin appeared in polysporangiate plants in Mid Silurian and was initially employed to strengthen aerial axes and increase plant sizes (Bateman 1998). The use of lignin in vascular tissue enabled plants to replace capillarity with the tension-cohesion mechanism for long-distance water transport. Lignin content in extant tracheophytes varies between 5% and 35% of dry weight. The nature and randomness of chemical bonds between monolignol precursors makes lignin one of the hardest biopolymers to degrade, its decomposition in soil being slower than for most other organic materials including cellulose. Although several organisms are able to attack lignin, the lignindegrading specialists in nature are white-rot fungi, a heterogeneous (continued)

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Box 11.1 (continued)

Fig. 11.23 (a) Normal cellulosic cell walls (in green) are resistant to tensile forces but not to bending, torsion or compression (arrows). (b) Lignified cell walls (in red) can withstand intense compression forces (up to some 10 atm). Living cells behave as an inflatable raft, whose stiffness depends on inner positive pressure. Dead cells with lignified cell walls, like xylem vessels, are more like a wood boat

assemblage of several thousand species, mostly Agaromycetes but also some Ascomycetes, responsible for white-rot wood decay (Tuomela et al. 2000). Lignin degradation produces humus, a complex mixture of molecules recalcitrant to further decomposition. Defined as the organic fraction of soil lacking recognizable cellular structures, humus is a brown, negatively charged colloid that enhances soil fertility by multi-faceted effects. For example, humus binds essential cations such as K+ and Mg++, reducing leaching by rain or irrigation; in addition, humus provides a favourable habitat to soil microorganisms and imparts the soil a crumb structure that favours aeration and root respiration and prevents erosion by the wind. A high humus content, due to accumulation during winter, accounts for superior soil stability in temperate areas versus tropical and equatorial areas. Coal accumulation in Permo-Carboniferous (about 350–250 MYA) (Chap. 5) has been ascribed to the presumed absence of lignin-degrading Agaromycetes at that time (reviewed by Beerling 2007). The hypothesis that lignin was the main precursor of coal and that Permo-Carboniferous coal accumulation was a sort of “global indigestion” has recently been refuted (continued)

11.7

Multiple Evolution of Leaves and Roots

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Box 11.1 (continued) because coal accumulation did not increase significantly with the transition from a lignin-poor lycopsid-dominated flora in the Early Carboniferous to a more lignin-rich fern and gymnosperm flora in Late Carboniferous and Permian. Extensive coal accumulation during this time span is now better explained as due to conditions that favoured organic matter burial, notably wet warm weather enhancing primary production and concurrent formation of wide sedimentary basins associated with the assembly of the Pangea supercontinent (Nelsen et al. 2016).

11.7

Multiple Evolution of Leaves and Roots

The ancestral sporophyte body plan of polysporangiophytes was a system of erect photosynthetic axes and prostrate axes attached to the soil by rhizoids (Hetherington and Dolan 2017; Fig. 11.5). Leaves and roots, generally considered essential parts of the plant body, were later additions. The leaves are flattened organs with determinate growth, usually with an upward side specialized for light interception and a downward side specialized in the capture of carbon dioxide from the atmosphere; besides photosynthetic tissue, the leaves contain vascular tissue forming a vein or a system of veins (Fig. 11.24). Botanists distinguish two major types of leaves, the microphylls and megaphylls (from Greek fillon, leaf). Microphylls are generally smaller but their most distinctive feature is a single unbranched vein. Megaphylls usually have a larger blade (or “lamina”) than microphylls, whence they are also called “laminate leaves” (Boyce and Knoll 2002), and branched or multiple veins (Fig. 11.24). Microphylls appeared in the Mid Silurian in lycopods; megaphylls probably evolved four times independently in the late Devonian to Early Carboniferous, in seed plants, progymnosperms (an extinct group of seedless heterosporous plants related to spermatophytes, e.g. Archaeopteris), sphenopsids (or Equisetopsida), and ferns (Boyce and Knoll 2002; Tomescu 2008; Harrison and Morris 2017). Plants with megaphylls are referred to as “euphyllophytes” (plants with true leaves) or “megaphyllophytes”. The gametophyte of mosses and “leafy” liverworts forms leaf-like structures called “phyllids”, which are not homologous with microphylls or megaphylls and evolved independently in the two lineages (Ligrone et al. 2012). The adaxial-abaxial patterning of megaphylls in seed plants is under the control of a set of genes (Class III HD-Zip transcription factors) that take no part in microphyll development (Floyd and Bowman 2006), thus confirming the independent evolution of the two types of structures. Further support to non-homology comes from differences in the developmental pattern. Lycophyte microphylls develop from two juxtaposed epidermal cells; fern megaphylls have a shoot-like structure developing from an apical cell, whereas seed plant megaphylls develop from a pool of cells

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Fig. 11.24 (a) Internal anatomy of a leaf. The photosynthetic tissue near the upper (ventral or adaxial) side consists of tightly packed cells elongated along the dorsal-ventral axis and containing numerous, large chloroplasts; the tissue near the lower (dorsal or abaxial) side consists of isodiametric cells with smaller chloroplasts, less dense cytoplasm and wider air spaces. Stomata are prevalently or exclusively located in the abaxial apidermis. (b) Vein system of a typical megaphyll (see main text)

recruited from the flanks of the shoot apical meristem (Harrison 2017). Despite independent evolution, leaf development in different lineages presents similarities, due to convergent evolution. For example, lycophyte microphylls and angiosperm megaphylls independently recruited repression of KNOX genes by ARP transcription factors, necessary to prevent indeterminate growth (Pires and Dolan 2012; Harrison and Morris 2017). The leaves certainly evolved by modification of pre-existing parts of the plant body, yet the pathways followed are still a matter of speculation. For example, it has been suggested that the microphylls derived from a flattened lateral branch or vascularized stem outgrowths (enations) or, alternatively, from sterilized sporangia (Crane and Kenrick 1997). The telome theory by German paleobotanist Walter Zimmermann envisaged megaphyll evolution as a more complex sequence of modifications of axial systems (Fig. 11.25). Given the independent evolution of megaphylls in separate lineages, the same sequence should have been recruited multiple times. Zimmermann’s model is still widely acknowledged today. Molecular and cellular evidence has identified plausible mechanisms in extant plants for overtopping and planation, less so for lateral outgrowth webbing (Beerling and Fleming 2007). The evolution of megaphylls proceeded in parallel with a reduction in atmospheric CD concentration from several thousand to a few hundred ppm (Fig. 11.26). The paleontological record documents a concomitant 100-fold increase in stomatal frequency (the number of stomata per mm2 megaphyll epidermal surface) relative to

11.7

Multiple Evolution of Leaves and Roots

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Fig. 11.25 According to Zimmermann’s telome theory, megaphylls evolved through three successive steps. Starting from an ancestral body plan based on equivalent, dichotomously branching axes (a), the branching pattern was modified to produce (b) a main axis with indeterminate growth and lateral, subordinate axes with determinate growth (overtopping). (c) Lateral branches switched from a three-dimensional to a flattened arrangement (planation). (d), complanate lateral branches merged to produce a laminate leaf blade with branched veins (webbing). Below are reconstructions of Devonian plants epitomizing the four evolutionary steps

Early Devonian leafless plants, probably reflecting a complex interaction between contrasting evolutionary pressures (Beerling 2005). Rhynia-like archaic plants with leafless photosynthetic axes intercepted a minimal fraction of solar energy, thus they could avoid lethal overheating despite possessing few stomata and relatively inefficient vascular tissue. The evolution of megaphylls amplified the plant surface active in CD uptake, but also raised problems of heat dissipation due to enhanced light

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Fig. 11.26 Paleozoic fluctuations of carbon dioxide (red) and oxygen atmospheric concentration (blue). Vertical bars indicate the timing of major events in the evolutionary history of land plants. Bars on the bottom indicate intervals of glacial (violet) or cool climates (orange). (Data from Berner and Kothavala (2001) for carbon dioxide, Berner et al. (2007) for oxygen, and Royer et al. (2004) for climatic changes)

interception per unit surface. Besides facilitating CD uptake, a rise in stomatal frequency afforded euphyllophytes a better control of temperature by facilitating heath dissipation by transpiration; this, however, was possible only with the evolution of more efficient vascular systems and rooting structures. Archaic tracheophytes had no roots; they explored the superficial layer of soil by means of creeping axes producing tufts of unicellular rhizoids (Hetherington and Dolan 2017). As discussed in the following section, these axes already hosted fungal symbionts that assisted in rock weathering and nutrient ion uptake. The evolution of proper roots was a major step in plant “terrestrialization” because it enabled plants to explore the soil and surface rock to greater depth. Like the leaf, the root probably evolved at least twice independently, in lycophytes and euphyllophytes (Raven and Edwards 2001; Pires and Dolan 2012; Kenrick and Strullu-Derrien 2014). Distinctive anatomical traits help paleontologists to distinguish roots from aerial axes in fossils (Raven and Edwards 2001). These include: (a) positive gravitropism (downward-directed growth), (b) a protective cap, or calyptra, covering the apex, (c) a lignified cellular layer (the endodermis) separating the vascular tissue from cortical parenchyma, (d) endogenous branching (lateral roots arising from an internal cellular layer known as pericycle. The earliest fossil roots are attributed to zosterophytes, the putative progenitor group of lycophytes (Fig. 11.27).

11.8

Land Plants Co-evolved with Symbiotic Fungi

385

Fig. 11.27 (a) Fossil specimen of Zosterophyllum shengfengense from the Xitun Formation (China), dated to about 413 MY. (b) Plant reconstruction. The bar is 20 mm in (a) and 10 mm in (b). (From Hao et al. 2010, licence number 4460350141825)

11.8

Land Plants Co-evolved with Symbiotic Fungi

About 85% of extant land plants host symbiotic fungi. The tracheophytes host fungal symbionts in their roots, whereby the name mycorrhizas (from Greek mykos, fungus, and riza, root) given to these associations. In most cases, the fungus provides the host plant with mineral nutrients from the soil, receiving sugar in exchange. Land plants are generally able to extract nutrients from the soil through their rooting systems; yet, essential ions such as phosphate are scarcely mobile and their concentration in the immediate proximity of the root apparatus may rapidly become insufficient. It is more convenient for the plant to invest organic matter to support symbiotic fungi, whose minute hyphae can explore the soil with great efficiency, rather than producing a larger radical system. This accounts for the widespread diffusion of mycorrhizas; in fact the lack of mycorrhizas in about 15% of living tracheophytes (e.g. the Brassicaceae and Chenopodiaceae) is most likely due to loss consequent to adaptation to nutrient-rich habitats where the symbiosis is no longer convenient (Brundett and Tedersoo 2018; Wang and Qiu 2006). About 74% of extant tracheophytes form arbuscular mycorrhizas (AM), a type of fungal symbiosis in which the fungus penetrates root parenchyma cells and forms intracellular hyphal systems called arbuscules for their similarity to diminutive trees (Fig. 11.28); the arbuscules are the main seat of exchange between the fungus and host plant. Fungal penetration does not cause any damage to host cells because a perifungal membrane continuous with the host plasmalemma maintains the fungus in a space that is functionally extracellular (Brundett and Tedersoo 2018; Bonfante and Genre 2010). The AM fungi are Glomeromycota, an ancient fungal lineage with

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Fig. 11.28 (a) Diagrammatic representation of root colonization by arbuscular fungi (a) and ectomycorrizal fungi (b). In arbuscular mycorrhizas, fungal hyphae (in blue) pass across the root epidermis and colonize the cortical parenchyma, forming intracellular arbuscules that are the main seat of exchange between the host plant and fungus. In ectomycorrhizas, the fungus (in brown) covers the root surface with a dense hyphal sheath, known as the mantle (a), which inhibits root hair development. Fungal hyphae from the mantle penetrate the root cortex (b, in yellow) and form an extensive network in intercellular spaces, known as the Hartig net (c), which is the main seat of exchange between the plant and fungus. (a) From http://jonlieffmd.com/blog/vital-plant-communi cation-with-bacteria-and-fungus. (b) From Landerweert et al. (2001), licence number 4460350525640

aseptate, multinucleate hyphae; unlike other mycorrhizal fungi, the Glomeromycota are so profoundly adapted to the symbiotic lifestyle that they are no longer able to live autonomously. Fungal symbioses closely similar to AMs have been observed in Early Devonian fossils (Field et al. 2015; Strullu-Derrien et al. 2016) as well as in extant bryophytes (see below), suggesting that AM-like associations evolved very early during plant terrestrialization. Arborescent lycophytes and primitive conifers of the Late Carboniferous (around 300 MYA), as well as cycads and conifers from the Mid Triassic (around 240 MYA) formed mycorrhizal symbioses similar to extant AMs (Martin et al. 2017). Genomic analysis of AM-forming plants has permitted the identification of a set of 174 highly conserved genes involved in the establishment and maintenance of the symbiosis; interestingly, these genes were selectively lost in fungal-free plant lineages such as the Brassicaceae (Delaux et al. 2014). Orthologs of tracheophyte AM genes were found in liverworts and hornworts (Wang et al. 2010) as well as in advanced charophyte algae, suggesting that the ancestral gene toolkit recruited for AM symbiosis during plant terrestrialization was already present in the algal ancestor (Delaux 2017). A second important class of mycorrhizas are ectomycorrhizas (ECM), in which the fungus (generally a member of the Basidiomycetes) forms a dense hyphal sheath, known as the mantle, investing the root surface. From the mantle, fungal hyphae penetrate the root parenchyma forming a system of intercellular hyphae known as the Hartig net, which is the main seat of molecular exchange between the ECM fungi and their host plants; unlike AM fungi, ECM fungi do not form intracellular

11.8

Land Plants Co-evolved with Symbiotic Fungi

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Fig. 11.29 Myco-heterotrophic plants have lost the photosynthetic apparatus and depend on fungal associates for organic matter. Well known examples are (a) the orchid Neottia nidus-avis and (b) the “ghost plant” Monotropa uniflora (Ericales). The Monotropoideae are often the only plants growing in the understory of dense evergreen forests, where light irradiation is too low to support photosynthesis. (a) http://www.floraitaliae.actaplantarum.org/viewtopic.php?t¼0. (b) http://www. denniskalma.com/flora/monotropa%20uniflora.html

structures (Fig. 11.28). ECM basidiomycetes are polyphyletic and interspersed with saprophytic relatives, with multiple lineages that have gained or lost the capacity to form mycorrhizas (Tedersoo et al. 2010; Brundett and Tedersoo 2018). ECMs are typical of arborescent plants in the gymnosperms and especially angiosperms, and are particularly efficient rock-weathering agents (Taylor et al. 2009; Quirk et al. 2012). The earliest ECM fungal species plausibly evolved with the Pinaceae in the Jurassic, 200 to 145 MYA (Martin et al. 2017). Among angiosperms, the Ericaceae evolved ericoid mycorrhizas, a symbiosis with ascomycetous fungi that permit the plant host to utilize organic nitrogen in soil; this is an advantageous adaptation in cold habitats with hypoxic soil, where organic matter mineralization is slow (Smith and Read 2008). Numerous plant lineages independently evolved myco-heterotrophy, a class of mycorrhizal associations in which the host receives organic carbon from the fungus, usually a saprotrophic basidiomycete feeding on decaying litter (Bidartondo 2005; Brundett and Tedersoo 2018). Some plant taxa are myco-heterotrophic only during their juvenile phase, for example most orchids and a number of ferns and lycophytes, the symbiosis later becoming more similar to ordinary mycorrhizas. Others are obligate myco-heterotrophs for the entire life cycle, for example some taxa in the Orchidaceae and Gentianaceae, and all members of Monotropoideae and Triuridaceae (Fig. 11.29). In some myco-heterotrophic plants, for example the Monotropoideae, the fungus is an ectomycorrhizal associate of a photosynthetic plant; the shared fungal symbiont establishes a physiological connection between the two host plants, a condition known as “myco-heterotrophic epiparasitism” (Bidartondo 2005).

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AM-like associations also occur in the gametophyte of liverworts and hornworts, as well as of ferns and lycophytes (Read et al. 2000). Some liverworts establish associations with ascomycetous fungi similar to ericoid mycorrhizas, whereas others host basidiomycetous endosymbionts similar to those present in myco-heterotrophic plants. The liverwort Aneura mirabilis lacks chlorophyll and lives embedded in forest litter or Sphagnum peat, where it obtains carbon by exploiting a mycorrhizal association between Tulasnella, an ectomycorrhizal basidiomycete, and a host tree (Wickett and Goffinet 2008). An experimental study in the liverwort Marchantia palaeacea demonstrated that the establishment of an AM-like association with a glomeromycotean fungus improves plant growth by enhancing nutrient uptake, especially phosphorus (Humphreys et al. 2010). This suggests that symbiotic interactions with fungi evolved in the bryophyte grade before the emergence of polysporangiophytes. The gametophyte of pteridophytes may be photosynthetic (e.g. Lycopodium cernuum, Gleichenia) or non-photosynthetic (Psilotum, Ophyoglossum, Botrychium) and in either cases may be associated with fungi identified either as glomeromycetes or basidiomycetes (Read et al. 2000).

11.9

The Impact of Land Plants on the Global Environment

Organic matter production by early land plants was probably a minor contribution to global productivity relative to cyanobacteria and eukaryotic algae living in the ocean. From the beginning, however, land plants increased rock weathering and riverine nutrient fluxes to the ocean (Mitchell et al. 2016), thus indirectly promoting global productivity (Lenton et al. 2012; Porada et al. 2016) and giving the start to the Paleozoic long-term shift towards high-oxygen and low-CD conditions (Taylor et al. 2009, 2012; Pires and Dolan 2012; Terrer et al. 2016; Mills et al. 2017). A particularly dramatic decrease in CD concentration occurred during the Devonian-Carboniferous, in concomitance with the diffusion of tracheophytes. From the geochemical perspective, the most significant innovations of tracheophytes were: (i) The roots, which permitted exploration of deeper soil and enhanced rock weathering and soil formation. (ii) Arborescence (tree stature), evolved independently in the course of Devonian (418–360 MYA) in several tracheophyte lineages, as a strategy for retrieving water and nutrients from deeper levels in the soil. (iii) The seed habit, which emancipated land plants from dependence on moist lowland habitats and allowed colonization of drier upland (Algeo and Scheckler 1998; Berner 2003b; Kenrick and Strullu-Derrien 2014). These innovations accelerated the silicate/carbonate cycle (Sect. 2.4) and contributed to reducing CD concentration to a few hundred ppm by the end of Carboniferous (Taylor et al. 2012; Fig. 11.26). After rising again to over 2000 ppm in concomitance

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The Impact of Land Plants on the Global Environment

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with the great extinctions of Permian-Triassic (Ogden and Sleep 2012) and TriassicJurassic (Smith 2011), CD concentration steadily decreased during the following 130 MY, reaching 200–280 ppm around 30 MYA and remaining stable in this range up to the “industrial revolution” about 300 years ago (Berner 2003a, b, 2006). The lower peaks in CD concentration were associated with a general cooling of the planet, with significant glaciation in the Carboniferous/Permian and a second cold phase started about 35 MYA and culminated in the Pleistocene (or Quaternary) glaciation (Ehlers and Gibbard 2007; Beerling and Royer 2011). In parallel with changes in CD, oxygen concentration rose to about 30% during the Carboniferous, plummeted to less than 15% during the Permian, and started rising again in the Jurassic, reaching the present atmospheric level (about 21%) in the Paleocene, about 30 MYA (Berner 2006; Berner et al. 2007; Fig. 11.26). Land plants were major drivers of the CD fall and oxygen rise during the Devonian-Carboniferous and the Jurassic-Cenozoic, yet the two episodes had different environmental backgrounds. The first probably reflected massive sequestration of organic material under the action of favourable sedimentary and climatic conditions (Box 11.1). The main drivers underlying the second episode were probably intense orogenic activity and the rise of angiosperms to dominance. The formation of large mountain complexes (including the Himalaya, Rocky Mountains, European Alps and Andes) in a relatively short interval enhanced the carbonate/ silicate cycle and consequent removal of CD from the atmosphere (Taylor et al. 2009; Lenton and Watson 2011). The global diffusion of angiosperms, initiated around 90 MYA, further contributed to the process. An additional factor probably involved in Cenozoic global cooling was the evolution of ectomycorrhizal fungi that associate with gymnosperm and angiosperm trees and are particularly effective rockweathering agents. The angiosperms distinguish themselves from the other tracheophytes in the superior efficiency of their vascular system and stomatal apparatus. These traits support high photosynthetic rates under non-limiting water supply (Brodribb et al. 2009, 2012; Haworth et al. 2011; McAdam and Brodribb 2012), thus increasing global productivity and favouring organic matter sequestration (Boyce and Lee 2011). A functional consequence of increased vascular efficiency in angiosperms is immediately visible in the inner anatomy of their leaves. Plant lineages with unicellular vascular elements (tracheids) have leaves with unbranched (lycopods) or scarcely branched veins (ferns and gymnosperms); in contrast, angiosperms have profusely branched leaf veins (Fig. 11.30). Vein density, expressed as the average vein length per unit leaf surface (mm/mm2), determines the distance between mesophyll cells and xylem elements; because water molecules move along this path essentially by diffusion, the length of the path strongly affects vein-to-mesophyll water flow (Boyce et al. 2009). The obvious consequence is that, in conditions eliciting strong transpiration, plants with low vein density may be obliged to close the stomata to prevent irreversible mesophyll dehydration even with non-limiting water availability. This may significantly reduce the potential photosynthetic yield. High vein densities enable angiosperms to maintain high transpiration rates and avoid photosynthesis-inhibiting cell dehydration. Comparative analysis of living

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Fig. 11.30 Leaf veins in a fern (a), a conifer (b and c) and a modern angiosperm (d and e). The dotted oval in (c) encloses the single vein in a Pinus leaf. Higher vein densities enable angiosperms to maintain high assimilation rates under strong evaporative demand (Boyce et al. 2009). Bars are 500 μm throughout

plants and fossils shows that the rise of angiosperms to dominance in most terrestrial ecosystems was associated with an increase in leaf vein density from about 3 to 9 mm/mm2 (Brodribb and Feild 2010). Producing highly branched leaf veins is not a difficult task in evolutionary terms; why did this advantageous trait fail to evolve for nearly 300 MY? Most likely, lycopods, ferns and gymnosperms never increased vein density because their unicellular vessels have relatively high resistance to water flow. In order to support higher vein density, viz. higher transpiration rates, the total amount of xylem in these plants should increase inordinately. Thanks to a more efficient xylem, high vein density and a stomatal apparatus with high conductance (Franks and Beerling 2009), the angiosperms are by far the most productive plants on the planet. By enhancing water evaporation, land plants are major drivers of the water cycle. Bare soil, or soil covered with poikilohydric plants or lichens, transfers only a minor part of rainfall to the atmosphere, the rest flowing back to the sea with rivers or through the underground. Tracheophytes initially colonized humid environments and spread to more stressful environments over time, capturing an ever-increasing part of the hydrologic cycle that passed through the soils of the planet. With the evolution of arborescence, the fraction of rainfall collected by plants further

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The Impact of Land Plants on the Global Environment

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Fig. 11.31 A representation of the increasing impact of land plants on the hydrologic cycle during their evolutionary history. Evaporation from bare soils after rain rapidly becomes negligible due to the formation of a dry surface layer (light brown). Poikilohydric plants covering the soil surface before 400 Myr ago did not substantially increase evaporation relative to bare soil. The first homeohydric plants with roots, stomata and a vascular system provided a way for water to bypass the dry soil layer and immediately return to the atmosphere. The evolution of trees with deeprooting systems through the Carboniferous-Permian dramaticallly increased transpiration and enhanced rainfall over continents. With the diffusion of angiosperm trees in the Cretaceous and Cenozoic, the fraction of precipitation recycled to the atmosphere further increased. (Redrawn from Berry et al. 2010)

increased, attaining a maximum with the rise of angiosperms to dominance (Fig. 11.31) By returning substantial amounts of water directly to the atmosphere, land plans increase local humidity, reduce the temperature and enhance precipitation (Berry et al. 2010). Simulation experiments with the current configuration of continents have shown that, if deprived of their plant cover, the continents would have summer temperatures up to 20  C higher, and precipitation would almost completely stop in regions that are presently quite humid, such as the Mid-western United States. Predictably, forest destruction will dramatically alter the high rainfall regimes in densely forested areas such as the Amazon Basin.

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Chapter 12

The Emergence of Humanity

The way people act reflects the kind of animal we are. . . The foundations of universally shared human behaviours must lie outside of human behaviour, in the constraints and channels laid down by our primate prehistory (Carel Van Schaik 2016)

Abstract Adaptation to the savanna and transition to bipedality probably triggered the divergence of the hominins from other apes in Central-Eastern or Southern Africa between 5 and 7 MYA. The human lineage (here identified with the genus Homo) appeared between 3 and 2 MYA, its earliest distinctive traits being increased brain size, the loss of fur, improved thermoregulation, and arm/torso anatomy adapted to high-energy throwing. Coercive suppression of conflict of interest probably favoured kinship-independent aggregation into cooperative groups. Cooperative hunting and social rearing improved the diet, thus providing the extra resources necessary for the development of larger and metabolically more active brains under selection pressure for higher cognition. In a complex network of mutual interactions, kinship-independent cooperation paved the way to the evolution of language and the emergence of culture, a body of shared knowledge and beliefs transmitted across generations. Culture accumulation and donated culture triggered cultural niche construction, the development of a continuously expanding environment, partly physical and partly cognitive and social, which was the main driver in the evolution of modern humans (Homo sapiens). Sapiens appeared in Eastern Africa around 200 KYA, migrated to the Middle East twice, around 100 and 70 KYA, and from there started a worldwide expansion about 60 KYA. Fire became embedded very early in human behaviour and was involved in almost all technological advances. The transition to food production, from about 11.5 KYA, was pivotal to the emergence of modern societies. Most genomic changes that distinguish humans from their primate relatives are in noncoding sequences with regulatory functions.

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_12

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Introduction

Just six million years ago, a single female ape had two daughters. One became the ancestor of all chimpanzees, the other is our own grandmother. Homo sapiens has kept hidden an even more disturbing secret. Not only do we possess an abundance of uncivilized cousins, once upon a time we had quite a few brothers and sisters as well. (Yuval Noah Harari 2015)

Homo sapiens was formally established as a species by Carl Linnaeus in 1758 and correctly placed (with remarkable audacity for the time) in the Primates, an order of primarily arboreal and vegetarian mammals encompassing lemurs, tarsiers, monkeys and apes, with a total of about 500 extant species (King 2016; Fig. 12.1). Humans share a number of distinctive characters with other primates, such as an opposable first digit for a firm grasp of tree branches, flat nails and sensitive fingertip pads for object handling, forward-facing eyes for a stereoscopic vision, and specific interspersed nuclear DNA sequences (Box 6.3) of about 300 bp, named Alu elements. The primate lineage encompassing the humans (Haplorrhini) differs from the rest of Primates in that it evolved routine trichromatic (three-color) vision, an adaptation considered useful for food detection (Surridge et al. 2003), whereas most other primates have mono- or dichromatic vision (Glen 2016; Herlyn 2016). Within the Primates, our species belongs to the Hominoidea, a group of large, tail-less primates commonly known as apes, native to Africa and Southeast Asia. In this scheme, Homo sapiens initially stood as the only species of its genus, but following the discovery of the original Neanderthal fossil in 1856 and of scores of other hominin species, Homo sapiens and a number of extinct relatives have been placed in the tribe Hominina (Fig. 12.2), with Central-Eastern or Southern Africa as the likely origin area (Tattersall and Schwartz 2009; Wood 2010; Wood and Baker 2011; also see the collection of articles in Guterl 2013). The emergence of the hominin lineage was most likely driven by a combination of climatic and biological factors. During the Cenozoic (an era initiated 66 MYA and still in progress), atmospheric carbon dioxide (CD) decreased from over 1000 ppm to around 200 ppm (Sect. 11.9). In parallel with decreasing CD concentration, the global climate became cooler and drier. In this scenario, around 8 MYA the rainfall regime in Central Africa turned more seasonal, inducing a reduction of rain forest and the emergence of savanna, a novel type of biome dominated by grasses (Poaceae) with C4 photosynthesis (Box 12.1). About 2.6 MYA, progressing global cooling triggered the Quaternary Glaciation, with cyclically expanding polar ice caps; Central Africa entered a phase of climatic fluctuation superimposed on a trend towards increasing aridity (Beerling and Royer 2011; Kaspar et al. 2010; Maslin and Christensen 2007; Maslin et al. 2015).

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Fig. 12.1 Primate phylogenetic tree. Letters in parentheses indicate apomorphies at different levels. (a) Flat nails (instead of claws), sensitive fingertip pads with tactile Meissner corpuscules, opposable thumbs. (b) Absence of L-gulonolactone oxidase, an enzyme necessary for synthesizing vitamin C from glucose. (c) Posteriorly closed eye sockets, unpaired uterus replacing ancestral bicornuate uterus. (d) Side-facing nostrils. (e) Downward-facing nostrils, routine trichromacy, loss of the third premolar. (f) Loss of the tail, forelimbs longer than hind limbs (lost in humans), delayed puberty. (g) Frontal sinuses, laryngeal sacs in males (lost in humans). Human apomorphies include obligate bipedality, larger brain sizes, an increase in the density of eccrine skin glands and a reduction of fur, loss of function (pseudogenization) of the MYH16 gene, loss of the os baculum (a penis bone present in most other primates including chimpanzees). For further apomorphies, see Herlyn (2016). There are 496 species of living primates. For further information, go to Primate Specialist Group at http://primates.squarespace.com/who_we_are_psg/

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Fig. 12.2 Taxonomy and interrelationships of Hominoidea, tail-less primates also known as apes. Numbers are estimates of the divergence time of the clades immediately downstream. The chimpanzee lineage (Panina) is sister to the hominin lineage (Hominina). Data from Glazco and Nei (2003). Other analyses give older divergence times (Langergraber 2012; Wilkinson et al. 2011). In contrast, Hobolth et al. (2007) dated the human-chimp split to about 4.0 MYA

Box 12.1: The Savanna The savanna is one of the world’s major terrestrial biomes, consisting of a dynamic mixture of trees and grasses (Beerling and Osborne 2006; Fig. 12.3). Savanna’s grasses are mainly Poaceae with C4 photosynthesis, a metabolic variant of photosynthesis that uses a cytosolic enzyme, phosphoenolpyruvate carboxylase (PEPC) for capturing CD from the atmosphere, reserving the canonical enzyme ribulose-bisphosphate carboxylase/oxygenase (RubisCO) only for the definitive fixation via the Calvin-Benson-Bassham cycle (Sage 2003). Spatial separation of the two enzymes is critical for the process; in most C4 plants, PEPC is expressed in mesophyll leaf cells, RubisCO in specialized cells associated with leaf veins (Fig. 12.4). In C4 plants, PEPC in mesophyll cells incorporates CD in a molecule of four carbon atoms (whence the name assigned to these plants), which is then transported to bundle sheath cells via plasmodesmata. Here the C4 compound is broken into a C3 compound and a molecule of CD that is fixed by RubisCO and converted into sugar through the Calvin-Benson-Bassham cycle. The sense of the process is that it maintains a high CD concentration in bundle sheath cells, thus enhancing carboxylase activity of RubisCO (Sect. 4.6). CD molecules that escape fixation by RubisCO in the bundle sheath can be captured again by PEPC in the mesophyll before being lost to the atmosphere via the stomata. (continued)

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Introduction

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Box 12.1 (continued)

Fig. 12.3 The savanna is an open-canopy ecosystem with a mostly C4 grass cover. C4 grasses are physiologically incapable of competing with C3 plants in the low-light environment of closed-canopy ecosystems (e.g. a forest) because of higher metabolic cost of C4 photosynthesis (Lehmann et al. 2011)

The carboxylation reaction by PEPC is strongly exergonic (△G
0 around 30 kJ mol1), thus enabling the enzyme to remove virtually all the CD molecules present in the air spaces. In contrast, at the present oxygen atmospheric concentration (which is much the same as 8 MYA), net fixation of CD by RubisCO becomes zero at a CD concentration of about 50 ppm. During the last 30 MY, C4 photosynthesis evolved several times independently in angiosperms as an adaptation to decreasing CD concentration, high temperature and drought (Sage 2003). C4 plants discriminate 13C vs 12C much less than C3 plants (Box 2.2), thus they have less negative δ13C values in their tissues. Since terrestrial animals obtain organic carbon from plants, either directly or indirectly, the δ13C in their body reflects the C3/C4 balance of their diet. Analysis of δ13C in fossil teeth has revealed that Australopithecus anamensis (~4 MYA) derived nearly all of its diet from C3 resources; Homo habilis (~2.3 MYA) had a diet with a predominance of C3-based resources, whereas nearly contemporary Paranthropus boisei had a diet predominantly based on C4 resources (Cerling et al. 2013; Lee-Thorp et al. 2010; Ungar et al. 2011). (continued)

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Box 12.1 (continued)

Fig. 12.4 Leaf anatomy in wheat (Triticum sp.) and corn (Zea mays), two members of the Poaceae using C3 and C4 photosynthesis, respectively. In the wheat leaf the photosynthetic function is restricted to mesophyll cells, which express the RubisCO but not the C4 key enzyme phosphoenolpyruvate carboxylase (PEPC); the cells surrounding vascular bundles are nonphotosynthetic. In the corn leaf, both mesophyll and bundle sheath cells are photosynthetic, the former expressing the PEPC, the latter the RubisCO. The two cellular types are connected by plasmodesmata and function cooperatively (see main text). (Picture from: http://www.faculty. umb.edu/yvonne_vaillancourt/Biology/C3%20C4%20images.htm)

About 8 MYA, C4 plants went through a worldwide expansion coincident with the emergence of the savanna, which replaced closed-canopy ecosystems (shrublands, thickets and forests) in large areas of Central and Southern Africa. Savanna-like biomes also appeared in Southern America, Asia and Australia (Fig. 12.5). The balance between closed-canopy ecosystems and savanna in Africa and the rest of the world depends on numerous factors including climate, hydrology, herbivory, fire and soil characteristics (Lehmann et al. 2011). The savanna thrive in areas with an average annual temperature above 10
C, abundant rainfall concentrated in a relatively short period, and a long dry season. The herbs survive the dry season in the form of seeds and underground perennial organs; savanna trees, instead, perform C3 photosynthesis and survive drought by reducing water loss and developing extensive root systems capable of exploring large volumes of soil. Under favourable conditions, C4 grasses are extremely productive, leading to rapid accumulation of highly flammable biomass. Because of this, savannas regularly experience wildfires, mainly started by lightning. As for drought, herbs survive a rapid passage of fire in the form of seeds or underground organs; the trees usually receive little damage because a thick layer of insulating cork protects the trunks, and the crown is too high to catch on fire (Fig. 12.6). Fire, in contrast, is a deadly enemy for closed-canopy communities (continued)

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Box 12.1 (continued)

Fig. 12.5 Present global distribution of savanna

Fig. 12.6 Wildfire is a fundamental factor for the persistence of savanna. (Picture by Carla Staver https://www.princeton.edu/main/news/archive/S32/00/77Q07/index.xml? section¼topstories)

(continued)

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Box 12.1 (continued) because here it easily propagates and intensifies. The Miocene shift towards a more seasonal pattern of rain distribution increased fire frequency; fire allowed grasses to invade the forests and the evolution of fire-resistant trees expanded the climate space that savanna could invade (Scheiter et al. 2012). On the other hand, regular fire of moderate intensity helps controlling the reproduction of trees, thus preventing the establishment of a tree canopy that would stop grass growth. Effort by Governments of several African Countries to reduce the frequency and extension of fire is currently causing a contraction of savanna and the return of forests. The emergence of savanna in Africa in the last 8 MY engendered the evolutionary diversification of numerous large ungulate herbivores (including horses, rhinos, antelope and elephants), and of their predators (hyenas, jackals and large cats). The expansion of savanna and concurrent reduction of forests were probably the main drivers of the divergence of Hominina; early humans successfully adapted to the savanna becoming opportunistic omnivorous foragers.

12.2

The Evolution of Bipedality

The Hominina is represented today only by our species, Homo sapiens, but in fact encompasses a number of other hominin species known from fossils. Australopithecus and Paranthropus as well as early Homo species were all habitual bipeds, as inferred from the skeletal anatomy of the pelvis, knee and feet and the forward position of the foramen magnum, viz. the hole at the base of the cranium that permits the passage of the spinal cord. In fact, bipedality (also referred to as orthogrady or, more informally, upright locomotion) is the first step towards “humanity” recorded in the fossil record, predating the increase of brain size, loss of body hairs and the use of stone tools (Harcourt-Smith and Aiello 2004; Crompton et al. 2010; Lequin 2018). More ancient putative hominins, for example Orririn tugenensis (dated to about 6 MYA) and Ardipithecus ramidus (4.4 MYA) were still prevalently arboreal, although they most likely used bipedal locomotion when moving on the ground. Sahelanthropus tchadensis, dated to about 7 MYA, may have appeared just after the divergence of Hominina from Panina, the chimpanzee lineage. The anterior position of the foramen magnum suggests that Sahelanthropus used bipedal locomotion, but the lack of postcranial fossils (i.e. parts of the skeleton below the cranium) has so far prevented confirmation of this hypothesis (Dale 2018). When moving on the ground, our closest ape relatives (the gorilla, chimpanzee and bonobo) use knuckle-walking, a form of quadrupedal locomotion in which the forelimbs support the body weight on the dorsal surface of middle phalanges. It has long been assumed that the concestor of Panina and Hominina used knuckle-walking

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The Evolution of Bipedality

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and that bipedality evolved in the hominins from this original type of locomotion (Stokstad 2000). There is no evidence, however, for a terrestrial knuckle-walking phase in hominin evolution at least from 4.4 MYA, because Ardipithecus ramidus and all later hominin species lack knuckle-walking features such as dorsal ridges on metacarpal bones like those present in chimps and gorillas (Lovejoy 2009; Lovejoy et al. 2009a,b). On the other hand, all great apes can use bipedality supported by the hind limbs alone, and most do so in an arboreal context; notably, it is the most arboreal form, the orangutan, which uses bipedal locomotion more often, for reaching fruits on thin peripheral branches (Crompton et al. 2010). The body of evidence accumulated in the last decade indicates that (a) the ability to use bipedality in special circumstances was already present in the ancestor of extant hominids; (b) the hominins evolved advanced bipedality without an intermediate knuckle-walking phase; (c) the chimpanzee lineage (Panins) evolved knuckle-walking after splitting from hominins (Gibbons 2009; Lovejoy 2009; Crompton et al. 2010). Knucklewalking also evolved in the gorilla, probably independently (Kivell and DanielSchmitt 2009). The hominins originated from an arboreal ancestor around 7 MYA. The persistence of arboreal traits including curved phalanges (the finger bones) and ape-like shoulders in early bipedal hominins such as Australopithecus afarensis, which lived between 3.9 and 2.9 MYA, indicates that the evolution of bipedalism was a slow, complex process. By the time Homo erectus appeared, around 1.8 MYA, the anatomical changes underpinning bipedality were complete, including a bowlshaped pelvis, stronger leg bones and arched feet for low-impact transfer of weight from leg to leg. Bipedality is a highly problematic type of locomotion for a body primarily built to move on four limbs, as is the case for all terrestrial vertebrates. The transition to bipedality required a complex remodeling of the skeleton, muscles and ligaments; in addition, the adoption of an orthograde (erect) posture affected blood circulation, requiring special adaptations to ensure blood flow from lower limbs back to the heart. Several theories have been proposed to explain why hominins adopted a bipedal locomotion (reviewed in Dale 2018). The most popular is the savanna theory, which proposes that bipedality evolved in response to environmental constraints of the savanna, a biome characterized by sparse trees and large open spaces. In these conditions, bipedality might have been a favourable adaptation affording improved locomotion on the ground and a better visual control of the territory, whilst helping to regulate body temperature by reducing exposition to solar radiation. An added advantage of bipedality might have been in facilitating food transport over long distances, a behaviour that is generally not feasible for an arboreal or quadrupedal hominoid and possibly favoured the emergence of male parental care in humans (Lovejoy 2009). Bipedality liberated the forelimbs from the constraints of locomotion (both arboreal and on the ground), thus permitting the evolution of anatomical and neural traits underlying manual dexterity (MacWhinney 2005; Hecht et al. 2014). Evolution, however, might also have proceeded the other way round, with better manual dexterity improving foraging on the ground and indirectly favouring the transition to

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bipedality. Dale (2018) argues that none of the theories proposed satisfactorily explains why bipedality emerged despite its conspicuous disadvantages, and suggests that the trait simply evolved as a fitness signal eliciting submissive behaviours in conspecifics. The complete abandonment of arboreality is probably easier to understand if accompanied by the control of fire, based on the supposition that, prior to the control of fire, early hominins almost certainly took to the trees at night like most other primates, as a defense against predators. The earliest evidence for controlled use of fire, however, is from about 0.8 MYA (Wrangham and Carmody 2010; Gowlett 2016), long after the transition to specialized bipedality. Whatever its origin, bipedality changed the way individuals fought, biting being replaced by punching and hitting with the hands and arms. The reduction in size of canines might be a consequence of this change in the way of fighting (Van Schaik 2016). More importantly, bipedality permitted the anatomical changes in the arms and torso that led to the evolution of high-speed throwing, an event that most likely had long-ranging consequences on subsequent human evolution (Sects 12.6 and 12.11). A positive feedback between manual dexterity and the development of dedicated cortical centers might have triggered the increase in brain size and cognitive abilities, which has been a dominant theme throughout the evolutionary history of hominins. Not all of this was obtained without costs. Painful occurrences such as herniated intervertebral discs, fractured vertebrae, spondylitis, scoliosis, and kyphosis are extremely common among humans but relatively rare in the other apes. Varicose veins and hemorrhoids are an unpleasant consequence of blood pressing down on the veins in the lower half of the body. Female hominins have paid an even heavier penalty to bipedality in the form of a reduction in the width of the pelvis and of the birth canal. Brain and cranial enlargement in human evolution exacerbated the problem, making the labor dangerous for both the mother and the infant. These negative outcomes were in part alleviated by a reduction of the relative sizes (and the degree of maturity) of human newborns relative to other primates (Gruss and Schmitt 2015).

12.3

Australopithecines

The Australopithecines, a group of hominins also dubbed “bipedal apes”, encompasses two genera, Australopithecus and Paranthropus (Hammond and Ward 2013; Foley et al. 2016). Well known to the public is the 3.2 MY-old specimen dubbed “Lucy”, found in Etiopia by paleoanthropologist Donald Johanson in 1975 and formally classified as Australopithecus afarensis (Fig. 12.7). Several fossils ascribed to the same species were found more recently, including a partial skeleton of a young individual (Walker and Stringer 2010). A. anamensis is a more ancient species dated to about 4.4 MYA, whereas A. africanus, A. sediba and A. garhi are more recent than A. afarensis. The genus Paranthropus encompasses three

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Australopithecines

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Fig. 12.7 (a) The skeleton of “Lucy”, a female specimen of Australopithecus afarensis. The brown pieces are casts of original fossils, the white ones are reconstructions. The anatomy of pelvis, knee and feet indicates that this primate used bipedal locomotion on the ground. The curvature of the phalanges (finger bones), the long arms and shoulder bone anatomy suggest that they were still partially arboreal. (b) Reconstruction of a male of A. afarensis. These primates weighted 30 (females) to 70 Kg (males) and were 1–1.5 m tall (a) Andrew from Cleveland, Ohio, USA - Lucy, CC BY-SA 2.0, https://commons.wikimedia.org/ w/index.php?curid¼3792582 (b) http://www.creativesculpture.com/IMAGES/blg_frm/bestof/lucy.jpg

formally recognized species, P. aethiopicus, P. robustus and P. boisei, dated from about 2.8 to 1.9 MYA (Tattersall and Schwartz 2009; Wood 2010). The forms ascribed to the genus Australopithecus have a relatively light skeleton, which earned them the name “gracile australopithecines”; because of bigger sizes and a more robust skeleton, Paranthropus species are known as “robust autralopithecines”. Gracile australopithecines were probably less specialized, had a more varied diet and spent a considerable part of their life on trees. They show clear adaptations to bipedal walking in the lower body (pelvis and legs) combined with “primitive” traits better suited to an arboreal existence in the upper body, such as long, curved fingers for grasping tree branches, shoulder blades with upward-facing sockets facilitating tree climbing, and inner ear anatomy similar to that of extant apes (Alemseged et al. 2006). Gracile australopithecines became extinct between 2.9 and 2.4 MYA, possibly because of further reduction of rain forest and expansion of savanna. This interval coincides with the appearance of Paranthropus, a genus adapted to a diet mainly based on C4 grasses (Box 12.1).

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Australopithecus and Paranthropus both possessed a brain volume of about 500 cm3, just above a chimpanzee’s. Rudimentary stone tools with an age of about 3.3 MY, possibly used for removing flesh or extracting the marrow from bones, have tentatively been attributed to Australopithecus afarensis (McPherron et al. 2010; Lewis and Harmand 2016). Another candidate toolmaker is a hominid referred to as Kenyanthropus platyops (Leakey et al. 2001), found in Kenia in 1999 and dated to 3.5–3.2 MYA. Some researchers consider this fossil an Australopithecus species, whereas others assign it to the genus Homo, with affinities with H. rudolfensis (Cela-Conde and Ayala 2003). Uncertainty in the interpretation of hominin fossils is a recurrent theme in paleoanthropological research, because these fossils are exceedingly rare and represent only a part, sometimes extremely small, of the complete skeleton.

12.4

The Genus Homo

The earliest populations of the Homo lineage emerged from a still unknown ancestral species at some point between 3 and 2 MYA. Between the latest known occurrences of “generalized” australopithecines (A. afarensis in Eastern Africa, A. africanus in Southern Africa) and the earliest known records of species attributed to the genus Homo, lies one million years of rare, isolated or fragmentary fossils with no clear-cut boundary between the two genera (Strait et al. 2015; Kimbel and Vilmoare 2016). Australopithecus sediba (dated to ~2 MYA) is thought to possess a unique relationship to the origin of Homo because of a number of Homo-like features in cranial and postcranial anatomy (Berger et al. 2010; Dembo et al. 2015). A partial mandible with teeth, discovered in the Ledi-Geraru area in Ethiopia and dated to 2.80–2.75 MYA, combines derived morphology observed in later Homo with primitive traits seen in early Australopithecus, and may therefore be a transition form between the two lineages (Vilmoare et al. 2015). The fossil and archaeological record suggests that Homo evolved against a background of long periods of habitat unpredictability superimposed on an underlying aridity trend (Antón et al. 2014). The earliest formally described Homo species is H. abilis (Fig. 12.8); the type-specimen was found in Tanzania in 1960 and proposed as a novel species under the name H. habilis in 1964 (Leakey et al. 2001). His anatomy is so primitive relative to other Homo species that it has long been debated whether the records should more correctly be ascribed to Australopithecus. Homo habilis lived in Central-Western Africa between 2.1 and 1.5 MYA; he was short, had disproportionately long arms (reflecting persistence of arboreal habits) and a cranial capacity about 50% larger (~640 cm3) than in australopithecines but much smaller than in modern humans (1350–1450 cm3). The hand had firm, short fingers with large thumbs, possibly an adaptation selected for ability in making and handling stone tools. Most significantly, the hand anatomy of H. habilis reflects the possession of the precision grip, a distinctive trait of humans essential to manipulate objects with dexterity (Fig. 12.9). Analysis of stone

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The Genus Homo

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Fig. 12.8 Facial reconstructions of three archaic species of Homo: H. habilis (female), H. rudolfensis (male), H. erectus (male). H. habilis: https://enigmadown.wordpress.com/category/ animais/page/3/ H. rudolfensis: http://www.avph.com.br/homorudolfensis.htm H. erectus: Smithsonian Museum of Natural History in Washington, D.C. Reconstruction by John Gurche, photographed by Tim Evanson

Fig. 12.9 The ability to put the tip of the thumb in contact with any of the other fingertips is a distinguishing trait of the human hand known as the precision grip, which reflects high manual dexterity. In addition, humans have wider pads than in other primates due to horseshoe-shaped edges of distal phalanges; in the grasping hand, larger distal pads can better conform to uneven surfaces, applying pressure more evenly

flakes provided evidence that H. habilis and the coeval species named H. rudolfensis (see below) were predominantly right-handed. In addition, cranial endocasts revealed that the brain of H. habilis and other more recent Homo species had distinct Broca’s and Wernicke’s areas, considered as the brain’s centres for speech capability in modern humans. Australopithecus endocasts show the Broca’s but not the Wernicke’s area, while the other hominids display neither (Schrenk 2013). Because of these major apomorphies, we will refer to the genus Homo as the “human lineage”. The occurrence

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in H. habilis of brain areas associated with speech in modern humans, however, is not a proof for the capacity of using language in this species (Sect. 12.9). Tooth anatomy, the δ13C value (Box 12.1), and routinary use of stone tools point to Homo habilis as a versatile and opportunistic species, with a diet based on both plants and animals. Ecological and behavioural versatility enabled H. habilis to survive a second major climate crisis between 1.9 and 1.6 MYA, which instead wiped away coeval Paranthropus species (Tattersall and Schwartz 2009). Homo habilis was able to struck pebbles of basalt, flint or chalcedony with a larger hammer stone, removing flakes from one side to obtain a sharp edge or a tip. This is known as the Oldowan technology from the Olduwai Gorge in Tanzania, where the first artefacts of this type were found (de la Torre 2011). Oldowan tools are also called “pebble tools” because they retain the shape of the pebbles used for their production (Fig. 12.10a). Other researchers prefer the denomination “mode 1” tools. The main use of Oldowan tools was most likely to open and disarticulate carcasses and to scrape the meat from the bones, although they might also have been employed to sharpen wooden implements and cut plants. The appearance of Oldowan technology is thus considered the earliest evidence for systematic consume of meat. Fossils found in Kenya in 1972 and initially ascribed to a novel australopithecine species (“Australopithecus rudolfensis”) were subsequently interpreted as a second archaic species of Homo coeval to H. habilis, named H. rudolfensis (Fig. 12.8). New fossils found more recently and dated to about 1.9 MYA suggest that H. habilis and H. rudolfensis co-existed in Eastern Africa between 2.0 and 1.5 MYA. The separation of the two species is controversial, as anatomical differences fall within the normal intraspecific variability. Even more uncertain is whether H. rudolfensis or H. habilis was ancestral to the later Homo line. A third species, referred to as Homo ergaster, lived in Eastern Africa between 1.8 and 1.4 MYA, and a fourth one, Homo erectus (Fig. 12.8), appeared around 1.5 MYA (Maslin et al. 2015). The anatomical transition from H. ergaster to H. erectus is so gradual as to raise again the suspicion that these are the same species. Because fossils ascribed to H. erectus also occur in Asia, paleoanthropologists prefer to use this denomination for the Asiatic lineage, whilst reserving H. ergaster for the African lineage. There is today almost general consensus about ergaster and erectus being two populations (African and Asiatic, respectively) of the species “Homo erectus sensu lato” (Maslin 2015), and we will do so in the following text. Homo erectus lived in Africa alongside with Homo habilis for several hundred thousand years and persisted in Asia up to at least 143 KYA, covering a temporal range of nearly two million years and thus being by far the most long-lived human species known. H. erectus was a large-bodied, dedicated terrestrial species that moved out onto the open savanna and most likely no longer slept in trees. From Africa, H. erectus migrated to Asia, arriving in China around 1 MYA. The most archaic African fossils of H erectus have an endocranial capacity of about 850 cm3, whereas later fossils from China have a capacity up to 1100 cm3. Distinctive traits of the cranium of H. erectus relative to australopithecines and H. habilis include a smaller dental arcade, large brow ridges fused together into a prominent supraorbital

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Fig. 12.10 a. Oldowan tool from Guelmim-Es Semara (Museo Arqueológico Nacional de Madrid). Oldowan tools were obtained by chipping off one or a few flakes from one side of a pebble with another stone. The Oldowan industry was used by early humans across much of Africa, South Asia, Middle East and Europe from 2.6 up until at least 0.45 MYA, being gradually replaced by the more sophisticated Acheulean industry appeared about 1.76 MYA b. Lithic artefacts of Acheulean industry, the first standardized tradition of toolmaking of Homo erectus and early Homo sapiens. Named for the type-site Saint-Acheul, in northern France, Acheulean tools were made by removing chips from both the front- and backside of chalcedony, jasper or flint cores, to obtain a sharper cutting edge. (a) By José-Manuel Benito Álvarez (España), https://commons.wikimedia.org/w/index.php? curid¼1892114 (b) By Didier Descouens, https://commons.wikimedia.org/w/index.php?curid¼11656505

thorus, a more orthognatic (less protrusive) face skeleton, a less sloped frontal bone, and a posterior projection of the occipital bone referred to as the occipital thorus or “chinon”. The emergence of H. erectus sensu lato in East Africa represents a fundamental turning point in hominin evolution. Along with a dramatic increase in brain size, the fossil evidence documents changes in life history (shortened interbirth intervals, delayed development), pelvic and shoulder anatomy (Sect. 12.6),

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increased body size, reduced sexual dimorphism, and longer hindlimbs for longdistance running, all pointing to increased ecological flexibility (Gruss and Schmitt 2015; Maslin et al. 2015). The artefacts initially associated with Homo erectus settlements are of the Oldowan type. As early as 1.7 MYA, this species developed a more sophisticated toolmaking technique known as the Acheulean technology (de La Torre 2016). Acheulean tools were flaked on both sides and could be handled more effectively than Oldowan tools (Fig. 12.10b). Circumstantial evidence suggests that H. erectus used fire occasionally at least 1.42 MYA; more robust evidence indicates that about 800 KYA H. erectus was able to control fire, and from 300 KYA he employed fire routinely for cooking, heating, and protection against predators (Wrangham and Carmody 2010; Gowlett 2016). During his long tenure, Homo erectus dramatically expanded his geographical distribution and, with time, diversified into populations with distinct anatomical traits which have been given names linked to the geographical origin, such as H. erectus georgicus (found in Georgia) and H. erectus pekinensis (the “Peking man”). The fossil known as the “Turkana boy” is the most complete early human skeleton ever found (Fig. 12.11). It belonged to an individual of 8–19 years, about 1.5 m tall, with an endocranial capacity of about 880 cm3 and signs of lumbar disc herniosis, possibly caused by a fall. The tooth structure suggests that the adolescence in this hominin was shorter than in modern humans, but longer than in chimpanzees (Graves et al. 2010). In 2003, a joint Indonesian-Australian research team found a nearly complete female skeleton of a tiny human that lived about 80 KYA ago on the island of Flores, Indonesia. The skeleton’s unique traits such as its small body and brain size (about 380 cm3) led the scientists to assign the find to a new species, Homo floresiensis. Other remains of H. floresiensis detected subsequently date to between 100 and 60 KYA. H. floresiensis individuals stood approximately 1.1 m, had large teeth for their small size, shrugged-forward shoulders, no chins, receding foreheads, large feet and relatively short legs. Despite their small body and brain size, H. floresiensis used stone tools, hunted small elephants and large rodents, coped with predators such as giant Komodo dragons, and may have used fire (Brown et al. 2004; Morwood et al. 2005). A number of explanations have been proposed for the unusual features of this species. H. floresiensis might derive from an Asian population of Homo erectus that arrived on Flores and adapted to the insular environment evolving smaller body sizes (van den Bergh et al. 2016). Alternatively, H. floresiensis might directly descend from an early Homo lineage with roots in Africa, such as Homo habilis (Dembo et al. 2015). The latter hypothesis is supported by a recent phylogenetic analysis of cranial, dental, and postcranial characters, suggesting an origin from an ancient (~1.75 MY) African lineage, sister to H. habilis alone or to a clade encompassing H. habilis, H. erectus, Homo ergaster, and H. sapiens (Argue et al. 2017). Humans colonized Europe much later than Asia. The oldest European fossils, found in the Sierra de Atapuerca region of northern Spain in 1994 and dated to about 900 KYA, present a combination of archaic and advanced traits. For example, they

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The Genus Homo

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Fig. 12.11 (a) The “Turkana boy”, a partial skeleton of Homo erectus found in Kenya near Lake Turkana and dated to about 1.6 MYA. The pelvis was narrower and the femur neck longer than in modern humans, affording H. erectus more efficient bipedality. (b) Forensis reconstruction of the Turkana boy (a) By Claire Houck from New York City, USA - Turkana Boy, CC BY-SA 2.0, https://commons. wikimedia.org/w/index.php?curid¼4295714. (b) By Cicero Moraes - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php? curid¼24806403

have a prominent supraorbital thorus, but the facial skeleton is relatively light and the endocranial capacity is around 1000 cm3 (Bermúdez de Castro et al. 1997). Stone artefacts associated with the Spanish settlement are of Oldowan technology (Carbonell et al. 2008). This unusual combination of characters led the discoverers to propose a novel species, Homo antecessor. A cranium found at Ceprano (Italy) and initially considered coeval with H. antecessor but subsequently dated to 430– 385 KYA, also presents a combination of archaic and advanced traits, including an endocranial capacity of about 1200 cm3. Although recognizing the affinity of this form with fossils found in South Africa (referred to as H. rhodesiensis or H. heidelbergensis, see below), the discoverers assigned it to a new species, Homo cepranensis (Mallegni et al. 2003), now treated as a synonym of H. heidelbergensis (Mounier et al. 2011).

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In 1907 an archaic jaw complete with teeth was detected near Heidelberg, Germany, and assigned to a new species, Homo heidelbergensis. Similar fossils were later discovered in Würtemberg (Germany), Greece, Italy and France. The volcanic complex of Roccamonfina in Italy retains footprints left by H. heidelbergensis in volcanic dust about 350 KYA. Most remarkably, fossils assigned to H. heidelbergensis were also found in Zambia (formerly Rhodesia) and at Bodo d’Ar (Ethiopia) in deposits dated to about 300 and 600 KY, respectively. Although the Acheulean industry had established in Africa long before, the Bodo cranium of H. heidelbergensis was associated with stone tools of Oldowan type. The same holds true for the 450 KY-old fossils of this species found at Arago, in France, but more recent strata in the same site showed a transition to Acheulean-type industry. Significant technological innovations are associated with later fossils of H. heidebergensis, although it is uncertain if these were developed autonomously or acquired from other human species (Tattersall and Schwartz 2009). A nearly complete skull from Kabwe (Northern Rhodesia, now Zambia), originally presented in 1921 under the name Homo rhodesiensis, is now attributed to H. heidelbergensis. The data suggest that H. heidelbergensis appeared in Ethiopia around 600 KYA and spread to South Africa and Northern Europe about 500 KYA. Considering the African origin and the fact that most of the definitions of H. heidelbergensis as an Afro-European taxon were from the Kabwe skull, Hublin (2009) proposed to drop the denomination H. heidelbergensis and reintroduce the original name H. rhodesiensis. In 1856, miners working in a limestone quarry in the Neander valley near Düsseldorf (Germany) discovered the bones of what they thought was a “cave bear”. The first scholars who examined the remains recognized that they were human but presented dramatic differences from modern humans. The skull had a pronounced supraorbital and occipital thorus, a sloped frontal bone and prognate face. When the bones were first exhibited to the public, it was common opinion that they belonged to a human affected by deformity. In the following years similar fossils were found in other European sites, and fossils detected in 1829 in the Engis caves (now in Belgium) and in 1848 in the Forbes’ Quarry, Gibraltar, were recognized as belonging to the same archaic lineage as the type specimen found in Germany. In 1963, William King proposed a new species that he called Homo neanderthalensis, the man of the Neander valley. Following the early discoveries, reports of H. neanderthalensis increased exponentially, with new sites found in Western, Central, Eastern, and Mediterranean Europe, as well as in Southwest and Central Asia (Krause et al. 2007). In spite of archaic traits including heavy brow ridges, an occipital thorus and the lack of a chin, an endocranial capacity equal or even superior to that of modern humans places H. neanderthalensis (Fig. 12.3a, c) in an advanced position relative to H. antecessor and H. heidelbergensis. The average stature and body mass were about 169 cm/78 kg for males and 160 cm/66 kg for females. The lower stature and bulkier body structure relative to early modern humans are interpreted as adaptations to the cold. A study of Neanderthal skulls suggests that their eyesight may have been better than in modern humans, owing to larger eye

12.4

The Genus Homo

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sockets and larger areas of the brain devoted to vision (Pearce et al. 2013). The Neanderthals (less commonly also spelled “Neandertals”) produced stone tools of Acheulean type, used fire, probably employed pigments for body decoration and buried their dead. From about 160 KYA, the Neanderthals employed the Mousterian (“or Mode 3”) technology, a more sophisticated lithic reduction than the Oldowan and Acheulean technology, so named after the type site of Le Moustier in France. The abundance of animal bones near Neanderthal settlement sites is evidence that meat from large mammals including woolly rhinos and mammoths was an important part of their diet (Tattersall and Schwartz 2009). The core of Neanderthals’ territory was in Europe, ranging from Spain and Italy to Russia. Yet, at times their overall geographic range expanded to the area of modern Turkey, Iran, Irak, Saudi Arabia and Central Asia up to the Altai Mountains. The southern peninsulas of Europe possibly acted as refugia for Neanderthal populations during glacial intervals, allowing survival during extreme climatic conditions when northern regions of the continent were uninhabitable (HarvatiPapatheodorou 2013). The fossils from the Sierra de Atapuerca in Spain, originally ascribed to H. antecessor, are now considered to be archaic Neanderthals (Meyer et al. 2016). Remains discovered in Spain near Gibraltar and tentatively dated to about 24 KYA possibly document the last occurrence of H. neanderthalensis; at this time, the Neanderthals had already disappeared from the rest of Europe for several thousand years (Hublin 2009; Harvati-Papatheodorou 2013). H. sapiens appeared in Europe about 45 KYA, implying that Neanderthals have co-existed in the same territory with Sapiens for many thousand years. The proposed date of Neanderthal extinction falls in a period dominated by climatic instability, suggesting inability to adapt to unfavourable climatic conditions as a possible reason. Climate change, however, is unlikely to be the sole cause for demise, because Neanderthals had successfully survived previous cold phases. A likely hypothesis is that competition for increasingly limited resources with modern humans gave the final blow to already stressed Neanderthal populations during the critical time interval of their co-existence in Europe (Harvati-Papatheodorou 2013). Sequence analysis has produced evidence for interbreeding between Neanderthals and Sapiens by showing that about 1.5–2.1% of DNA in non-Africans modern populations is of Neanderthal origin (Sect. 12.5). In March 2010, scientists announced the discovery of a finger bone fragment of a juvenile hominin in the remote Denisova Cave in the Altai Mountains in Siberia, dated to about 41 KYA. The cool climate of the cave had preserved DNA, permitting a team of scientists at the Max Planck Institute for Evolutionary Anthropology in Leipzig (Germany) to sequence DNA extracted from the fragment. The results revealed that: (a) The Denisovan lineage is genetically distinct from both H. neanderthalensis and H. sapiens (b) Denisovans, Neanderthals and Sapiens derive from a common ancestor dated to about 800 KYA.

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Fig. 12.12 Taxa recognized in a typical speciose hominin taxonomy and their time span as inferred from fossil evidence. Redrawn from Wood (2010)

(c) The Denisovans interbred with Neanderthals and Sapiens, about 3–5% of the DNA of extant Melanesians and Aboriginal Australians being of Denisovan origin (Meyer et al. 2012; Reich et al. 2011; Prüfer et al. 2014; Vernot et al. 2016). The scientific community has not unanimously accepted all the species mentioned above, nor numerous other species proposed over the time (Fig. 12.12). Particularly radical is the position of paleoanthropologist Tim White, who maintains that all hominin fossils of an age between 2 MY and 300 KY should be lumped together within a single species, H. erectus, with pronounced regional variability (White 2003). White suggests that H. erectus made the transition from the Oldowan to Acheulean technology and spread throughout the “Old World” (Asia and Europe) without producing any other species before the divergences of H. neanderthalensis and H. sapiens. Other researchers find White’s scenario an excessive taxonomical simplification, but there is no agreement about the species to retain or reject. The discovery of past interbreeding events between formally distinct species makes the issue even more problematic. Because these events left a recognizable fingerprint in our genome, the hybrids must have been fertile. Based on the biological definition of species (Chap. 7), inter-fertile taxa such H. neanderthalensis and H. sapiens are not separate species. Paleoanthropologists, however, are traditionally reluctant to apply the biological notion of species, in part because of its psychological impact when dealing with humans. Species separation in anthropology will remain a matter of personal opinion as long as there is debate among scientists about how to define and

12.5

Origin and Diffusion of Modern Humans

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circumscribe a human species (Box 12.2). Critical information might come from molecular analysis, yet difficulties in sequencing ancient DNA have so far hampered phylogenomic characterization of earlier hominin lineages (Sect. 12.5).

12.5

Origin and Diffusion of Modern Humans

The history of Homo sapiens as described by fossils begins in the Kibish Formation, an area located in the basin of the Omo River, Ethiopia. Here, in 1967, paleontologists found two partial skulls, four jaws, a femur, around two hundred teeth and numerous fragments of other parts of the skeleton, with an age initially estimated at about 160 KY. Despite a few archaic traits, for example a continuous supraorbital thorus, an occipital thorus and a moderately sloped frontal bone, the skulls presented characters unique to modern humans such as a projecting chin, a widening at the level of the parietal bones and, most notably, an endocranial capacity of over 1400 cm3. More recently, these fossils were argon-dated to about 195 KYA (McDougall et al. 2005). Three similar skulls dated to about 160 KYA were subsequently discovered at Herto Buri, also in Ethiopia (White et al. 2003). These fossils are interpreted as an archaic form of Homo sapiens (“H. sapiens idàltu”), appeared in Ethiopia around 200 KYA and probably ancestral to modern humans. About 100 KYA, H. sapiens was present in two South-African sites and in the Middle East, the latter already inhabited by H. neanderthalensis. The anatomy of these more recent populations was completely modern, with innovative traits including a tall globular braincase, a straight forehead, a delicately built and distinctly retracted face, separate supraorbital ridges, a prominent chin (Fig. 12.13), and a slender postcranial skeleton (Tattersall 2009; Hublin 2013; Stringer 2016). H. sapiens subsequently disappeared from the Middle East, to re-appear in that area about 70 KYA. Around 45 KYA, H. sapiens was present in Europe, Asia including large islands, and Australia. The European lineage of H. sapiens is traditionally known as the Cro-Magnon man (Fig. 12.13c, d) from the site in France where its remains were found first; this term is now replaced in the scientific literature with the less evocative acronym EEMH from “Early European Modern Humans”. The earliest Cro-magnon fossils have been found in Italy and dated to about 45 KYA (Benazzi et al. 2011). By 33 KYA the Cro-Magnons had arrived in Britain, then connected to the continent by a land bridge, and around 25 KYA they were present in the territory of modern Czech Republic. Cro-Magnons were tall compared to coeval Neanderthals (the males about 1.80 m), had an endocranial volume of about 1650 cm3 (slightly larger than extant humans) and were excellent hunters capable of killing large prey including mammoths. Early Cro-magnons produced Mousterian tools up to about 41 KYA, when they developed a novel, more sophisticated technology known as the Aurignacian culture, or “Mode 4” (Hoffeker 2009; Benazzi et al. 2015). Aurignacian finds encompass well-finished bone and flint tools such as arrows, spear points and axes, as well objects of art like pendants, bracelets, ivory beads, and animal figurines

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Fig. 12.13 Facial reconstruction and skull anatomy of Homo neanderthalensis (a, b) and Homo sapiens Cro-Magnon (c, d) a: http://humanorigins.si.edu/evidence/human-fossils/species/homo-neanderthalensis b, d: https://annoyzview.wordpress.com/tag/homo-neanderthalensis/ c: Bettmann Archive

including anthropomorphized depictions that may be evidence of symbolic behaviour (Fig. 12.14). The oldest undisputed musical instrument is the Hohle Fels Flute discovered in the Hohle Fels cave in Germany’s, made from a vulture’s wing bone and dated to approximately 35 KYA. From about 17 KYA, the Cro-Magnons produced magnificent cave paintings such as those in the Lascaux cave, in southwestern France. Historically, most of the debate on the origin of modern humans has focused on two competing hypotheses: the multi-regional or anagenetic theory and the Out-ofAfrica (OOA) theory (Liang and Nielsen 2011). According to the multiregional theory, multiple lineages of H. sapiens evolved independently in different regions of the world from local populations, in sequences such as H. ergaster ! H. heidelbergensis ! H. sapiens in Africa, H. erectus ! H sapiens in Asia, H. heidelbergensis ! H. nearderthalensis ! H. sapiens in Europe.

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Fig. 12.14 An ivory figurine representing a “lion-man”, found in Germany and dated to about 32 KYA, is one of the earliest expressions of human ability to imagine things that do not exist

This theory explains the high genetic homogeneity of extant humans by assuming high levels of gene admixture (interbreeding) between African, European and Asiatic populations. The OOA theory, primarily based on fossil record documenting the appearance of modern humans first in Ethiopia and then elsewhere in the globe, proposes a single recent origin in Africa. Before the application of molecular phylogeny to paleoanthropology, the choice between the two theories depended essentially on the interpretation of the fossil sequence. The molecular approach has revolutionized paleoanthropology, meeting so great a success that it has rapidly become an autonomous discipline: molecular anthropology or, more generally, molecular paleontology (Pääbo 2014; Llamas et al. 2017). Mitochondrial DNA (mtDNA, Chap. 6) is particularly useful in human phylogenomics. Unlike nuclear DNA, mtDNA is present in cells in multiple copies, thus facilitating extraction and cloning, is not subject to meiotic recombination and is transmitted only across the maternal lineage (Chap. 7). In addition, mtDNA encompasses non-coding sequences with a high frequency of mutation due to the relatively inaccurate mechanism of duplication of mtDNA. Note that mutations of coding sequences are as frequent as for non-coding sequences, but they rapidly disappear because of natural selection, so we do not see them. The high mutation rate of noncoding mtDNA permits the reconstruction of relatively recent cladogenesis events such as those involved in human evolution. The main limit of molecular paleontology is post-mortem dearth of genetic material. Despite being a relatively stable molecule, DNA slowly degrades after death by breaking into fragments and going through chemical changes that alter the original sequence. For example, deamination

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of cytosine residues causes C to acquire the base-pairing properties of T, and G the base-pairing properties of A. Because of this, DNA sequencing is currently feasible only for relatively recent fossils, viz. H. heidelbergensis, H. neanderthalensis, the Denisovans and H. sapiens. Contamination from contemporary human DNA is a further potential source of errors in molecular anthropology. To avoid contamination, sample preparation and DNA extraction must be done in dedicated clean rooms. The introduction of high-throughput technologies (Green et al. 2010; Liang and Nielsen 2011) has dramatically improved the sequencing performance, permitting sequence determination of about 60% of the genomes of Neanderthals and Denisovans to a quality as high as for extant humans (Pääbo 2014; Prüfer et al. 2014, 2017). Analysis of DNA markers in modern humans throughout the world has revealed that the overall African population exhibits the maximum genetic variability (Tetuskin 2001; Tishkoff et al. 2009). Total genome analysis traced extant humans back to an ancestral African population dated to about 200 KYA (Mallik et al. 2016). Mitochondrial genome analysis indicates that the modern mtDNA haplogroup L3, a very large proportion of contemporary humans, derive from an ancestral population that lived in Africa around 60 KYA (Fu et al. 2013). Two main groups of contemporary people trace their matrilineal ancestry to this population, namely (a) a significant proportion of Africans (approx. 70% of North Africans, 50% of East Africans, 40% of West Africans, 25% of Central and Southeast Africans and approximately 10% of South Africans, and (b) the rest of the world population (Mirazòn Lahr 2016). These results are incompatible with the multiregional hypothesis and clearly support the OOA theory. The nuclear genome of non-African modern humans averagely contains about 1.2% of sequences of Neanderthal origin, slightly more in Middle Eastern and Asian populations (Sankararaman et al. 2014; Prüfer et al. 2017), reflecting past interbreeding events between H. neanderthalensis and non-African Sapiens (Fig. 12.15). The lack of Neanderthal genes in African modern humans is evidence that interbreeding occurred when modern humans carrying Upper Paleolithic technologies encountered Neanderthals after migrating out of Africa. Based on the frequency of non-random associations of allelic sequences (a phenomenon known as linkage disequilibrium) in the genomes of present-day Europeans, the last gene flow from Neanderthals into Europeans was circumscribed to 37–86 KYA (Sankararaman et al. 2012), in line with paleontological evidence for Neanderthals and Cro-Magnons living in the same territory between 45 and 24 KYA. The low level of gene admixture between the two lineages may reflect low inter-fertility or strong cultural barriers. As already mentioned, Australo-Melanesians and to a lesser extent other eastern non-Africans possess genes of Denisovan ancestry. At odd with former inferences, the Simons Genome Diversity Project demonstrated that indigenous Australians, New Guineans and Andamaneses derived from the same source population as other non-Africans (Mallick et al. 2016). Evidence from multiple lines of research suggests that (a) Neanderthals and Denisovans were more closely related to each other than to modern humans and diverged from an ancestral population between 381 and 473 KYA; (b) the last

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Fig. 12.15 Gene-flow events documented by genome analysis of ancient and contemporary humans. The numbers indicate the approximate average fraction of the genome contributed to each receiving group. X denotes an unknown Neanderthal group that admixed with Eurasian modern humans, and Y the Denisovan group that admixed with the ancestors of present-day people in Oceania. People on mainland Asia also have small amounts of DNA from Denisovans (dotted arrow). From Pääbo (2014), license number 4461990523349

concestor of H. sapiens and Neanderthals/Denisovans was a population of H. heidelbergensis living in Africa at least 600 KYA (a) H. sapiens diverged from an African population of H. heidelbergensis about 200 KYA (Hublin 2013; Prüfer et al. 2014, 2017; Meyer et al. 2016; Stringer 2016; Fig. 12.16). Climate change was probably the main environmental driver of the expansions and contractions in the size and geographical distribution of human populations. The demographic history of modern humans encompasses four major transitions (Harcourt 2016; Mirazòn Lahr 2016). Sapiens originated in East Africa about 200 KYA and from there he started a first major expansion across Africa and to neighbouring Western Asia 130–100 KYA. A second migration out of Africa occurred between 70 and 45 KYA, replacing indigenous populations with extremely limited interbreeding. Local/regional diversification took place between 45 and 25 KYA. The last transition involved widespread dying out of hunter–gatherer populations, expansion of farming and emergence of complex societies, from about 11 KYA to present. In the course of the second migration, Sapiens first settled in the Middle East around 70 KYA, and from there he started worldwide expansion about 60 KYA. A migration wave circumscribed the Black Sea, passed across Greece and the Balkans, and arrived in Western Europe around 45 KYA; a second wave crossed the Arabian

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Fig. 12.16 Provisional phyletic tree of the human lineage as inferred from paleontological and genomic evidence. Dubious species are omitted for clarity. The symbol X indicates extinct lineages. H. habilis is treated as the stem lineage. H. ergaster (considered the African sister group to H. erectus) diverged from H. habilis about 1.8 MYA and remained stable in Central Africa for about 400,000 years before disappearing from the fossil record about 1.4 MYA. Much later evidence of H. heidelbergensis in the same region indicates that there is a hole in the paleontological record or that an intermediate species is still missing in the record. The Neanderthal/Denisovan lineage probably diverged from a non-African population of H. heidelbergensis around 400 KYA, whereas H. sapiens diverged from an African population of H. heidelbergensis about 200 KYA. The concestor of H. neanderthalensis and H. sapiens is dated to about 600 KYA

coast and proceeded through Iran, Pakistan and India up to East Asia (Hoffeker 2009). In concomitance with the coldest peaks of the Quaternary glaciation, with a sea level up to 100 m lower than today, Sapiens moved southward along the Indonesian islands and arrived in Australia about 45 KYA. In the Neolithic, a second wave of Sapiens arrived in Europe from the area known as the “Fertile Crescent” (Sect. 12.8). These were farmers that moved to find cultivable land, carrying domesticated plants and animals and assimilating indigenous populations of hunter-gatherers along the way (Fu et al. 2012). Genetic evidence suggests that farmers coming from Anatolia (Turkey) settled in the Aegean area and from there resumed migration towards Western Europe (Hofmanovà et al. 2016). By around 5000 KYA, almost all populations in mainland Europe had moved from a hunter-gatherer life style to agriculture. Comparison of DNA sequences from ancient and modern Europeans points to admixture of at least three well-differentiated populations: (i) West European hunter-gatherers, (ii) early European farmers mainly of near-eastern origin, and (iii) ancient northern Eurasians (Lazaridis et al. 2014). There is broad agreement that the Americas were settled by migrants from northeastern Asian populations via Beringia, the land bridge that connected northeast Asia with northwest North America during the Pleistocene. Yet, the chronology

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Death from a Distance: High-Speed Throwing

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of migrations, the source populations and the routes followed remain uncertain. Phylogenomic analysis (Moreno-Mayer et al. 2018) suggests that a single founding Asian population settled in Beringia around 36 KYA, becoming genetically isolated from the Asian pool around 26 KYA. The Beringians entered Alaska before 20 KYA, and around 17.5–14.6 KYA they gave rise to a northern and a southern Native American lineage, to either of which all modern Native Americans belong. There is evidence of a second gene flow from a Siberian population to northern Native Americans some time before 11.5 KYA. This scenario contrasts with earlier models assuming several independent migrations from Asia. Homo sapiens has long been described as ‘polytypic’ by traditional anthropology, because of evident morphological differences among populations. Phenotypic diversity, however, depends on a restricted number of genes affecting characters such as the color of the skin, facial features and the hair. The global genetic diversity of contemporary humans in fact has turned out to be only slightly higher than variability within individual populations. A comparative analysis of the genomes of 185 individuals from African populations and 184 people from European and Chinese populations detected single nucleotides variations at 38,877,749 positions. Of these, not a single nucleotide difference was unique to Africans or Eurasians, and only 12 differences occurred in 95% or more of Africans and in 5% or less of Eurasians, or vice versa (Pääbo 2014). Genetic homogeneity suggests that at a point in the past, the worldwide population of modern humans reduced to extremely small size, thus experiencing a loss of genetic diversity, a form of genetic drift known as the “founder effect” (Bamshad et al. 2004). The hypothesis that this hypothetical reduction of the ancestral population followed the catastrophic eruption of Toba volcano in Sumatra about 75 KYA is now dismissed (Lane et al. 2011). Comparative genomic analysis of autochthonous modern populations also documents a decline in genetic and phenotypic diversity with distance from Africa; this is interpreted as a “serial founder effect” that cumulatively reduced diversity during out-of-Africa expansion (Li et al. 2005; Ramachandran et al. 2005; Stringer 2016).

12.6

Death from a Distance: High-Speed Throwing

The abundance of animal bones near ancient settlements indicates that meat has been an important part of the hominin diet from at least 2.6 MYA, thus supporting the evolution of a more active metabolism than in other primates (Stanford and Bunn 2001). Hominins were physically much weaker than other predators, lacking natural weapons such as fangs and claws. They, however, managed to become excellent hunters using their ability to throw projectiles, initially just pebbles but later replaced with tools manufactured on purpose. High-speed throwing depends on a set of derived anatomical features that permit elastic energy storage and release at the shoulder (Roach et al. 2013). The largest contribution to projectile velocity comes from internal (medial) rotation of the humerus around the long axis. Shoulder anatomy permits the humerus

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Fig. 12.17 High-speed throw in humans depends on complex interaction of muscles and ligaments in the arm, shoulder and torso, wich accumulate elastic energy during the preparatory arm-cocking phase and rapidly release it during the acceleration phase. The arm-cocking phase starts with a large step towards the target; as the foot hits the ground, the arm has already been rotated externally, extended horizontally, and abducted nearly 90
at the shoulder, with forearm flexion approaching 90
at the elbow. A rapid rotation of the torso towards the target and the contraction of pectoralis major, the major shoulder horizontal muscle, generate large torques. In the acceleration phase, these stretched elements recoil, releasing energy and helping to power the extremely rapid internal rotation of the humerus, which transmits kinetic energy to the projectile. The mechanics of these movements is deeply wired in the human neuromuscular system, being spontaneously produced without specific training. From Roach (2013), license number 4461991007030

to align with the main pectoral muscle (pectoralis majus), which is the most important muscle involved in the operation. This not only produces the maximum angular momentum possible, but stores elastic energy in the articulations of the shoulder, elbow and wrist before the throw; further elastic energy accumulates with torso rotation (Fig. 12.17). The forward rotation of the humerus during the throw takes a few milliseconds, being the fastest movement that the human body can produce (more than 9000
per second). The chimpanzee anatomy lacks the traits necessary for high-speed throwing, being more suitable for arboreal locomotion. Morphological traits that help human throwers to store elastic energy have gradually evolved in hominins, some pre-dating Homo. A tall, decoupled waist first appeared in Australopithecines as an adaptation for bipedal locomotion. A degree of humeral torsion was present in both Australopithecines and early Homo. The anatomical architecture underpinning throwing performance, including shoulder orientation, long legs, and hyper-extendable wrists, was entirely in place in Homo erectus (Roach et al. 2013). The invention of the bow and sling (at least 9 KYA) increased the distance between the hunter and prey, making hunting a less dangerous occupation and probably contributing to the reduction in the robustness of the human body recorded in fossils (Ruff 2002). The mechanics of manual throwing is still important today in several working activities as well as in sports such as baseball, tennis and basket. The evolution of high-speed throwing not only gave early humans access to a richer diet, but also enabled them to kill or injure conspecifics from a substantial distance. According to the scenario proposed by Bingham and Souza (2009), this

12.7

Why Don’t Humans Have Fur?

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triggered positive selection for individuals who actively suppressed conflicts of interest with conspecifics, thus paving the way to the evolution of cooperation (Sect. 12.12).

12.7

Why Don’t Humans Have Fur?

Among the traits that distinguish humans from the other primates is an almost naked skin. Modern humans have hairs only on the head, pubis and armpits, as well as on the face as a secondary sexual character in males, the rest of the body being completely naked or covered only with sparse hairs. Anthropologists have been puzzling about the reason for this bizarre evolutionary choice. Fossils give us information on the skeleton, muscles, even brain anatomy, but cannot tell us anything about the skin and hair. The hairs are an apomorphy of mammals. Thick fur insulates the body, affording protection against cold or excessive heating by solar radiation. Fur also protects the body from abrasion and humidity, may provide camouflage to confound predators or preys, and may send signals for conspecific recognition and communication. Despite these important functions, fur was lost in a number of mammals, notably in aquatic (e.g. the cetaceans) or soil-dwelling species (e.g. the naked mole rat). Some largesized mammals such as the elephants and rhinoceroses lost the fur to facilitate dissipation of excess metabolic heat in warm environments; in contrast, elephants and rhinoceroses living in Europe and Northern Asia during the Quaternary glaciations had dense furs. Fur loss in humans is probably also linked to thermoregulation, although humans are not particularly large mammals. As discussed above, the hominin lineage evolved under selective pressure of climate change driving rain forest replacement by savanna in large areas of Central Africa. Living in the savanna as a forager is problematic; firstly, foraging usually requires covering large distances under high air temperatures; secondly, it may be necessary to run for hunting animal prey or escaping predators. An increase in core body temperature of 1
C is sufficient to trigger the onset of heat stroke in modern humans. Model previsions (Ruxton and Wilkinson 2011) indicate that, in the typical conditions of a hot cloudless day in an open equatorial region, hair-covered hominins could not maintain heat balance while walking for sustained periods even if they travelled near dusk or dawn. Based on the assumption that early hominins had similar reactions to hyperthermia as modern humans, it has been estimated that furred individuals would walk in full bright sunshine for only 10–20 min before overheating. On the other hand, hair-less hominins would receive a higher heat load than furred individuals in the middle of the day, due to the lack of the thermal buffering effect of hair; therefore, in the absence of other adaptations, hair-less hominins would be obliged to restrict active life to the early and late hours of the day. The human solution to these contrasting forces was a dramatic improvement in the mechanism of evaporative cooling by perspiration, i.e. the secretion of water through the skin.

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The mammal skin contains three types of glands, all stimulated by a rise in the body temperature: sebaceous, apocrine and eccrine glands. Sebaceous and apocrine glands predominate in most mammals. These glands are associated with hair follicles and produce an oily secretion that is of limited efficiency in heat dissipation, because it tends to gather on the surface of the fur, where evaporation does not cool the skin effectively. Eccrine glands are not associated with hairs and produce a watery solution (the sweat) containing sodium chloride and nitrogenous waste (urea) that directly wets the skin, favouring heat dissipation. In primates, eccrine glands predominate over sebaceous and apocrine glands and are concentrated in areas with less dense fur such as the abdominal area and pits. In adapting to savanna conditions, humans improved this cooling system by losing the fur and increasing the number of eccrine glands, which ranges in adults between two and four millions (Jablonski 2013; Bovell 2015). The excess heat load that must be dumped through sweating in the middle of the day is higher for quadrupeds than bipeds, because a higher fraction of a quadruped’s body surface is exposed to direct sunlight. Yet, a thermoregulatory advantage to bipedality probably arose only after the loss of the fur and the evolution of high sweating rates (Ruxton and Wilkinson 2011). When did the hominin lineage loss the body hair cover? Several lines of evidence suggest that the australopithecines lived at altitudes higher than 1000 m above sea level, with an average daily temperature estimated at about 25
C. When these conditions are introduced in model previsions, the heat load during the day is strongly reduced even for furred animals, implying low or no selection pressure for fur loss; indeed, low nocturnal temperatures at the altitudes where australopithecines lived probably required the maintenance of fur. The consensus is that the fur was probably lost later, in the genus Homo, with the exploitation of open habitats at lower altitudes (Dàvid-Barrett and Dunbar 2016). The combination of bipedality and efficient thermoregulation enabled early humans to run over long distances without suffering from overheating, an ability known as endurance running that was most likely pivotal to their biological success (Ruxton and Wilkinson 2011). The only “fur” that modern humans retained is the hair on the head, which protects the skull and its precious content from sunlight, hairs in the pubic area and armpits, which are associated with pheromone-producing apocrine glands, and the hairs on the face, arms, thorax and legs in the adult male. Body hairs in human males are a secondary sexual character under the control of testosterone, probably signaling sexual maturity (Van Schaik 2016). The interest of human females to choose a sexually competent partner (testosterone is required for sperm development and maturation) might have positively selected hirsutism in males. Having hormonedependent traits that exaggerate differences between males and females may be adaptive, as hormonal imbalance is a common cause of infertility in each sex. Significant beard growth is largely absent from human populations in Africa, suggesting that the trait only appeared after the major migration out of Africa. Beard growth, however, is almost absent also in natives from East Asia and America. This variation is consistent with the hypothesis that beard growth only emerged after complex clothing (see below in this section) became an established habit, as a signal of

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gender identity and condition where clothing hid body shape and hairiness. The habit of shaving or not shaving and shaving styles are subject to cultural evolution. There is evidence that mustaches and beards tend to be more common when men outnumber women and therefore must compete for mates; this suggests that being bearded signals proneness to violence and masculine behaviour, whereas being clean-shaved is a sign of reduced violence and proneness to invest in offspring (Van Schaik 2016). Why are females less hairy than males? One of the hypotheses put forward proposes that human males preferred females with a juvenile aspect (thus more likely to be fertile), and this might have negatively selected body hair production in adult females (Jablonski 2013). Because a juvenile aspect also promises that fertility will persist for a longer time, the persistence of childish features in adult females might have a link with pair bonding and male involvement in offspring care (see next section). As for all evolutionary transitions, the loss of fur was certainly a gradual process. The MC1R allele present in dark-skinned modern African populations appeared around 1.2 MYA (Roger et al. 2004). Because this allele controls constitutive synthesis of eumelanin, a pigment that protects the skin from ultraviolet radiation, its diffusion in human populations probably accompanied fur reduction. The process presumably attained completion in H. erectus, whose body proportions reflect adaptation to endurance running (Ruxton and Wilkinson 2011). A major difference between the human and chimpanzee genome is in genes affecting skin properties. Human variants of these genes encode for proteins that make the skin particularly waterproof and resistant to abrasion; in contrast, body hairs are extremely fragile in humans compared to chimpanzee, due to differences in keratin structure (Moll et al. 2008). The glabrous skin on fingers and toes of primates including humans forms wrinkles in response to immersion in water. Water-induced wrinkling is not due to osmotic swelling of the outermost layer of skin, as initially thought, but in fact is caused by vasoconstriction under the control of the autonomic nervous system. The reaction is fast enough to happen in response to dew or rainy conditions but not so quick that casual contact with water (like when eating fruit) will elicit it. Waterinduced wrinkling improves the grip on wet or submerged objects, possibly due to a water-channeling effect as in tires (Kareklas et al. 2013). Wrinkling occurs more quickly under hypotonic conditions, consistent with the freshwater wet conditions relevant to primate grip (Changizi et al. 2011). Wrinkled fingers could be an adaptation to wet conditions that helped our ancestors to gather food from wet vegetation or streams; the analogous effect in the toes could help to get a better footing in the rain. If a wrinkled skin affords a better grip, why is it not retained all the time? A possible reason is that wrinkles may cause increased vulnerability to skin damage under dry conditions. Despite the paucity of hair, modern humans host three different lice: Pediculus humanus capitis that lives on the head, Pediculus humanus humanus that lives on clothes and moves to the skin only for feeding, and the crab louse Pthirus pubis that associates with body hair, primarily in the ano-genital area (Phthirus pubis is an etymologically correct but taxonomically invalid synonym). Because lice

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co-speciate with their hosts, each terrestrial vertebrate having its own species, louse phylogeny may provide valuable insight into host evolution. Molecular analysis has shown that Pediculus humanus as a species diverged from the chimpanzee louse (Pediculus schaeffi) 5 to 7 MYA, a dating consistent with the divergence of the chimpanzee and hominin lineages. The head lice variant (P. humanus capitis) evolved around 1.5 MYA (Light and Reed 2009), in good agreement with the likely dating of fur loss in the human lineage. The clothing louse, instead, evolved from the head louse only 170–80 KYA, perhaps concurrently with the introduction of clothing (Toups et al. 2013). The human crab louse seems to have diverged from the gorilla crab louse (Pthirus gorillae) about 3.3 MYA (Reed et al. 2007), being possibly acquired by pre-human hominins with the use of gorilla shelters. Clothing has its origin in protection from cold, symbolic and decorative implications being later additions. Protection from cold was probably already necessary in original African habitats, at least during the night, but clothing probably became indispensable only when humans moved to higher latitudes, where they experienced the effects of Pleistocene glaciations. Clothing may be “simple” or “complex”, the former consisting of a single layer of material (e.g. the furred skin of an animal) wrapped around the body, the latter of several layers of which at least the innermost one is fitted or “tailored” to the body shape. Simple clothes made from animal hides require basic skin-preparation techniques, mainly cleaning and scraping. The production of complex garments requires the skins to be carefully shaped by cutting and the pieces joined together by laces or sewing. Where multiple layers are used, the inner garments must be carefully prepared, with finer cutting and sewing to achieve the necessary close fit (Gilligan 2007). There are no physical remains of clothing from prehistory. Indirect evidence includes awls, likely used to pierce holes in furs, eyed bone needles and even buttons, as well as artistic depictions proving that early European populations of H. sapiens made use of complex clothing. The absence of equivalent evidence for Neanderthals suggests that these never developed the technology necessary for the production of complex garments. The bulky body shape and the lack of frostbite signs in the fossil record of Neanderthals suggest that these were biologically cold adapted, whereas the opposite is true for Sapiens. Pre-adaptation to cold enabled the Neanderthals to cope with moderately cold weather, although they apparently never expanded to resource-rich northern areas that were later successfully colonized by Sapiens. A possible reason for Neanderthal extinction was the lack of complex clothing, which prevented them from coping with climate worsening between 40 and 30 KYA (Gilligan 2007).

12.8

Reproductive Biology of Humans

Parental care is an ancestral trait in mammals, which own their name to the special glands used for feeding the young in the first phase of post-natal development. During gestation and lactation, the females are unfertile and sexually non-

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Reproductive Biology of Humans

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responsive, due to a suspension of ovulation. Because of this, in most mammal species it is advantageous for males to seek multiple mating opportunities, entirely leaving parental care to the mothers. It is thus not surprising that monogamy, a type of sexual interaction in which each male and female in a population mates with one partner of the opposite sex at a time, occurs in only about 10% of mammals. Even fewer mammal species (about 3%) evolved social monogamy, an exclusive partnership involving not only joint reproduction and biparental (maternal plus paternal) offspring care, but also social living arrangements such as shared use and joint defense of a territory. In contrast, over 90% of bird species are engaged in social monogamy, the reason most likely being the bird combination of a high metabolic demand, the need for egg hatching and the lack of mammary glands (Reichard 2003; Opie et al. 2013). Monogamy has costs and benefits for both genders, with a potential reduction in the genetic fitness of the progeny as the main cost, and increased chances of offspring survival as the main benefit. Potential reduction in offspring number is an extra cost for males, although monogamy may compensate for this by alleviating the cost of mate search and of competition with other males. It has long been maintained that evolutionary pressure for the emergence of social monogamy was mainly from fitness benefit associated with biparental care. It is now clear that, far from being a unitary phenomenon with a simple evolutionary explanation, social monogamy followed multiple pathways in different animal lineages (Reichard 2003). For example, direct male care is absent in 41% of socially monogamous mammals (Lukas and Clutton-Brock 2013) and, among primates, 15–29% of species form stable couples yet far fewer commit in biparental care (Fernandez et al. 2009). Multi-male/multi-female groups with polygynandrous sexuality (males and females mating promiscuously) and a rigid social hierarchy is a highly stable, probably ancestral organization in social primates, secondarily replaced in some lineages by single-male harems or monogamy as a response to ecological stress (Gintis et al. 2015). Modern humans practice a particularly advanced form of social monogamy in which the partners not only cooperate in rearing the offspring and acquiring material resources, but also share a social life. Human monogamy is often imperfect, with recurring infidelity and abandonment, polygyny (more than one wife at a time) being admitted in several societies now and in the past, and polyandry (more than one husband at a time) emerging occasionally. Nevertheless, polygamy remains an exception even when it is allowed, most modern human societies being based on the assumption that their members engage in enduring, sexually exclusive partnerships officially formalized and explicitly signaled. The flexibility of the human mating system contrasts markedly with reproductive strategies in other social primates. The two living chimpanzee species (Pan troglodytes and Pan bonomus) are our closest living relatives, but they could not be more different from us in sexual habits. The female has a menstrual cycle of 36 days and conspicuously advertises the brief phase (estrus) when she is fertile. When in estrus, the female engages in multiple copulations with several males, so that it is virtually impossible to establish the paternity of the young even in captive groups. Females become fertile at the age of

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10–13 years, and have an infant on average every 5 years; therefore, they can procreate a maximum of six offspring in a lifespan of 30 years. Infanticide by adult males is frequent, being generally directed at eliminating the offspring of rivals and inducing estrus in lactating females. Unlike the chimpanzee, the gorilla is polygynic, with dominant males forming a harem of 3–5 females. Females start ovulating at the age of about 8 years and signal ovulation mainly through pheromones, much less conspicuously than in chimpanzees. Female gorillas give birth every 6–8 years, for a maximum of five offspring in a lifespan of about 40 years. Only the mothers provide direct parental care, the main role of the father being protection against predators and other males. Replacement of a patriarch by another male generally entails the killing of the young already in the harem. Both in chimps and gorilla, multiple births are virtually absent: two infants depending on the same mother would have no chance of survival. Similar gestation periods in chimpanzees (237 days), gorilla (257 days), orangutan (260 days) and humans (266 days) suggest that the approximate 9 month gestation period predated the emergence of the hominin lineage. The human female traditionally stops lactation 2 to 3 years after giving birth, and multiple births are less rare (about 2%) than in other apes, thus she is potentially able to procreate over 10 offspring in a fertile lifespan of about 30 years. It is broadly agreed that the increase in the reproductive rate of humans is an outcome of monogamy, ensuring paternal care to the offspring, combined with alloparenting, i.e. additional offspring care by other individuals besides the parents. In fact, the human monogamous couple is the basis of a unique social network that extends to include other families and even unrelated groups (see below in this section). There is much debate about when and why social monogamy evolved in the hominin lineage. Hypotheses about “when” vary from very early, e.g. just after the divergence of early hominins from the Panina or even before, to relatively late, e.g. as an apomorphy of Homo erectus around 1.5 MYA, or even later as an effect of cultural evolution. For example, the lack of large canines and the reduction of sexual dimorphism in Ardipithecus ramidus (dated to about 4.4 MYA), have been interpreted as evidence of reduced male competition and an early origin of monogamy (Lovejoy 2009). Concerning “why”, three hypotheses of general application for the emergence of monogamy in non-human animals, notably primates (King 2016), are considered. The biparental care hypothesis proposes that increased need for parental care under harsh environmental conditions created selective pressure for paternal investment. Offspring care pays off in terms of reproductive fitness only if the male attendant is the real father; thus, this model holds that monogamy evolved in parallel to male parental care, to prevent reproductive exploitation among males (Lovejoy 2009). Although bipaternal care and social monogamy are usually associated, a comparative analysis of reproductive strategies in mammals suggests that male care in humans is probably a secondary effect rather than the key to the emergence of social monogamy (Lukas and Clutton-Brock 2013; Opie et al. 2013). Biparental care, however, helps explaining why a species, including ours, stays monogamous.

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Reproductive Biology of Humans

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The territorial dispersal (or female-spacing) model considers that adaptation to savanna dramatically expanded the territory to explore for foraging, and consequently reduced population density. This possibly favoured the transition from multiple mating, considered the ancestral condition, to mate guarding and eventually to monogamy, large territorial ranges preventing effective defense of more than one female. Settling down with a single partner reduces male risk of aggression by other males while patrolling his territory and increases the chances that his mate’s offspring are his own. The territorial dispersal model works well with several monogamous mammals but it hardly fits with the strong sociality of humans (Edgar 2014). The infanticide avoidance model assumes that social monogamy evolved in humans from female selection of males more inclined to protect their mates and the young from attack by other males. From the male perspective, infanticide is adaptive because it stops lactation-induced amenorrhea, thus stimulating the female to start a new pregnancy (Van Schaik 2016). The risk of being killed by members of the same species is particularly high for young primates because of slow development, due to larger brain sizes than in other mammals (Kuzawa et al. 2014). In the infanticide avoidance scenario, the males need to mate-guard to prevent females from mating with other males and to offspring-guard to prevent infanticide. This imposes a heavy load on the male, especially if combined with offspring feeding requiring longer foraging times. Statistical analysis across 230 primate species pointed to male infanticide as the most compelling explanation for the appearance of social monogamy in humans (Opie et al. 2013). In contrast, a research extended to 2500 mammal species found no significant correlation between social monogamy and a reduction in the frequency of infanticide, instead pointing to territorial dispersal as the most likely explanation for the evolution of social monogamy in mammals (Lukas and Clutton-Brock 2013), a conclusion independently confirmed by a third study (Schacht and Bell 2016). The lack of evidence favouring any of the mechanisms proposed suggests that the evolution of social monogamy in humans followed a different pathway from that in the majority of birds and mammals, perhaps under the effect of multiple pressures within the whole spectrum of possibilities considered. Modern humans are sexually dimorphic, adult men being 10–20% heavier on average than adult women, with wide variation among populations. Sexual dimorphism was clearly present in our likely ancestors, notably H. erectus. It is well known that, if a species shows sexual dimorphism in body mass, it is never entirely monogamous. Human sexuality is thus a mix of signals that suggest ancestral promiscuity with male-male competition, and a derived state with stable pair bonds and male care (Van Schaik 2016). de Waal and Gavrilets (2013) suggest that human social monogamy did not evolve with a Darwinian mechanism (i.e. through changes in genes controlling the reproductive behaviour), but only culturally. Bingham and Souza (2009) propose a partially different scenario in which humans are innately both monogamous and promiscuous, shifting from one to the other reproductive strategy according to circumstances. In their perspective, the predominance of monogamy in historical times is mostly an effect of normative enforcement. In fact, normative monogamy exerts a stabilizing influence in large societies by reducing sexual competition among males and the size of the pool of unmarried men (Henrich et al. 2011).

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A second puzzling trait of human reproductive biology is hidden ovulation, or ovulatory crypsis. Human females do not signal the phase of the estral cycle during which they are fertile (about 7 over 28 day) and they themselves are usually unaware of being or not being fertile. Hidden ovulation might have evolved as a means for human females to keep their partner close to them for protection; if ovulation were signaled, males would have interest in guarding their mate only during the fertile phase to prevent approaches by other males, but would go wandering for other females in the rest of time. A second benefit of hidden ovulation to females could be the possibility to escape guard by the partner and engage in extramarital sex during the fertile phase, thus increasing the genetic variability of their own offspring (Schoroder 1993; Diamond 1997). If so, the trick was not completely perfected, as there is evidence that the human female ovulatory cycle is not fully concealed; besides affecting internal physiology, ovulation may affect behaviour including preference for masculine features, choice of dress, sexual desire, and even women‘s facial attractiveness (Carmen et al. 2012). Human females show near-continuous receptivity, even when they do not experience ovarian cycles during pregnancy and lactation; in this respect, humans resemble bonobos rather than any other primate. The hypothesis that hidden (or unpredictable) ovulation and prolonged mating receptivity reflects some level of polyandry in the reproductive strategy of human females finds support in womens’s ability to reach an orgasm. Female orgasm, signaled by muscular contractions in the anogenital area, a strong increase in heart rate, body tension and rapid breathing, also occurs in other primate species, all showing female polyandrous mating. There is evidence suggesting that female orgasm is an adaptation favouring sperm retention and paternity of a preferred male partner (Van Schaik 2016). Several distinctive traits in human anatomy may function as sexual tools reinforcing pair bonding (Morris 1967; Diamond 1997). For example, the displacement of the vagina in an anterior position favours face-to-face sexual intercourse, thus intensifying the emotional involvement known as “love”. Other tools for reciprocal seduction are prominent lips and earlobes with high sensitivity to tactile stimuli, lacking in other primates. Both the breasts and penis are oversized in humans relative to the other apes, probably due to mutual sexual selection. Humans lack the baculum, the penis bone present in all other great apes. The function of the baculum is to produce an immediate erection; the human penis achieves erection more slowly, by filling with blood. Greater sizes afford the human penis a sufficient rigidity, compensating for the loss of the baculum and probably enhancing vaginal wall stimulation during mating. Among primates with promiscuous sexual lifestyles, males select mates based on features signaling proximity to ovulation. Human females lack these signals but instead display prominent breasts and buttocks. Breasts are generally absent in other primates, except in great apes, where they become prominent only during lactation. There is evidence that large breasts and buttocks in humans are perceived as indicators of long-term fertility, thus their evolution is probably associated with the establishment of lasting pair bonds and male investment in offspring (Van Schaik 2016).

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Reproductive Biology of Humans

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Human reproductive biology features a third major innovation besides social monogamy and biparental care. A human individual consumes an average of about 13 million kcal from birth to complete autonomy, a significant part of which is used for brain development and functioning. Not only is the human brain much larger than in other primates relatively to body size, but also is metabolically more active, accounting for about 2% of total body mass but 20% of the resting metabolic rate, with energy demand peaking to a maximum during childhood (FonsecaAzevedo and Herculano-Houzel 2012). The unusual length of human childhood and the need for prolonged care probably reflect a slowing down of body growth necessary to compensate the high cost of brain development (Kuzawa et al. 2014). Such a heavy demand might exceed the capability of even a well-integrated monogamous couple engaged in biparental care. Thanks to their highly developed sociality, humans evolved social rearing (also known as alloparenting or cooperative breeding), a novel, flexible child-rearing system that is absent in the other primates or is present in much simpler forms (Chapais 2013). Alloparents include grandmothers, sisters, cousins, older siblings, non-reproductive adults and pre-reproductive children. Human mothers are willing to let others hold their babies right from birth, a behaviour highly unusual in other mammals including apes. According to Blaffer Hrdy (2009), social rearing provided the evolutionary foundation for bigger brains, longer lifespans and language by making an extended childhood and a high caloric input possible in spite of harsh life conditions. A second fundamental outcome of social rearing was the development of empathy, namely the ability to perceive the perspectives of others and to interpret their motivation and intentions. Instead of relying on the single-minded dedication of their mothers, human youngs had to interact with multiple caretakers as well. Other apes possess a rudimentary cognitive wiring for “reading” mental states (theory of mind), but with social rearing this potential was more fully expressed in early humans and thus exposed to natural selection. Blaffer Hrdy suggests that the ability to “mind read” other people, to wonder and care about what other people think and to spontaneously share emotions and material objects with them was pivotal to all later cooperative human ventures, from big-game hunting to creating increasingly complex social systems. Bingham and Souza (2009) maintain that social rearing was essential for brain enlargement, but consider this trait of human biology as the output of a more fundamental innovation unique to humans, kinship-independent cooperation. We will return to this scenario in Sect. 12.12. An increase in brain mass is positively correlated with increased longevity in homoeothermic vertebrates. Increased longevity, however, comes with slower offspring maturation and longer inter-birth intervals; therefore, the brain mass negatively correlates with reproductive rate (Isler and Van Schaik 2009). Comparative data on extant mammals and computer simulations suggest that hominoids face a grey ceiling that limits their brain size, with larger values leading to demographic non-viability (Isler and Van Schaik 2012). This supports the hypothesis that alloparental care, alias social rearing, was essential for the evolution of a large brain in humans.

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Menopause, a prolonged post-reproductive phase following the cessation of ovulation is a trait almost unique to humans. In most wild animals, fertility decreases with aging but a prolonged post-reproductive phase is lacking. Menopause may have evolved in humans as a way for grandmothers to help their daughters in offspring breeding, especially in the first years after birth, thus enabling them to successfully rear more children. Evidence from modern hunter-gatherers points to the critical role of grandmothers in supplying food to just-weaned juveniles (Kim et al. 2012). In natural populations of some cetaceans and insects, reproductive senescence occurs much faster than somatic aging and females exhibit prolonged post-reproductive lifespans. As for humans, these instances are interpreted as forms of inter-generational cooperation (Croft et al. 2015). In addition, postmenopausal longevity was found to have the potential to determine an elongation of the average lifespan through transmission of longevity to both sexes (Kim et al. 2012). Male/female differences of cultural origin among humans are usually referred to as gender differences, in order to distinguish them from ancestral, genetically based sex differences.

12.9

The Evolution of Language

Major distinctive traits of humans, including a large brain, language, social skills, cooperation, cultural transmission and genetics, are entangled in such a complex network of mutual interactions that even defining the order of discussion is problematic. We start here with language, with the premise that some of the ideas presented will recur in the following sections, a degree of redundancy being unavoidable. Language is a system of communication that deploys a finite number of elements to express a potentially infinite amount of information. As such, language only exists in humans, with music as its closest possible homologue (Brandt et al. 2012). Language is the greatest of all social tools: it is what lets people share and cooperate, divide labor, make plans, transmit knowledge, tell stories. There are about 7000 extant languages, some with just a dozen sounds, others with more than a hundred, some with complex patterns of word formation, others with simple words only, some with the verb at the beginning of the sentence, some in the middle, and some at the end. Understanding this diversity and the underlying systematic constraints is the central goal of linguistics (Dunn et al. 2011). In contrast, animal communication is essentially a non-creative, stereotyped activity that fundamentally depends on genetic determinants. Although there is a degree of freedom that permits the appearance of local variants, animal ways of communicating are restricted to a limited, species-specific repertoire of informational units. A major property of human language (hereafter simply language) is its symbolic nature, namely the arbitrary association of sounds with meanings, and its ability to refer to things or states that are not in the immediate realm of the speaker and the recipient; these properties make the messages conveyed through language

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The Evolution of Language

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potentially unreliable because not immediately verifiable. Animal communication needs to be reliable because it has a cost to the sender and is immediately verifiable by the receiver (Zahavi 1993). For example, vervet monkeys emit a “leopard call”, a “snake call” or an “eagle call” when they spot one of these predators; these calls trigger defensive responses in the other monkeys, but are potentially dangerous to the caller by rising the attention of predators. The gene(s) responsible for this behaviour fixed in the population because the benefit from receiving the same alert by other monkeys was higher than risk (Dawkins 1976). This advantage would be lost if the calls were not reliable and selection would eliminate the gene(s) responsible for the behavioural trait. Because of its symbolic nature, language has no cost for the user. The propensity of language to be used for deceiving rather than informing the recipient is a major obstacle to attempts at explaining language evolution with a Darwinian mechanism, as unreliability would rapidly lead to the emergence of “defence” mechanisms preventing the spread of symbolic communication (Bingham and Souza 2009; Knight 2016). Language, therefore, might have emerged only within a kind of society where symbolic communication could be established and maintained through collective social endorsement. In other words, a high level of sociality and cooperation was a necessary premise for the evolution of language. This is at odd with the well-substantiated notion that language in turn was essential for the emergence of advanced sociality and cooperation (MacWhinney 2005; Pinker 2010; Leung et al. 2017). To solve this dilemma, Bingham and Souza (2009) assume that language evolved in parallel with kinship-independent cooperation as a means for members of cooperative groups to exchange increasing amounts of information, with coercive control of conflicts of interest ensuring reliability (Sect. 12.12). “Continuity theories” build on the idea that language exhibits so much complexity that it must have evolved from earlier pre-linguistic systems used by our ancestors (Cheney and Seyfarth 2005). Everett (2017) argues that humans were already using a means of communication recognizable as language in the times of Homo erectus, thus pushing back the starting date for the emergence of language to about 2 MYA. In contrast, “discontinuity theories” maintain that language, because of its unique properties, must have appeared suddenly when cognitive ability was sufficiently advanced, with no link with former systems of communication (Bolhuis et al. 2014; Berwick and Chomsky 2016). Berwick and Chomsky (2016) argue that language emerged approximately between 200 and 60 KYA, viz. the time lapse between the appearance of the first modern humans in Eastern Africa and the onset of spread to the rest of the world. It is debated whether language is an innate genetically encoded faculty, or a communication system learned through social interaction, alias a product of cultural inheritance. The truth is probably in the middle. Anatomical constraints place an upper limit on the number and variety of sounds that non-human primates (and other mammals) can produce (Cheney and Seyfarth 2005). The human ability to modulate sounds when speaking depends on a combination of anatomical traits not present in other apes. These include a supralaryngeal vocal tract with a 1:1 oral-to-pharyngeal proportion, the shape and placement of the tongue, a relatively low position of the

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larynx, neck elongation, and increased innervation of intercostal muscles controlling pulmonic pulsing (MacWhinney 2005; Liebermann 2007). Laryngeal descent during development in humans is accompanied by the descent of the hyoid bone, which disrupts the normal separation of the respiratory and digestive tracts during swallowing, thus increasing the risk of choking. These anatomical traits reflect selection pressure for improved sound modulation and therefore must have been closely associated with language evolution. In line with the discontinuity model, Liebermann (2007) reports that fully modern speech anatomy is lacking in H. neanderthalensis and even in archaic forms of H. sapiens, appearing in the fossil record only after 50 KYA. Whether or not Neanderthals could speak, however, remains a contentious issue. A recent study demonstrates that the hyoid bone from two Neanderthal individuals displays very similar internal architecture and micro-biomechanical properties as in modern humans; because these traits reflect the loadings to which a bone has been subjected during life, the finding suggests routine use of speech in Neanderthals (D’Anastasio et al. 2013). This conclusion is consistent with the presence of the modern human variant of the FOXP2 gene in the Neanderthal genome (Sect. 12.14). Based on a broad range of evidence, Dediu and Levinson (2013) argue that language is an ancient feature of our genus, most likely already present in the concestor of modern humans and Neanderthals about half a million years ago. When people using different native languages and no knowledge of each other’s language routinely interact because of migration or commercial relationships, they tend to develop simple forms of language known as pidgins, consisting of elements from both original languages and of new elements added along the way. Pidgins consist of nouns, verbs and adjectives, with few or no articles, prepositions, conjunctions or auxiliary verbs. If the children of a pidgin-using generation adopt pidgin as their native language, this develops into a creole language, which becomes fixed and with time acquires more complex grammar, syntax and morphology. Creole languages independently evolved in different areas of the world display remarkable similarities in grammar and syntax, suggesting the existence of universal language rules deeply wired in the human brain. The analysis of phonemic diversity in modern languages has produced important novel insight on the history of language. Phonemes are sound units that distinguish the words from each other in a particular language. A survey of over 500 languages throughout the world has shown that African languages have the largest number of phonemes, and that the number of phonemes in languages declines with distance from Africa, being the lowest in Oceania and South America (Atkinson 2011). This finding points to parallel mechanisms shaping genetic and linguistic diversity and supports an African origin of modern human languages. Perreault and Mathew (2012) calibrated the rate at which phonemic diversity increases with time by comparing phonemic diversity of languages of Mainland Southeast Asia and Andaman Islands, known to have diverged between 65 and 45 KYA. The value obtained was used for estimating how long African languages must have been in existence to reach their current phonemic diversity. The results

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suggest that modern languages diverged from an ancestral language starting from 350 to 150 KYA, which is significantly earlier that the 200–60 KYA interval proposed by Berwick and Chomsky (2016) but still probably restricted to the Sapiens lineage.

12.10

The Cognitive Revolution: Interplay of Genetics and Culture

As a species we are endowed with Paleolithic emotions, medieval institutions, and neargodlike technological capacity (Edward O Wilson 2012)

The Oldowan technology made its appearance around 2.6 MYA, and since remained virtually unchanged for one million years (Stout et al. 2010). About 1.7 MYA, H. erectus developed the Acheulean technology by discovering how to flake flat pebbles on both sides. This second level made only limited progress in the following 1.3 million years, to be replaced about 200 KYA by the Mousterian technology, attributed to Neanderthal populations in Europe and Asia and to both Neanderthals and Sapiens in North Africa and Middle East. The Mousterian technique, roughly covering the interval between 200 and 30 KYA, is a step forward from Lower Paleolithic Acheulean hand-held axes to hafted tools, i.e. stone points or blades mounted on wooden shafts. Each time, the transition from a more primitive to a more advanced technology entailed a phase of rapid increase in the diversity and complexity of the artefacts, followed by a long stasis (Fig. 12.18). The production of tools enabled humans to improve their diet by increasing caloric intake and protein intake in the form of meat, an achievement probably essential for sustained evolution of a large brain. Yet, technological progress proceeded at an extremely slow pace until about 50 KYA, when almost suddenly Sapiens started producing innovative and complex artefacts. Between 50 and 30 KYA, Sapiens invented boats and navigation, the bow and arrows, oil lamps, needles and musical instruments, and produced the first objects of art and symbolic representations in the history of humanity. In the same time interval, Sapiens expanded from Africa to the Far East and Europe, rapidly replacing earlier populations, and to Australia, America and Polynesia, still unknown to humans. Strikingly, none of now-extinct hominins that have coexisted with Sapiens, including Neanderthals, appears to have produced symbolic artefacts, despite Neanderthals having a larger cranial capacity than Sapiens. These unprecedented accomplishments were not associated with changes in brain size or anatomy, modern human anatomy appearing in the record significantly earlier than any evidence of symbolic behaviours (Tattersall 2009). It is widely maintained, therefore, that the transition was the product of a revolution in Sapiens’ cognitive abilities, a new way of thinking and communicating (Fisher and Ridley 2013). Based on the assumption that biological changes must precede cultural changes, it has been

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Fig. 12.18 Technological advance in human history encompasses four major transitions, separated by long periods of stasis. The last episode, initiated about 50 KYA, is still expanding at an explosive rate

suggested that the original cause of this cognitive revolution was a genetic change affecting brain wiring and physiology (Vallender et al. 2008). A number of mutations have been identified in the modern human genome which could underpin the evolution of larger brain size and more complex behavioural traits (Somel et al. 2011, see also Sect. 12.14). In most cases, however, these mutations are shared with Neanderthals, thus they predated the cognitive revolution. The Simons Genome Diversity Project found no evidence for mutations associated with rapid developments in human behaviour in the last 50 KY, thus supporting the view that the cognitive revolution was due to cultural innovation rather than genetic change (Mallik et al. 2016). Compelling evidence from molecular research, in fact, points to cultural innovation as a powerful driver of genetic change (Fisher and Ridley 2013). Particularly provocative is the idea that cultural innovation has driven major changes in anatomical and behavioural traits that reproduce in part the changes induced in animals by domestication (Gibbons 2014). Genetic change stemming from cultural innovation, however, reflect selection of allelic forms already present in the original populations (a process known as polymorphism selection) rather than the appearance of novel genes. This drives us back to the question why a “cognitive revolution” occurred in Sapiens around 50 KYA and not in Neanderthals or before.

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Sterenly (2011) proposes that humans became behaviourally modern when they could reliably transmit accumulated “informational capital” to the next generation through the construction of “learning environments”. Although born with a much bigger brain, children do not perform better than chimpanzees when tested on spatial reasoning, the ability to discriminate quantities or the understanding of cause-andeffect relationships. In contrast, children outperform chimps in the ability to communicate, learn from others, and evaluate another being’s perceptions and wishes (Herrmann et al. 2007). This suggests that human children are not born with better cognitive skills for dealing with the physical world but rather are endowed with a special set of abilities, called “cultural intelligence,” that prepares them for learning from parents, teachers and playmates. Superior cognitive skills in humans largely develop during the learning process underlying intergenerational transmission of knowledge within social groups. In this perspective, the phenomenon we call “cognitive revolution” may be viewed as the “obligate” outcome of knowledge accumulation and social evolution. If correct, this hypothesis implies that a child of Sapiens from a hundred thousand years ago, or perhaps even a Neanderthal, would develop “normal” cognitive abilities if raised in a modern society. Anthropologists traditionally focus on language as an instrument necessary to codify and transmit complex information. An obvious interdependency connects language and expertise. Language permits the transmission of knowledge and skills to other individuals, who therefore do not need to recapitulate the trial-and-error process, lucky accidents, or strokes of genius underlying technological progress (Pinker 2010). An additional function of language is in exchanging social information through “gossiping”, an activity considered essential for the establishment of functional groups. By using language to establish social relationships, in a similar way as other primates use grooming, modern humans became able to establish larger and more stable bands than those formed by their ape relatives and probably by earlier hominins (Dunbar 1998). Psychological research suggests that there is a maximum threshold of about 150 individuals for human groups based on direct personal acquaintance; this is currently known as the Dunbar number, from the British anthropologist Robin Dunbar (Hernando et al. 2009). Within this size limit, human groups do not need hierarchies or formal rules to remain stable. Personal acquaintance is not effective for the establishment of larger groups, which therefore requires other means of aggregation. Yuval Noah Harari (2015) conjectures that, besides language, a second fundamental change underpinned the cognitive revolution: Sapiens became able to codify and describe not only the real world but also imaginary things. This led to the birth of myths, inventions of the mind transmissible from one individual to another and from one generation to the next through language. Harari suggests that shared myths are the psychological key enabling humans to interact and cooperate not only with relatives, friends or acquaintances but also with perfect strangers. Indeed, shared myths influence our lives, our way of thinking and acting, in such a pervasive way that we are usually unaware of their symbolic and imaginary nature. Shared myths include “moral rules” and “justice”, religion in all its forms, patriotism, honour, social behaviour and, going down the staircase of values, wealth, career, fashion,

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movie and music stars, football, stamp collecting and so on. As Harari puts it, “you could never convince a monkey to give you a banana by promising it limitless bananas after death”. In contrast, it is possible to persuade a male human to blow himself in a suicide bombing by promising him paradise with exclusive use of scores of perpetual virgins, or a female human by explaining her that suicide bombing is the best way for her to acquire a value. Fiction permits humans not to merely imagine things individually, but to do so collectively. Perfect strangers can successfully cooperate by believing in common myths. Research in comparative primatology, sociobiology, neuroendocrinology, human behavioural ecology and cognitive psychology points to the ability to participate with others in collaborative activities with shared goals and intentions as the most distinctive behavioural trait of humans. Harari argues that shared myths are the cement that aggregates humans into peoples, enabling them to work together for common goals that transcend individual capabilities. We will return on this point in the next section. Transgenerational cultural transmission is a form of epigenetic inheritance that, although present in non-human animals, only in humans has become a major player in evolution (Rendell et al. 2011). According to Harari’s analysis, myths not only paved the way to the development of complex societies, but also provided a form of inheritance and change completely independent of genetics and much faster than biological evolution. Before the cognitive revolution, behavioural and technological innovation was essentially dependent on random genetic change (mutations) and natural selection of genetic variants. This is possibly the reason why it took a million years to progress from Oldowan to Acheulean and one and half million years from Acheulean to Mousterian. With the cognitive revolution, humans acquired the ability to consciously modify their behaviour at an extremely fast pace, and transmit novel models to next generations. Myths make a substantial contribution to building and maintaining a collective identity, yet the emergence of novel myths has the potential to drive extremely fast and radical changes in human society. History provides countless examples of sudden ideological transitions, from the French and Russian revolutions to the fall of communism, the abolition of slavery and racial segregation and changes in the treatment of homosexuality, each leading to radical changes in social structure and social rules. As Harari observes, “the immense diversity of imagined realities that Sapiens invented, and the resulting diversity of behaviour patterns, are the main components of what we call cultures. Once cultures appeared, they never ceased to change and develop, and these unstoppable alterations are what we call history”. The concept of myths by Harari appears to be a variant of Richard Dawkins’ “memes” (Dawkins 1976). The term “meme” is a neologism from the Greek word “mimeme”, imitation. A meme is an idea that is born in the mind of an individual and can replicate by spreading to other individuals’ minds through communication or imitation. By modifying the mind where they settle in, memes can change the behaviour and therefore may affect the fitness of the host. Likewise, memes shared by the members of a group - not necessarily myths but also notions of practical nature - may influence the behaviour and the fitness of the group. While spreading from mind to mind, memes can mutate; like genes, therefore, memes may compete

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with related variants, thus becoming prone to Darwinian evolution: successful memes remain and spread in the meme pool, whereas unfit ones stall and go into oblivion. Memes, moreover, tend to aggregate in combinations that are transmitted en-bloc; examples of “multimemetic complexes” are political doctrines, religions or economic systems. Like genes, memes can be transmitted vertically from parents to offspring, or horizontally between non-kin individuals (Blackmore 1998). Unlike genes, no precise definition of memetic unit has been agreed. Moreover, genes are digital strings of information, easy to replicate with a high level of fidelity, whereas memes have analogic structure and are copied as a whole, then tested and “amended” according to need, thus they may evolve with a Lamarckian besides Darwinian mechanism (Blackmore 1998). The notion of meme has been viewed by several researches as a useful philosophical perspective within which to examine human cultural evolution, yet it has received severe criticism by others (Mayr 1997; Benitez-Bribiesca 2001; Fracchia and Lewontin 2005).

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Cooperation and Culture

The worst in our nature coexists with the best, and so it will ever be. To scrub it out if such were possible, would make us less than human (Edward O. Wilson 2012)

Cooperation is the process by which organisms of the same species (conspecifics) operate together for mutual benefit. Sharing the same ecological niche, conspecifics compete for resources; thus, in the absence of other forces, natural selection favours non-cooperative, selfish behaviours (defection). Cooperation implies costs, the first of which lies in affording a benefit to a potential competitor. Natural selection weeds out predispositions toward providing help that end up in systematically reducing an individual’s inclusive fitness. Cooperative (prosocial) behaviours can emerge only when the ensuing benefits to either side outweigh the conflict of interest (see Hamilton’s equation Sect. 8.1). Barring instances in which a benefit is immediately available to both sides, thus virtually eliminating the risk of defection, cooperation in non-human animals is most often linked to genetic relatedness. This ensures that advantages afforded to the recipient improve the chances of survival of the gene pool shared with the actor, a mechanism known as kin selection and based on kin recognition (West et al. 2011; Dawkins 2012; Rand and Nowak 2013). Mechanisms for assessing genetic relatedness occur in many species, and there is evidence for unconscious kin detection in humans as well (Lieberman et al. 2007). An extreme example of kin selection is complex sociality (eusociality) in termites, ants and bees (Crozier 2008). Kinship-independent cooperation, also referred to as reciprocal cooperation or reciprocity, is rare and extremely limited among non-human animals, with DNAbased evidence available only for chimpanzees (de Waal and Suchak 2010).

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The ability to cooperate with conspecifics on a large scale, independently of genetic relatedness and without any immediate benefit for single individuals, is a unique trait of humans (Fehr and Fishbacher 2003). Modern humans are able to form large groups of genealogically distant individuals, whose existence depends on cooperative enforcement of rules preventing defection. Data from extant forager communities (the closest proxy of ancient human societies) show that cooperation among foragers is much stronger than in any other non-eusocial species, being based on a network of obligate sharing relations involving non-kin bonds. As such, human cooperation is sharply distinct from individual, empathy-based instances of reciprocal cooperation observed among primates and other mammals (de Waal and Suchak 2010; Van Schaik 2016). How humans developed social cooperation not linked to kin selection is one of the most hotly debated issues in modern anthropology. Altruistic behaviours towards non-kin individuals have an immediate cost to the actor without enhancing longterm fitness. Theoretical studies provide an evolutionary explanation for cooperative behaviour by showing that punishment reduces gain to free riding, so groups with more punishers can sustain more cooperation. On the other hand, in experimental models in which punishment is uncoordinated, the sum of costs to punishers and their targets often exceeds the benefits of increased cooperation resulting from the punishment of defectors (Boyd et al. 2010). Thus, in the absence of other players, human cooperation could hardly have emerged. The most popular hypothesis for the origin of reciprocal cooperation among early humans is the cultural group selection model (Fehr and Fishbacher 2003; Richerson & Boyd 2005), which maintains that social groups emerged from the acceptance and transmission of normative rules. Simply put, groups with more successful social norms outcompeted groups with less successful social norms. Norms enforcing group-level cooperation and group coordination were most successful, thus fixing these traits in the cultural/genetic network. An alternative paradigm of human evolution recognizes bipedality as the ultimate cause of reciprocal cooperation (Bingham and Souza 2009). The narrative of this scenario starts from considering that, by liberating the forelimbs from locomotion constraints, bipedality permitted the evolution of high-speed throwing (Sect. 12.6). While the ability to throw objects is not unique to humans, this adaptive trait is so highly skilled in humans as to be comparable to a cheetah‘s running or dauphin’s swimming ability. In the same way that the anatomy of these animals reflects their extreme ecological specialization, the anatomy of the human torso and arms, as well as the complex neural circuitry necessary for calculation of weight, speed and distance and for movement control, were built by selection for stronger and more precise throwing. Bingham and Souza argue that enhanced throwing skill created a novel ecological niche in which there was a high return for cooperation. The core of their reasoning is that the ability to throw projectiles not only afforded early hominins a potentially lethal weapon, but also enabled them to kill at a substantial distance, avoiding direct physical confrontation. Relatively weak individuals thus became potentially dangerous adversaries, especially when operating collectively. An early consequence of this was replacement of the ancestral hierarchical

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organization based on alpha-male supremacy with a lasting egalitarian organization, or “social dominance hierarchy” based on cost-effective coercive enforcement of cooperation (Bingham and Souza 2009; Shultz et al. 2011; Gintis et al. 2015). In eusocial insects, only a few or even a single member of the group (the queen, in the case of eusocial hymenoptera) reproduces. Because of this, Darwinian selection operates only on reproductive members, the rest of the colony being just an “extended phenotype” of these individuals (Wilson 2012). In contrast, all individuals retain at least the ability to reproduce in human societies and, if the success of the group depends on how well its members work together, the fitness of single individuals largely depends on the success of the group. From this, Wilson (2012) argues that human cooperation emerged from “multilevel selection” targeting traits of individual members and traits of the group as a whole. Dawkins (2012) harshly questions the idea that differential group survival drives genetic evolution as differential individual survival does. He points out that the only unit of Darwinian selection are the genes, because neither individuals nor groups make copies of themselves as genes routinely do. The core of the issue is probably that the evolution of human sociality simultaneously involved (and still does) genetic inheritance and cultural transmission, the first subject to Darwinian selection, the second to both Darwinian and Lamarckian selection (Sect. 12.10), in a network of mutual interactions still largely unexplored. Whatever its origin, kinship-independent cooperation greatly enhanced the fitness of individuals living in social groups, thus fixing in the population and since dictating the direction of human evolution. In this perspective, other unique human traits such as language, social breeding, increased brain size, cognitive and social skills and complex social organization are regarded as evolutionary aftermaths of cooperation rather than causative agents (Bingham and Souza 2009; Gintis 2013; Gintis et al. 2015). Collective defense against predators and confrontational scavenging (stealing the prey to large predators) were probably the immediate outcomes, followed by cooperative hunting. An improvement in the diet, mainly due to enhanced access to meat, was the premise for increased brain size under selection pressure for better social skill. Collective breeding further improved the diet of the young and paved the way to systematic education, i.e. trans-generational transmission of knowledge and behavioural rules. Life within stable cooperative groups removed the obstacles to the evolution of language stemming from potential for hostile manipulation (Sect. 12.8), as individuals reporting false information for selfish purposes faced social ostracism or even death. Thus, language immediately emerged in cooperative social groups under selection pressure for enhanced communication. Bingham and Souza (2009) consider information encryption a second major function of language besides communication; they suggest that sound modulation (speech) was preferred to the use of signs as the main instrument of language at least in part because sound facilitated encryption, helping to prevent the spread of useful information to competing groups. Language led to the emergence of culture, a body of skills, knowledge and normative beliefs socially transmitted across generations (Gintis 2011). Because of dependence on language, culture is traditionally viewed as the “major dividing line”

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between human and non-human animals. Key elements of culture, namely innovation and social transmission, are present in non-human animals, especially primates. Culturally transmitted knowledge in non-human animals includes feeding and hunting techniques, ways of making nests and even the use of medicinal plants; a wellknown example is nut cracking by stone tools in certain populations of capuchin monkeys and chimpanzees, reported 500 years ago and still present now, the latter also documented by archeological record dated to 4000 years ago. Human culture, however, displays three unique properties that set it apart from non-human cultures. The first is cumulative cultural evolution, namely knowledge accumulation paving the way to the development of skills and artefacts that are beyond the inventive ability and cognitive range of single individuals; cultural innovations in animals are always within the cognitive reach of individuals. The second difference is in the extensive use of symbolism by human culture (alias Harari’s myths), usually with normative functions. The third unique feature of human culture is a process dubbed donated culture: the transmission of ready-made products, either artefacts or patterns of organization, which permits beginners to bypass the long sequence of steps necessary for their development from zero (Van Schaik 2016). The unique properties of human culture triggered the process known as cultural niche construction: the creation of a novel, continuously expanding environment, partly physical and partly cognitive and social (Laland and Brown 2006; Gintis 2011; Sterenly 2011; Rendell et al. 2011; O’Brien and Laland 2012). Within this novel environment, reciprocal cooperation was enforced by punishment of defectors and “moral rules”. Human-behaviour modelling suggests that the total cost of punishing a free-rider declines as the number of punishers in the social group increases, the average benefits exceeding the cost when the number of punishers exceeds a threshold (Boyd et al. 2010). Cooperation-enforcing moral rules include a strong sense of fairness in resource allocation (and thus cheater detection), a concern for reputation and proneness to conform to the majority (Van Schaik 2016). The internalization of “moral” norms presupposes a genetic predisposition, i.e. a deeply wired “moral code” that was probably positively selected by cultural niche construction through a process similar to domestication (Gintis 2011; Gibbons 2014). The default genetic predisposition to selfish behaviours was never lost, remaining an adaptive option under local conditions (Wilson 2012). Faults in the hard-wired moral code or in the educational process may result in anti-social behaviour (sociopathy). Primary achievements of cultural niche construction were tools for food preparation, enhanced hunting techniques, the control of fire. Fire became embedded in human behaviour very early and was involved in almost all technological advances (Gowlett 2016). Notably, the use of fire for cooking meat, tubers and seeds strongly improved their digestibility, thus increasing the amount of energy extractable from food; this favoured brain size increase and affected other traits as well, for example inducing a reduction in the masticatory and digestive apparatus (Wrangham and Carmody 2010). Structural analysis of fossil teeth permits an estimation of the age at the time of death. The data indicate that the frequency of individuals of at least twice the age of reproductive maturation, i.e. the age at which they could first become grandparents,

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increased at a slow pace during hominin history from Australopithecines to modern humans, but suddenly shifted to much higher values in Cro-magnon populations of the Upper Paleolithic, about 30 to 20 KYA. This change in longevity shows no correlation with anatomical changes, because anatomically modern populations of H. sapiens living in the Middle East around 100 KYA had relatively low rates of longevity. Thus, the observed demographic shift probably had a cultural origin from enhanced protection and integration within socially structured groups (Caspari and Lee 2004, 2006). In conclusion, human history appears to encompass two distinct phases: an early phase in which genetic change was virtually the only driver of innovation, and a later phase in which cultural change became predominant. The genetic phase covered the early evolution of Hominins after divergence from Panins, its major outcomes being bipedalism, more efficient thermoregulation and high-speed throwing. Following these fundamental innovations, reciprocal cooperation and cultural niche construction became the main drivers of human evolution, producing fundamental human traits such as hyper-cognition, language and technological innovation, which deeply affected the trajectory of human evolution but apparently involved relatively minor changes in genetics (Sect. 12.14).

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The Transition to Food Production

During most of their history, humans lived as foragers (or hunther-gatherers), feeding on wild plants and animals. This holds true not only for early Homo species but also for Sapiens, which lived exclusively as a nomadic forager until 30–20 KYA, shifting in part to a sedentary foraging lifestyle after this date. Agriculture, namely food production from domesticated plants and animals, first appeared around 11 KYA, marking the end of Pleistocene and the start of Holocene (conventionally placed at 11,650 years ago). The two styles of life are profoundly different. Living as foragers requires a large territory; farming permits humans to live on a smaller territory through intensification, an increase in the amount of food produced per land unit surface. Population densities of foragers are rarely over one individual per ten square miles, while farmers may be over 100 times as many. In both cases, survival is at stake during cyclic or occasional unfavourable phases, which foragers can overcome more easily than farmers by migration. The transition to agriculture proceeded across intermediate stages, each probably reversible. There is evidence of the use of grain seeds as food from at least 20 KYA. Either deliberately or unknowingly, foragers may have spread seeds or rhizomes, favouring the diffusion of plants used as a source of food. Agriculture as the main way of life emerged independently in several areas of the world. The first documented appearance of agriculture roughly coincided with the end of the last glacial phase and the beginning of an interglacial phase with warmer and drier weather favourable to annual plants (Richerson et al. 2001; Bettinger et al. 2009). There have been, however, analogous warm phases in the course of Pleistocene

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(starting 2.58 MYA) which did not elicit a stable transition to farming. The fact that about 20% of the world population persisted as foragers until nineteenth-century European expansion suggests that, even under permissive conditions, the transition to agriculture is not a likely event (Bettinger et al. 2009). Price and Bar-Yosef (2011) argue that the transition necessitates a third condition besides technology and a favourable territory: an increase in human population density. Foraging populations fluctuate in density in accordance with climatic conditions. An increase of population density during favourable climatic phases may dictate partial reliance on farming if less favourable conditions follow. For a while, the balance between the two strategies may move back and forward, permitting populations to adapt to environmental fluctuation. If dependence on farming lasts too long, however, population density may rise to a level no longer supported by foraging as the main way of life. The return to foraging, therefore, would entail the death of a part of the population by starvation or tribal wars; alternatively, the population might be forced to intensify food production, thus becoming irreversibly bound to farming as the main livelihood. This is probably what happened in several areas of the world, during the chronological window between 11,500 and 3500 years ago. Essential for a stable transition to farming are plant and animal species suitable to domestication, as well as favourable environmental conditions, notably fertile soil and access to water. It is not surprising, therefore, that farming first appeared in few scattered areas and spread from there to the rest of the world (Fig. 12.19). The most important cradle of agriculture was the Fertile Crescent, a vast area covering a part of the territory of modern Egypt, Israel, Syria, Iraq, Iran, Turkey, and subject to inundations from Nile, Tigris and Euphrates rivers. Quite a few of the most important plants and animals employed in agriculture today come from this area. The Fertile Crescent is the homeland of wheat, barley, peas, lens, and flax, as well as of pigs, sheep and goats among domesticated animals (Lev-Yadun et al. 2000). Cattle derived from wild aurochs in Pakistan and Turkey, the donkey in NorthEastern Africa, and the horse in Caucasus. Rice and soy come from China, chicken and aubergine from India, sugarcane from New Guinea, sorghum from the Sahel area of Africa; the squash, sunflower and tobacco plants from North America, corn from Central America, potatoes, tomatoes, beans and peppers from South America (Kris 2018). The only animal domesticated in the pre-farming world is the dog, derived from a wolf lineage at the end of Pleistocene and initially employed for guarding the camps at night. From the beginning, the adoption of agriculture as the main way of life involved a reciprocal evolutionary pressure between humans and domesticated organisms. When collecting wheat or barley ears, for example, early farmers selected plants with an axis (or “rachis”) less prone to breakage under the effect of its own weight or of the wind, thus favouring the diffusion of “dwarf” varieties (Fuller et al. 2014). Increased dependence on certain plants such as wheat or rice as the main source of food in turn exerted a strong evolutionary pressure on humans, who physiologically adapted to the novel diet. In addition, by providing food, plants selected the human groups that more efficiently ensured their diffusion. Thus, if humans domesticated

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Fig. 12.19 World areas where agriculture appeared independently. As Jaris Diamond (1991) observed, the spread of domesticated plants is relatively easy along the same latitude band, for example from China to Europe and vice versa, but much more difficult between areas at different latitudes. This prevented diffusion of important crops, such as wheat and rice, from temperate to tropical or equatorial areas, with major economic and social consequences

plants and animals, the opposite is also true for plant species on which human populations substantially depended for sustainment; examples include wheat, rice, corn, potatoes, millet. The shift from foraging to food-producing societies, known as the Neolithic or Agricultural Revolution, is signaled by a sudden worldwide increase in the proportion of juvenile skeletons in archaeological sequences, reflecting an estimated increase in average fertility rate of two more births per woman (Bocquet-Appel 2011). The increase in population size linked to the establishment of agriculture accelerated human evolution and created novel niches, both biological and cultural (O’Brien and Laland 2012). For example, agriculture substantially modified selection pressure on alleles expressed in the human ability to process novel diets or to face attack by pathogens. Foragers typically eat fiber-rich plant material such as seeds, roots and rhizomes, whereas farmers prefer soft food such as porridge, legumes and vegetables. This change in the diet elicited a reduction of the masticatory apparatus that added to the reduction due to food cooking (von CramonTaubadela 2017). The distribution of lactose tolerance is a further remarkable instance of adaptation to farming. Lactose, a disaccharide present in milk, is not absorbed as such but needs hydrolysis into glucose and galactose by lactase, an enzyme produced by the intestine. The young of mammals including humans express the gene for lactase during lactation but stop with weaning. Five mutations are known in humans, which keep the lactase gene active in adults. These were positively selected in populations in which milk from domesticated ungulates and

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dairy products are an important part of the diet in adults. Three of these mutations occur in Africa, where cattle rearing is much diffuse; one is in the Arabian peninsula, where it associates with the use of camel milk; the fifth is distributed in a large area extending from Europe to India, with maximum peaks of frequency (above 80%) in North Europe (Great Britain, Ireland, Germany, Sweden) and Pakistan (Gerbault et al. 2011). A long-ranging effect of agriculture was food surplus utilizable for exchanging, which fostered the emergence of secondary activities and services and increased social structuring. One might think that farming was so advantageous as to make the abandonment of the foraging lifestyle unavoidable when culture accumulation had put in place the necessary technological background. This view is today largely dismissed. The abundance of food in the modern world, or rather in the richer part of it, might present agriculture as a blessing. Actually, compared with foraging, farming entailed a worsening of average life conditions for the greatest part of human history, the only exception being perhaps the last 100 years, and only in a part of the world. In fact, the diet turned less varied, nutritional deficiencies became commonplace, and dependence on a few crop species increased the frequency and severity of famine. The rise of dense and sedentary populations, extensive ecological disruption, and high contact rates with domesticated animals and wild pest species such as rodents provided new opportunities for pathogens to cross species boundaries, leading to the appearance of novel, highly virulent “crowd” infections such as measles, smallpox and tuberculosis (Chisholm et al. 2016). Not the least, agriculture needs extremely hard work that has undermined the health of workers for thousand years. Foragers employ less than half the average time required in traditional farming (i.e. without the use of machines and chemistry) for the same energy input, and may use the rest of the time for social interaction, leisure or rest. Comparison of Neolithic skeletons shows that foragers were taller, lived longer and were healthier than farmers (Diamond 1991). The view that farming requires a wider expertise than foraging is incorrect as well. Present-living communities of foragers routinely use and transmit a huge amount of knowledge about wild plant properties and distribution, food collection and preparation, hunting techniques, disease and wound treatment and much else. Individual property most likely predated agriculture, becoming a landmark of human societies with the transition from nomadic to sedentary foraging. The transition to food production most likely entailed the emergence of a new system of property rights including land owning and long-term food storage (Bowles and Choi 2013). The possession of land and its use for food production enhanced social stratification to a level unknown in foraging communities, rapidly leading to the separation of privileged elites and masses of population living in conditions of mere subsistence, an outcome regularly recurred worldwide since the beginning of the “Agricultural Revolution”. In striking contrast to foraging societies, which are inherently stable, the history of the last 11 thousand years does not suggest that agriculture is an effective strategy for achieving demographic and political stability in the world's farming population (Rowley-Conwy and Layton 2011).

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Fig. 12.20 Conditions fostering the shift from foraging to farming and major consequences of the emergence of agriculture in Neolithic societies

Neil Diamond defined agriculture “the worst mistake in the history of the human race”. In his words, “having to choose between being a peasant farmer in Ethiopia or a bushman gatherer in the Kalahari, which would be the wiser choice?” That said, one could not forget the achievements made possible by agriculture. Surplus production indirectly led to inequalities, violence and injustice, but also produced the resources necessary for a systematic pursuit of knowledge. A world in which foraging had remained the only style of life would never have produced Saint Peter’s cathedral or the Relativity Theory. Figure 12.20 summarizes main benefits and disadvantages of the emergence of agriculture in Neolithic societies.

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The Evolution of Social Organization

Our deeply rooted preference for an egalitarian decision-making process may be a case of mismatch, reflecting an adaptation to a now largely defunct lifestyle. . . .. Deep down, we may still be foragers (Carel P. Van Schaik 2016)

There are currently two main scenarios for the early evolution of human social organization: the multi-level society (MLS) and the large fission-fusion society (LFFS).

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The MLS model, favoured by Chapais (2013), assumes that ancestral human communities were aggregations of social units, each made of a single adult male and one, more commonly several, adult females and their offspring (stage 1). In this scenario, a second layer developed by aggregation of groups that shared foraging space and sleeping sites (stage 2). The emergence of monogamy converted these associations into multi-family aggregations (stage 3) which then evolved into more cohesive communities, or tribes, with the establishment of kin-like bonds by interfamily marriage (stage 4). An increase in the capacity to recognize membership and adjust individual behaviour for maximal benefit from social interaction led to the emergence of federations of multifamily groups (stage 5), which remained the highest level of social organization until the establishment of food production. The MLS model implies that hominins ancestrally had a polygynic gorilla-like sexuality, assumes an early evolution of monogamy and infers a late division of labor between males and females. The LFFS model (Van Schaik 2016) assumes that early humans lived in large promiscuous (polygynandrous) groups subject to fission and fusion depending on contingent conditions as in extant chimpanzee populations. In this scenario, the first step towards closer social integration was male bonding for communal defense from predators in the savanna environment; this paved the way to cooperative hunting, both activities stemming from the ability to throw stones and use sticks or clubs as weapons. Concurrent development of pair bonds and family bonds led to the establishment of collective breeding, with adult males providing meat and protection, and females gathering vegetable food and caring the youth. The LFFS model explains why humans, despite being pair bonded, retain a sexuality characteristic of polygynandrous species; in addition, by assuming an early gender-linked division of labor, the LFFS model explains why humans are still sexually dimorphic. In the LFFS scenario, higher levels of social organization could arise by splitting of larger communities into smaller communities that expanded the territorial range whilst remaining connected through exchange of goods and mates, thus retaining a common language. Humans have lived as nomadic foragers for the great majority of their evolutionary history. This lifestyle is dramatically different from that of our common ancestor with chimpanzees and critically relies on cooperation. Foraging groups probably were egalitarian as long as they remained nomadic, each member having fair access to the resources available. Decision-making was essentially based on collective discussion, leadership depending on the capacity to motivate, persuade and gain consensus rather than physical strength. The most important individual resource in nomadic foraging societies was personal reputation, i.e. the reliability perceived by other members of the group in coordinated social actions (Van Schaik 2016). This still holds true now: in all occasions in which the anonymity protection afforded to individuals in large modern societies ceases to operate, humans are extremely attentive to their reputation from a very young age. Social structure became more stratified and less egalitarian when foraging societies settled down, permitting storage of food and other resources and creation of

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substantial personal property. Foragers that have become more sedentary, because they live in resource-rich territories, are called collectors or complex foragers. The establishment of agriculture was pivotal to the establishment of complex forms of social organization that culminated in the emergence of modern states encompassing hundreds of thousands to millions of individuals (Table 12.1). The general preference humans express for democracy as the best system for making collective decisions is probably a legacy of social egalitarianism practiced as nomadic foragers for nearly two million years of our history (Van Schaik 2016). Nonetheless, the emergence of elites and the loss of democracy appear to be an obligate step in the evolution of human societies. The unavoidable tendency of wealth to distribute unevenly is the prime cause for the emergence of dominating elites. This notion conveys a strong feeling of “unrightness”. Coercive power, however, is essential for enforcing cooperation in groups too large for individual members to know each other personally. Large groups stabilized by coercive power tend to outcompete smaller groups, the latter persisting only in remote areas protected by geographic barriers. Bingham and Souza (2009) argue that the ultimate driver dictating the scale and political organization of human societies at any time is the type of weapons available. In their scenario, weapon technology determines the range of projected coercive threat and this in turn controls the sizes of societies, the greater the range, the larger the social group. Social scale, in turn, dictates the level of complexity of cultural niche construction including technological advance, thus establishing a positive feedback on weapon technology. In line with their model, Bingham and Souza view the cognitive revolution recorded around 50 KYA (Sect. 11.10) as the effect of a social transition triggered by the invention of the long-range spearthrower or atlatl (Shea and Sisk 2010). This device amplifies the normal human throwing motion, permitting a bolt or dart to be lofted to a distance of over a hundred metres (Fig. 12.21). Bingam and Souza’s model associates the persistence of an egalitarian organization in nomadic gathering societies to rudimentary weapon technology: because weapons were easy to produce and to use, each individual had access to coercive power. The development of weapons at the same time more effective, more costly to produce and requiring longer training resulted in the emergence of elites that used coercive power to exploit weaker members of the society and deploy resources to their own benefit. Social group enlargement catalyzed by the introduction of wider-ranging weapons unavoidably resulted in competition between independent groups. War can be defined as organized violence carried out by political units against each other. As such, war is present in some social primates such as chimpanzees (but not bonobos) and several platirrhine monkeys, all presenting male bonding and simple forms of social rearing. Anthropologists debate whether warfare was common from the beginning of our history as a cooperative species or only appeared among sedentary societies. There is no clear evidence for warfare predating the onset of sedentism, because known examples of violent death inferred from damaged skeletons might be due to other causes than war, from homicide to hunting accidents. In contrast, archaeological evidence for war becomes overwhelming after the

Chiefdom (~11 KYA) Archaic state (5–6 KYA) Early modern State (~800 years ago) Modern State (~200 years ago)

Social group Band and macroband Tribe

Political system Egalitarian Partial despotism Despotism Despotism Partial despotism/ democracy Democracy

Population size range 102–103 103–104 103–105 104–106 105–107 106–108

Main subsistence Nomadic foraging Sedentary foraging/ horticulture Agriculture Agriculture

Agriculture & Industry

Agriculture & Industry

State

State

Elite State/Elite

Ruling component on force Individual Individual

Absent

Present

Predominant Predominant

Patriarchy None Present

Monotheism/ none

Polytheism Polytheism/ Monotheism Monotheism

Dominant religion Animism Animism

Table 12.1 Main types of human social organization. The dates in parentheses indicate the likely time of first appearance. Chiefdoms and States appeared only in the post-farming world. All types of social organization have been in existence until recently. From Van Schaik (2016), and Bingham and Souza (2009)

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The Evolution of Social Organization

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Fig. 12.21 Design and technique of use of a traditional atlatl

appearance of sedentism (about 13 KYA), including remnants of fortifications, large numbers of violently killed peoples buried together, weapons, and depictions of war scenes on murals. War was probably present at some level well before sedentism, but its nature changed dramatically over time, accompanying the transition from nomadic to sedentary foraging, from foraging to agriculture, and from egalitarian tribal societies to hierarchically organized chiefdoms and states. The turning point in warfare came with the spread of agriculture leading to higher population densities and competition for cultivable land. Warfare probably favoured the emergence of despotic social organizations, with elites taking the role of warriors defending the populace against outside threats and then turning into a force of oppression (Van Schaik 2016). Expanding their hypothesis of a direct link between weapon technology and social evolution, Bingham and Souza (2009) propose that: (a) The invention of the bow and the introduction of horses (cavalry) in warfare were key events underlying the emergence of “archaic states”. (b) The introduction of gunpowder artillery in the thirteen century AD in China, the Islamic world and Western Europe fostered the evolution of “early-modern states”. (c) The invention of repeating rifles in the nineteen century was pivotal to the development of “modern states”. (d) The appearance of easy-to-use and relatively cheap gunpowder weapons at the end of the nineteenth century gave common people potential access to coercive power, thus paving the way to the emergence of modern democracies (Table 12.1).

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Following the above reasoning, one might foresee that the development of intercontinental missiles and nuclear bombs will trigger the rise of a global human federation, a process probably already initiated with the current shift to global economy and real-time information sharing through Internet. This, in the hope extant states do not mess up too much before. As Bingham and Souza (2009) put it, “all our social traumas result from elite domination or self-interested manipulation of more democratized societies. Democratization is the antidote for the first problem, aggressive knowledge seeking by the global wise crowd is the cure for the second”.

12.14

The Search for “Human” Genes

The human genome consists of about 3235 Mb, for a total of nearly 110 cm of DNA subdivided into 23 chromosomes. The Human Genome Project, started in 1990 and officially completed in 2003, determined the DNA sequence of the human genome. A complex annotation (Glossary) work has since been in progress to identify single genes. Protein-coding genes account for less than 1.5% of total human genome, nonprotein-coding genes (or “RNA genes”) probably for less than 0.5%, the rest being introns, pseudogenes and seemingly meaningless repetitive sequences (Box 6.3). Gene identification in the human genome is far from a simple task, the estimated number being still uncertain, despite considerable effort, 15 years after the completion of sequencing. Considering only protein-coding genes, estimates range from 22,233  1000 (Pertea and Salzberg 2010), ~20,500 (Clamp et al. 2007), down to ~19,000 (Ezkurdia et al. 2014), i.e. less than half the genes in barley (Oryza sativa) and not much more than in a tiny nematode worm. In 2005, the Chimpanzee Sequencing and Analysis Consortium published the complete genome sequence of the chimpanzee (Pan troglodytes), one of the two closest living relatives of humans, followed a few years later by genome sequencing of the macaque (Gibbs et al. 2007), orangutan (Locke et al. 2011), gorilla (Scally et al. 2012), and bonobo (Prüfer et al. 2012). Comparison with other primates’ genomes is yielding fundamental insight into the evolution of the human genome (Pääbo 2014). Humans, chimpanzees and bonobos turned out to have nearly 99% identical genomes (by comparison, the average sequence homology between different individuals in our species is 99.5%). The differences observed between humans and their ape relatives, about 35 Mb, are distributed unevenly, the great majority of genes being identical or nearly identical and a minority being highly divergent. Of particular interest is a sequence of 118 base pairs dubbed HAR1 (Human Accelerated Region 1), showing high divergence in humans vs. other species (Fig. 12.22). In the human genome, HAR1 is located in two partially overlapping non-coding RNA genes, HR1A and HR1B, in chromosome 20. HAR1A is expressed in a particular lineage of neurons fundamental to the development of the cerebral cortex; disturbances in these neurons are associated with a severe, often deadly, congenital

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The Search for “Human” Genes

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Fig. 12.22 Differences in HAR1 sequence of humans vs. chimpanzee and of chimpanzee vs. chicken. The data show that HAR1 went through fast mutation rate in the hominin lineage after separation from Panins; in contrast, it remained remarkably stable for at least 300 MY after the divergence of mammals from their common ancestor with birds. The accelerated evolution of human HAR1 and similar sequences probably reflects positive selection. Redrawn from Pollard (2013)

disorder known as lissencephaly (“smooth brain”), in which the cortex lacks its characteristic folds and exhibits a reduced surface area (Pollard et al. 2006). Following the discovery of HAR1, the human genome turned out to contain a great number of other HAR sequences (Pollard et al. 2006; Bush and Lahn 2008; Franchini and Pollard 2017). Most of these do not encode for proteins or RNAs, but appear to function as regulatory DNA sequences controlling the expression of a number of genes. More than half of these genes code for transcription factors involved in brain development and function. Thus, although HAR sequences account for a very small fraction of the human genome, changes in these sequences might have profoundly altered the functional anatomy of the brain by influencing whole networks of regulatory genes (Pollard 2013). Comparative genomic analysis has shown that at least 84% of changes in human HAR sequences occurred before the divergence of modern humans from the Neanderthals and Denisovans, whereas about 8% occurred afterward, confirming these sequences as a hot spot in human evolution (Burbano et al. 2012). Other genes displaying prominent sequence divergences relative to homologues in other primates are ASPM, Microcephalin (MCPH1) and FOXP2. ASPM and Microcephalin genes control brain development, their null (loss of function) mutations being associated with microcephaly, a serious developmental disorder in which the brain size is reduced by up to 70% without other gross abnormalities in the rest of the body (Vallender et al. 2008). Phylogenetic analysis points to strong positive selection of these genes in the primate lineage leading to Homo sapiens, with sequential accumulation of amino acid changes accompanying the increase in brain size, the last ones dated to 6 KYA in ASPM and 40 KYA in Microcephalin (Evans et al. 2004; Mekel-Bobrov et al. 2005; Shi et al. 2013). Mice carrying a truncated ASPM protein have reduced brain and testis size; the human ASPM gene was able to rescue the normal phenotype in transgenic mice but did not induce any further brain enlargement relative to normal mice (Pulvers 2010).

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The FOXP2 (Forkhead box Protein2) controls fine movements employed in humans for speaking, and is shared by many vertebrates, where it generally plays a role in communication (for instance, the development of bird song). Null mutations of this gene cause a severe speech disorder in humans but do not affect cognitive abilities. FOXP2 has been dubbed the “language gene”, although this gene is necessary but crucially not sufficient for the mechanics of human speech. FOXP2 encodes a transcription factor of 715 amino acids that is highly conserved among mammals but in modern humans carries two amino acid substitutions at positions 303 and 325 (Enard 2011). Both mutations also occur in Neanderthal and Denisovan genomes, so they probably appeared in a common progenitor at least 500 KYA (Green et al. 2010). A more recent study, however, identified several nucleotide substitutions in a regulatory (not translated) region of the gene, which are present in modern humans but absent in a 49,000-year-old Iberian Neanderthal (Maricic 2012). Additionally, Neanderthals (and Denisovans) probably lacked other alleles (CNTAP2, ASPM, MCPH1, PCDH11Y and X) suspected to be associated with language (Hauser 2014 and references therein). Steady progress is being made in building a more complete “parts list” of genomic changes that ultimately generated the substrate for human cognitive abilities, including those in regulatory genes. Nonetheless, even in the best-understood cases, the genotype/phenotype gap remains large. Molecular biology still has a long way to go before it can illuminate the evolution of cognitive abilities in humans. An important insight from molecular research is that the vast majority of all genomic changes that distinguish the human lineage from its primate relatives are in noncoding regions with regulatory functions (Franchini and Pollard 2017). Box 12.2: “Human races” From the use of polygenism to justify slavery, to the use of eugenics to justify unfair immigration laws, sterilization of the poor, and even genocide, biological anthropology has had a sordid history (Wolpoff and Caspari 2013).

The concept of human races is deeply rooted in most human cultures and has long been employed to justify war, genocide and slavery or, under milder social conditions, systematic inequality. In 1758 Linnaeus introduced the genus Homo in his Systema Naturae, distinguishing “Homo sylvestris” or “Homo troglodytes”, a mixture of known facts about the orang utan and myths, and Homo sapiens, the latter with four geographical variants from Africa, America, Asia and Europe. When human variants were considered in a taxonomic context, their relationships started being described in phylogenetic terms in the framework of polygenism, an interpretation of biological diversity as the expression of independent ancestry. In pre-Darwinian times, human variation was generally assumed to reflect the existence of races created separately by God. After the (continued)

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Box 12.2 (continued) acceptance of Darwinian evolution, many scientists shared the idea that human races diverged from a common ancestor but evolved “humanity” independently. Others such as Ernst Haeckel (1834–1919) went so far as to postulate a polyphyletic origin from different ape ancestors. In the twentieth century the dominant view was that human races were different subspecies evolved in parallel, with little or no contact, from geographically disperse populations of Homo erectus. Substantial similarities in anatomical and behavioural traits, most of which were by any account far more relevant than diagnostic race traits such as skin pigmentation or hair texture, were explained as due to parallel evolution from “interracial competition” or to no better defined “intrinsic evolutionary direction”. Haeckel applied Darwinism to theorize that the extermination and exploitation of other human racial groups (species, for him) were the inevitable and desirable consequences of natural selection. These ideas were widely adopted by both the public and the scientific community, especially in Central Europe, and became the central paradigm of National Socialism in Germany in the early 1930s. Polygenism remained strong throughout the first half of the twentieth century, the race concept losing its primacy in the interpretation of human variation only after the rise of population genetics. The most influential polygenist of twentieth century paleoanthropology was Carleton Coon (1904–1981). In the book “The Origin of Races” appeared in 1962, Coon argued that the humankind encompassed five major geographic races, or “human subspecies”, isolated from each other for most of their history: the Congoids and Capoids in Africa, the Caucasoids in Europe and Western Asia, the Mongoloids in Central, South, and East Asia, and the Australoids in Greater Australia. The core of Coon’s polygenism is that these races stemmed from five distinct subspecies of Homo erectus, which he regarded as a prehuman species, and acquired “humanity” independently. In Coon’s perspective, differing cultural achievements and cognitive abilities of extant human races reflect the fact that these emerged independently and have been “human” for different lengths of time. Coon’s book generated enormous controversy and was generally poorly received by the anthropological community. The multiregional hypothesis of human evolution proposed by Wolpoff et al. in 1984, now discredited, was in some respect a revival of polygenism (Sect. 12.5). The idea that different racial groups acquired humanity independently has very little currency in modern anthropology. Although fully espousing the view that there are no human races today, Wolpoff and Caspari (2013) point out that human races have co-existed in the past. Two of these were probably the Neanderthals and the Denovians, both co-existing with Sapiens for several thousand years and interbreeding with them (Sect. 12.4). Likewise, the genome (continued)

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Box 12.2 (continued) of sub-Saharan African populations contains about 2% of DNA acquired about 35 KYA from an archaic population that split from the ancestors of anatomically modern humans about 700 KYA, suggesting interbreeding events between genetically differentiated lineages (Hammer et al. 2011). Modern scholarship views racial categories as a social construct lacking any biological foundation. Overall, the DNA sequence database points to extant humans as an exceedingly homogeneous species with a monophyletic recent origin in Africa. The most significant cause of current human variation is natural selection of pre-existing polymorphisms (alleles) rather than new mutations. For a more comprehensive review of the topic, see Wolpoff and Caspari (2013).

12.15

Is Humankind Compatible with the Global Ecosystem?

Is there anything more dangerous than dissatisfied and irresponsible gods who don’t know what they want? (Yuval Noah Harari, 2015)

Niche construction theory places emphasis on the capacity of organisms to modify their environment and to affect natural selection acting on their own evolution and that of other organisms. In no other species is niche construction as powerful and pervasive as in humans. Humans are unique not only in the use of tools but also because they employ external sources of energy. The complexity of the tools and of energy-transforming devices has increased steadily in the last 50 KY, from stones to nanomaterials and from animal work to nuclear plans, closely mirroring advances in social organization. Humans have been powerful ecological agents long before becoming farmers. Dramatic and well-documented examples are the extinctions of numerous animal species following the arrival of hunter-gatherer populations in the American continent, Australia and much more recently in New Zealand. About 90 genera of megafauna genera (animals weighing above 44 Kg) extinguished within 2000 years after the arrival of humans in America; these include giant sloths, saber-toothed cats like Smilodon, camels, horses (re-introduced by the Spanish in the sixteenth century), mammoths, mastodons, and at least two species of bison (https://en.wikipedia.org/wiki/Quaternary_extinction_event). The human impact on global environment increased by orders of magnitude with the transition to agriculture from about 11 KYA. Replacement of natural ecosystems such as forests, prairies and savannas with cultivated fields was an unprecedented manifestation of niche construction. Through agriculture and associated urbanization, humans have deeply modified environmental conditions and habitats worldwide, causing a dramatic expansion of a few domesticated and commensal species (e.g. rats, flies, mosquitos) and a similarly dramatic reduction, or even the extinction

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Is Humankind Compatible with the Global Ecosystem?

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of numerous wild species, a process currently referred to as the Sixth Extinction (Kolbert 2015). A second dramatic increase in the impact of humans on the planet initiated about 300 years ago with the industrial revolution and large-scale use of fossil fuels. This date is now considered the start of a new geological era, the Anthropocene, in which humans are the major drivers of environmental change. With a global population approaching 8 billion, the environmental impact of humans has never been as mighty and pervasive as in the present. The main factors at play are over-exploitation of resources, habitat degradation and loss, climate change, diffusion of invasive species, and pollution (Pelletier and Coltman 2018). In 2017, 15,364 scientists from 184 countries signed a warning asserting that human population growth is the primary driver behind many ecological and even societal threats (Ripple et al. 2017). Much earlier calls, notably The Limits to Growth in 1972 and the United Nations report Our Common Future in 1987 caused much public debate but had very little effect on economic and social policies (Spier 2010). There are many examples from the past of societal collapse arising from overpopulation and environmental mismanagement, such as Easter Island, the Norse settlement in Greenland, and the Mayan Empire (Diamond 2005). Despite these warning episodes, there is no sign of a change in the current globe wide trend towards self-destruction. Mainstream economics is obsessed with the myth of “economic growth”, basically meaning a steady increase in the amounts of raw materials transformed into goods for human consumption. These materials, including food, timber, oil, water and minerals, must obviously come from Nature, which economists perversely keep thinking of as an infinite repository (Harding 2006). Tragically, so-called “externalities” such as pollution, ecological destruction and disease are never seriously considered in the calculation of Gross Domestic Product, nor is a more vague but fundamental parameter, human happiness. In the absence of a radical rethinking of the human position in the planet and of our duty towards future generations, the only predictable outcome is ecological disaster followed by war for increasingly rarer resources. A recent study estimated that land use and related pressures have already reduced local biodiversity intactness (the average proportion of natural biodiversity remaining in local ecosystems) beyond the proposed “safe limit” of 20% across 58.1% of the world’s land surface, where 71.4% of the human population lives. Beyond this limit threshold, human intervention is necessary to ensure adequate local ecosystem function (Newbold et al. 2016). What is going to happen if humans fail to tune with Earth’s pace? A likely outcome is a burst of global extinction, most certainly including our own species. The Earth has already gone through several planetary catastrophes, with five major extinctions involving the disappearance of up to 95% of animal species in the Phanerozoic. Yet, each time the planet managed to resume ecological equilibrium and biological diversity. Thus, there are good reasons to believe that the wound we are leaving behind will heal very quickly, deleting all signs of our short passage. Humankind ultimately owes existence to “moral codes” ruling within-group cooperation but more or less explicitly enforcing hostility towards the “others”. In order to survive, we now need new rules enforcing supra national, global cooperation: a novel major transition for our species and for the planet as a whole.

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Chapter 13

Synopsis

Started on a barren planet with no ocean and a thin unbreathable atmosphere, our long journey terminated on a life-teeming planet with an oxygen-rich atmosphere, an ocean covering over two thirds of its surface and large chunks of continental crust hosting a diversity of terrestrial ecosystems and a particularly invasive primate species. Let us look back and make a take-home overview of the story. According to the alkaline vent hypothesis, life was a product of chemical disequilibrium between the mantle and the surface of the Earth, built by tectonic activity and ultimately arising from gravitational separation of an iron/nickel core. The elementary unit of life is the cell, a self-reproducing chemical system that exists in a condition far removed from thermodynamic equilibrium by increasing entropy in the environment. The first cells possibly originated as early as 4 GYA from organic-mineral complexes in submarine geothermal vents; the participation of transition metals such as iron, nickel, manganese and zinc in fundamental processes of modern life might be a legacy of this ancestral past. The information underpinning cell reproduction is stored in DNA and membranes, both functioning as templates for new copies. Extant life arises from the Last Universal Common Ancestor (LUCA), an organism possessing universally shared traits, notably DNA, ribosomes, a genetic code, a chemiosmotically active bounding membrane and a basic gene set. It is widely maintained, although not universally agreed, that the bacterial and archaeal domains directly diverged from LUCA and that the eukaryote domain appeared later. Probably borne in the dark of the ocean bottom, early life turned to sunlight as a source of energy, thus gaining access to the surface of the planet. Photosynthesis ancestrally employed reduced compounds of geochemical origin as a source of electrons for carbon dioxide reduction. Combining type-1 and type-2 photosystems, the cyanobacteria became able to extract electrons from water, thus putting an end to life dependence on geochemistry. About 2.4 GYA, free oxygen started accumulating in the atmosphere-ocean-crust system, an event known as the Great Oxygenation. Life adapted to the novel conditions and exploited the oxidative properties of oxygen by evolving high-energy-yielding aerobic respiration and novel biosynthetic pathways. © Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9_13

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When and how eukaryotes evolved is not resolved yet. Mitochondrion-late models assume that the eukaryotic cellular organization, epitomized by the ability to perform phagocytosis, emerged before the evolution of the mitochondrion by endosymbiosis. Mitochondrion-first models, in contrast, propose that eukaryotes derive from the fusion of an archaeon with a proteobacterium and that the conversion of the latter into a mitochondrion was the very event that triggered the evolution of all other traits distinguishing the eukaryotic cell. The genome of eukaryotes has a chimeric structure encompassing scores of genes of archaeal and bacterial affinity, besides genes unique to eukaryotes. Some archaeal lineages have genes encoding for “eukaryotic-signature” proteins, including homologs of actin, tubulin, GTPases and components of the endomembrane system. Phylogenomic analysis of these protein data sets consistently places the eukaryotes within the Archaea, thus indirectly supporting the fusion model of eukaryogenesis. The most popular version of this model explains the presence of bacteria-like membranes in eukaryotes by assuming that the archaeal host switched from archaeal to bacterial membranes in consequence of the evolution of the mitochondrion. The mitochondrion-late “neomuran” model holds that the eukaryotes and archaea are sister groups diverged from a common bacterial ancestor, with the eukaryotes retaining ancestral G3P-ester lipids and evolving phagocytosis, and the archaea evolving novel G1P tetraether lipids as an adaptation to hyperthermal acidic conditions. Molecular-clock analysis dates the first appearance of eukaryotes to at least 1.5 GYA and the last common ancestor of modern eukaryotes (LECA) to 1.5–1.0 GYA. Molecular phylogeny currently resolves extant eukaryotic diversity into two major lineages: the Amorphea (essentially the Animals, Fungi and Amoebozoa), and Diaphoretika (Rhizaria, Archaeplastida, Chromista and Alveolata); the affinities of Excavates, currently considered paraphyletic, and some minor groups remain to be determined. Sex, defined as the mixing of DNA from different cells in the same genome, is a universal property of life that is essential for long-term conservation of biological information. At the same time, sex promotes the formation of new genes and new genomes, thus acting as a powerful agent of genetic change. Exploiting their unique ability in membrane budding and fusion, eukaryotes evolved meiotic sex involving wholesome cellular fusion and genome recombination, a mechanism possibly emerged under fluctuating environmental conditions and fixed as a means for the maintenance of multi-chromosome, large-sized genomes. Meiotic sex sets sharp interspecific boundaries, yet at the same time it favours speciation by blocking gene flow between populations. Because of high-frequency horizontal gene transfer, species boundary in prokaryotes is conventional and the total number of genes assigned to species (known as the pangenome) is usually much larger than the number of genes present in single cells or strains. Multicellularity evolved multiple times in both prokaryotes and eukaryotes. A complex multicellular organization with a controlled internal environment independently evolved in brown and red algae, fungi, animals and plants. The main driver of multicellularity is increased fitness from cellular specialization and co-operation; in order to prevent the emergence of cellular competition, this requires that the cells of multicellular organisms share the same genome. Probably for the same reason, most

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sexually-reproducing multicellular eukaryotes evolved mechanisms of uniparental transmission of DNA-containing organelles (mitochondria and chloroplasts). Specialized cellular junctions enhancing cellular adhesion and communication independently evolved in several lineages, often following convergent paths. Complex multicellular organisms deeply affected global geochemistry, increased ecosystem complexity, and changed the face of the planet. The last common ancestor of extant eukaryotes (LECA) was a heterotrophic aerobic microorganism feeding on microbial prey by phagocytosis. Around 1 GYA, a member of the Diaphoretika acquired photosynthesis by converting a cyanobacterial endosymbiont into a primary chloroplast, thus generating the photosynthetic lineage known as Plants or Archaeplastida. The primary chloroplast was horizontally transferred to other eukaryotic hosts by secondary endosymbiosis, generating the Euglenophyceae (Excavates), Chlorarachniophyceae (Rhizaria) and a heterogeneous group of photosynthetic eukaryotes named “Chromoalveolata”. The secondary chloroplasts of Euglenophyceae and Chlorarachniophyceae independently derived from two distinct unicellular green algae. Chloroplast DNA analysis strongly supports a monophyletic ancestry of chromoalveolate chloroplasts from a red algal endosymbiont. In contrast, phylogenetic analysis of nuclear genomes and the lack of clear evidence for chloroplast loss in non-photosynthetic chromoalveolates argue against chromoalveolate monophyly, thus lending support to hypotheses of horizontal chloroplast transmission by serial endosymbiosis. The transfer of photosynthesis from bacteria to eukaryotes enhanced global productivity, organic carbon burial and oxygen accumulation in the ocean and atmosphere, thus giving a fundamental contribution to the establishment of conditions favourable to the emergence of complex multicellular forms. Multicellular fossils unambiguously identified as animals formally mark the transition from the Proterozoic to Phanerozoic eon at 432 MYA. The ancestral body plan of the animal clade was a bag-shaped structure in which coordinate activity of flagellate cells enhanced feeding on unicellular prey by phagocytosis. The evolution of an intestine and extracellular digestion enabled the animals to feed on macroscopic prey. The intestine hosts a symbiotic microflora, known as the gut microbiota, which participates in extracellular digestion. Traditional systematics divides the animals into the Parazoa (sponges) and Eumetazoa (including the Ctenophores), based on the absence/presence of an intestine and neurons. In some analyses, molecular inference places the Ctenophores at the base of the animal tree, suggesting that regressive and parallel evolution may have blurred the phylogenetic signal from morphological traits. The diffusion of pelagic animals in Cambrian oceans increased the amount of organic carbon sequestered in sediments, due to relatively rapid sinking of feces and carcasses, probably contributing to the rise of oxygen to nearly modern levels. Widespread diffusion of filter-feeding nutrition among animals reduced the bacterial component of plankton and favoured the evolution of larger-celled eukaryotic phytoplankton. No other organism, barring perhaps the cyanobacteria, has exerted so a profound impact on the planet as land plants. Appeared in the Ordovician, around 480 MYA, land plants probably inherited somatic desiccation tolerance (poikilohydry) from

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Synopsis

their green algal ancestor (most likely a member of the Zygnematophyceae). All land plants are haplodiplontic organisms, with a haploid sexual phase (the gametophyte) alternating with a diploid sporophyte that reproduces by meiotic spores. Primary events in plant terrestrialization were the insertion of a sporophyte in an ancestrally haplontic life cycle, and symbiotic association with fungi ancestral to modern mycorrhizas. A second major transition in plant terrestrialization was the evolution of a free-living homeohydric sporophyte with stomata and a waterproof cuticle (both sporophytic innovations) and rhizoids (deployed from the gametophyte). Competition for light and mineral nutrients favoured larger body sizes, leading to the evolution of lignin and xylem. A third critical advance was the evolution of the seed in the late Devonian, which ultimately severed the ancestral dependence of land plants on liquid water for sexual reproduction. Massive sequestration of organic matter from land plants in the Permo-Carboniferous (350–250 MYA) caused a drop in atmospheric carbon dioxide (CD) concentration from several thousand to a few hundred ppm. In parallel, land plants evolved megaphylls (leaves with multiple veins) with increased stomatal frequency. CD returned to thousand-ppm levels in concomitance with the Permian-Triassic Extinction, and started decreasing again from about 130 MYA, at least in part because of increased silicate weathering due to the uplifting of Himalaya and other large mountain complexes. The rise to dominance of flowering plants (angiosperms) about 90 MYA further contributed to the fall in CD concentration, which plummeted to about 200 ppm around 30 MYA and remained relatively stable thereafter. The planetary thermostat reacted to falling CD concentration by setting the average global temperature at lower levels. About 2.6 MYA, the temperature drop triggered the Quaternary Glaciation, with polar ice caps cyclically expanding at time intervals of about 40 and, more recently, 100 KY. Because of cooler and drier weather, from about 8 MYA, large areas of rain forest in Central-Eastern Africa were replaced by savanna, a novel grassland biome with widely spaced trees. The expansion of savanna triggered the divergence of hominins, a novel ape lineage that evolved bipedality under selection pressure for foraging on the ground. Early in evolution, the hominins lost most of their body hair and improved their thermoregulation system, thus becoming enduring runners capable of withstanding prolonged exposition to high temperature. Freed from locomotion constraints, the forelimbs developed enhanced manipulation ability. Anatomical changes in forelimbs and torso enabled early hominins to throw projectiles with high speed and precision. The ability to project coercive threat on conspecifics favoured the emergence of reciprocal (or kinship-independent) cooperation as a more convenient strategy than selfish behaviour. Reciprocal cooperation paved the way to a series of interdependent changes, most notably collective breeding, language, knowledge transmission, which in turn triggered a positive feedback on brain size and cognitive and social skill. The accumulation of shared knowledge created a novel evolutionary landscape known as cultural niche construction that became the dominant driver of human evolution. Modern humans migrated from Central Africa to the Middle East around 70 KYA and spread to Europe and Asia from about 60 KYA. A sudden improvement in artefact technology and the first appearance of

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Synopsis

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symbolic representations around 50 KYA is interpreted as evidence of a cognitive revolution, yet it is uncertain whether this had genetic or cultural roots. Starting 11 KYA, agriculture increasingly replaced foraging as a stable lifestyle. The transition to food production worsened the average living conditions, yet it became irreversibly fixed because of increased population size. Surplus production favored social structuring but also led to unequal wealth distribution and the emergence of dominating elites, in sharp contrast with the substantially egalitarian structure of foraging societies. Unsustainable impact on planetary resources urgently demands a radical change in human cultural niche.

Glossary

Albedo From Latin albus, white, the fraction of incident radiation reflected by a surface. Used with no other annotation, the albedo refers to visible radiation. α- and β-polysaccharides Polysaccharides in which the glycosidic linkages of the polymer backbone have α or β stereochemistry. The prefixes α and β refer to the stereochemical configuration of the carbonyl-bearing carbon of monosaccharide subunits after cyclization. In Haworth projections, the hydroxyl group bound to this carbon in α- and β-monosaccharide subunits lies below or above the molecule plane, respectively. Alleles Gene variants that control the same function and are located at a specific locus in a specific chromosome. The existence of more than one allele for the same gene in a population is referred to as ! gene polymorphism. Amenorrhea The interruption of ovulation, usually in response to unfavorable conditions. In mammals in which lactation is longer than pregnancy, temporary post-partum amenorrhea prevents offspring conflict. Angular momentum From latin “momentum”, movement, the angular momentum is a vector quantity that represents the product of a body’s rotational inertia and rotational velocity about a particular axis. The magnitude of the angular momentum of an orbiting object is equal to its linear momentum (product of its mass m and linear velocity v) times the perpendicular distance r from the center of rotation to the object’s center of gravity, or simply mvr. SI units for the angular momentum are kgm2/s. Annotation (of genes) The process of identifying the locations of coding regions (genes) in a genome (structural annotation) and determining what those sequences do (functional annotation). Once a genome is sequenced, it needs to be annotated to make sense of it. Autotrophy From Greek autòs “self” and tròphein “to nourish”). Organisms that obtain more than 50% of their carbon from CO2 (or bicarbonate) are autotrophs. Organisms that obtain less than 50% of their cellular carbon from CO2 are

© Springer Nature Switzerland AG 2019 R. Ligrone, Biological Innovations that Built the World, https://doi.org/10.1007/978-3-030-16057-9

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478

Glossary

heterotrophs (Schönheit et al. 2016, reference in Chap. 3). This conventional definition takes into account the fact that carboxylation reactions are universally present in all extant organisms, including humans. Biofilm A consortium of microorganisms whose cells are embedded in an extracellular polymeric conglomeration of polysaccharides, proteins, lipids and DNA. Biome Biological community with distinctive properties developed in response to specific environmental conditions, mainly physical climate. Examples of biomes are the tundra, taiga, savanna and mediterranean shrubland. Clone Asexually-produced progeny of a single parent. Barring mutations, the members of a clone are genetically identical. Codon Information unit of the genetic code. A codon consists of a triplet of adjacent nucleotides in the messenger RNA, and codes for a specific amino acid in the cognate protein. Because RNA is made of four different nucleotides (adenosine, guanosine, cytidine and uridine phosphate), there are 64 (43) codons, of which 61 code for 20 amino acids (sense codons) and three are stop signals (nonsense codons). The existence of more than one codon for each amino acid is known as “codon degeneracy”. Transfer RNAs contain a triplet of nucleotides, known as the “anticodon”, that is complementary to each of the 61 sense codons. Concestor Common ancestor of related evolutionary lineages Diffusion Net movement of free particles (usually atoms or molecules) in a gaseous or liquid phase, driven by energy differences depending on uneven concentration, temperature, pressure or, for charged particles, electrical potential. Diffusion tends towards an equilibrium characterized by uniform energy distribution, i.e. maximum ! entropy; at the equilibrium, particles continue moving but their net flow in any direction is zero. Diffusion is governed by Fick’s laws and by the diffusion constant D. The diffusion constant has dimension m2/s and varies with the mass and shape of particles and with the phase (gas or liquid). Diffusion rapidly becomes less efficient over increasing distances; because of this, numerous multicellular organisms of macroscopic sizes have evolved systems for ! mass transport. Donnan equilibrium The development of an electrical potential difference that counterbalances ion diffusion along a concentration gradient across a membrane. Entropy From Greek “entropia”, transformation, entropy (usually indicated with the symbol S) is the quantitative expression of the degree of disorder or randomness of a thermodynamic system, or the amount of thermal energy not available to do work. Any spontaneous transformation involves an increase in entropy (ΔS > 0). Isolated systems spontaneously evolve towards thermodynamic equilibrium, defined as the state of maximum entropy. Non-isolated systems, such as living organisms, may lose entropy provided that entropy in their external environment increases by a greater amount, so that total entropy in the Universe increases. In statistic mechanics, entropy expresses the number Ω of different configurations that a system defined by macroscopic variables may assume. Under the assumption that all configurations are equally possible, entropy is the natural logarithm of Ω multiplied by the Boltzmann constant Kb [S ¼ Kb  lnΩ]. Both entropy and the Boltzmann constant have dimensions of energy divided by temperature.

Glossary

479

Food chain The unidirectional transfer of energy from a trophic level to another in ecosystems. Fundamental trophic levels are: producers (photo- and lytoautotrophs), consumers (heterotrophs), and decomposers (heterotrophs). The number of trophic levels increases with ecosystem complexity. Because each organism interacts with several trophic levels (for example its wastes feed the detritivores and decomposers, whereas its body may feed a predator), energy transfer usually follows a reticulate pathway known as “food web”. Genome and genotype The genome is the DNA sequence that characterizes a species in the haploid condition. The term genome generally refers to both coding and non-coding sequences, as well as mitochondrial and plastid DNA in eukaryotes. Genome size may be expressed in picograms (1 pg ¼ 1012 g) or megabases (1 Mb  106 base pairs), 1 pg corresponding to about 978 Mb. The term “genotype” usually indicates the genome of an individual organism or its allelic state relative to certain genes. Habitat The type of natural environment in which a particular species or organism lives. Ecologists call “biotope” a habitat shared by two or more species. Heterotrophy By convention, heterotrophy is a form of metabolism in which less than 50% of total carbon is obtained from CO2 or bicarbonate (cf. autotrophy). Homeobox genes Genes coding for transcription factors containing a DNA-binding domain of about 60 amino acids. These transcription factors, known as homeodomain proteins, regulate the expression of targeted genes during development and cellular differentiation in eukaryotes. Homologous genes Genes derived by mutation from the same ancestral gene. Homologous genes are recognizable from !sequence homologies. Horizontal gene transfer (HGT) Transfer of genetic material from organism to organism independent of kinship relationships. HGT is distinct from parent-tooffspring gene transfer, or vertical inheritance. Genes acquired by HGT do not reflect common ancestry, thus they cannot be used for phylogenetic inference. HGT frequency is relatively high in prokaryotes, much lower in eukaryotes. Hypha Filamentous cellular structure typical of fungi, consisting of a continuous multinucleate cytoplasmic mass, or of uni-, bi or multinucleate cells separate by septa. The hyphae typically elongate by tip growth and branch profusely, forming a hyphal system known as the mycelium. The terms “hypha” and “mycelium” are traditionally also used for actinobacteria. Introns Non-coding intragenic sequences interspersed with coding sequences. Introns are abundant in the eukaryotic genome, but relatively rare in prokaryotes. Introns are transcribed into RNA and removed by RNA splicing in the nucleus before RNA translocation to the cytosol for translation. Kinetochore Disc-shaped protein structure found at the centromere of chromosomes, to which microtubules anchor during mitosis. Lamarckian inheritance Also known as “Lamarckism” or “inheritance of acquired characteristics”, it is the hypothesis that an organism can pass on characteristics acquired through use or disuse during its lifetime to its offspring. Attributed to French biologist Jean-Baptiste Lamarck (1744–1829), the hypothesis has actually been put forward in several forms long before.

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Glossary

Light-year The distance that light travels in vacuum in one Julian year (365.25 days). A unit of length used to express astronomical distances. Lytoautotrophy A form of autotrophic metabolism in which the energy and reducing power employed for carbon dioxide reduction are obtained by (aerobic or anaerobic) oxidation of inorganic compounds such as sulphur, hydrogen sulphide or ferrous iron. Mass transport/mass flow Unidirectional flow of a gaseous or liquid fluid down a pressure or temperature gradient. Messenger RNA (mRNA) RNA molecule encompassing one (eukaryotes) or several sequences (prokaryotes) that ribosomes translate into proteins. Neoteny A developmental alteration causing a slowing down of somatic development. Neoteny entails the retention in adults of traits normally present only in the young. See also ! paedomorphism and progenesis. Niche (ecological niche) The position of a species/population within an ecosystem, namely its lifestyle and physical, chemical and biological conditions that permit its existence in a particular habitat. Orogeny Deformation of lithosphere at convergent plate margins. Orogeny is the primary mountain-building process. Orthologous genes (orthologs) Genes derived from a common ancestral gene by a speciation event. Orthologous genes control the same function in different species. Osmolarity The number of osmoles of solute per litre of solution. An osmole is a number of Avogadro (N ¼ 6.0221023) of solute particles. For calculating osmolarity it is necessary to take account of the degree of ionic dissociation of the solute considered. Osmotrophy A feeding method based on the uptake of organic compounds present in solution in the environment, by means of transmembrane carriers. Organisms feeding by osmotrophy are osmotrophs. Opposite to osmotrophy is phagotrophy, i.e. cellular feeding by phagocytosis of particulate organic matter including entire cells. Heterotrophic bacteria are osmotrophs; the fungi are typical osmotrophic eukaryotes. Paedomorphism The retention of juvenile traits in the adult, sexually mature phase; paedomorphism may result from ! neoteny or ! progenesis. Pangenome The full complement of genes present in a taxonomic group, including genes not shared by all individuals. The pangenome does not consider allelic variants of the same gene. Because of relatively high frequency of horizontal gene transfer, the pangenome of prokaryotic species may be orders of magnitude larger than the genome of single cells or clones. Meiosis demands a high level of homology in the genome of conspecifics; consequently, in sexually-reproducing eukaryotic species, the pangenome has much the same sizes as the genome of single individuals. When referred to taxonomic groups above the species level, for example a phylum, kingdom or domain, the pangenome encompasses all homologous gene variants present in the whole spectrum of organisms belonging to that group.

Glossary

481

Paralogous genes (paralogs) Genes derived by duplication of an ancestral gene in the same organism and diverged for novel, sometimes subtly different functions. Genes coding for different forms of tubulin in eukaryotes or of collagen in animals are examples of paralogous genes. Molecular phylogeny compares the sequences of orthologous genes, but tends to exclude paralogous genes. Periplasmic space The space between the plasma membrane and cell wall or, in gram-negative bacteria, between the inner and outer membrane. Photic zone The upper layer of the ocean or a lake that receives sufficient sunlight for net photosynthetic production. The photic zone corresponds roughly to the water layer above the photosynthetic compensation point level, where photosynthetic oxygen production is equal to oxygen consumption. Plasmids Small accessory chromosomes present in prokaryotic cells besides the main chromosome. Plasmids contains genes that may benefit the survival of the organism, for example antibiotic resistance, but are generally not essential. Plasmids replicate independently of the main chromosome and are prone to ! horizontal gene transfer. Polymorphism (gene polymorphism) More than one allele of the same gene within a population. By convention, each allele must occur in the population with a frequency of at least 1% to be considered polymorphic. Progenesis An acceleration in sexual development; progenetic organisms develop sexual competence and reproduce before completing somatic development. Humans are progenetic organisms. Prostetic group A non-protein compound that covalently binds to an enzyme and participates in catalysis. The heme group of cytochromes is an example. Unlike prostetic groups, co-enzymes (e.g. NAD or FAD) bind to enzymes by non-covalent bonds. Protein domain A part of a polypeptide chain that folds autonomously from the rest of the filament. A domain may comprise 40–350 amino acids. Single polypeptide chains may comprise one to several domains. Domain shuffling, i.e. random recombination of gene sequences coding for domains belonging to different proteins, is an important mechanism of biological innovation. Most existing proteins are mosaics of domains from other proteins. Proteome The entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time. Proteomics is the study of the proteome. Pseudogene Non-coding DNA sequence derived from a gene by loss-of-function mutation. Ribosomal RNA (rRNA) The RNA component of ribosomes. Ribosomes are about 60% rRNA and 40% protein. Sequence homology Similarities in the sequence of genes or proteins, reflecting origin from the same ancestral sequence. Sn position In order to designate the configuration of glycerol derivatives, the carbon atoms of glycerol are numbered stereospecifically. The carbon atom that appears on top in the Fischer representation showing the central hydroxyl group

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Glossary

to the left is designated as C-1. The prefix ‘sn’ (for stereospecifically numbered) is used to distinguish such numbering from conventional numbering conveying no steric information. Spliceosome Complex molecular machine unique to eukaryotes, which removes the introns from mRNA precursors (“spliceosomal introns”). A spliceosome consists of small nuclear RNAs and approximately 80 proteins. Strain A low-level taxonomic rank used at the intraspecific level. In microbiology, a strain is a genetic variant or subtype of a microorganism with some level of genetic definition, such as plasmids conferring specific traits. Unlike a clone, a strain is not necessarily made of genetically identical organisms. The process of separation of a strain from a natural mixed population is known as “isolation”. An “isolate” is the output of a single isolation procedure. Different isolates can belong to the same strain and different strains can belong to same species. Structural homology (in proteins) Proteins in phylogenetically distant groups may have no recognizable sequence homology but display similar molecular architectures. Well known examples are FTsZ and MreB, two bacterial proteins considered structural homologues of eukaryotic tubulin and actin, respectively. Structural homology independent of sequence homology may reflect remote origin from an ancestral protein but suggests that divergence occurred a long time ago or that there was an anomalous acceleration in evolutionary rate after divergence. The conservation of structural homology probably reflects strong functional constraints allowing amino acid replacement only with amino acids sharing similar properties. Subaerial A subaerial organism lives under conditions of continual exposure to the air. Symbiosis From Greek “sun” together, and “bìos” life, the term indicates long-term functional associations of different organisms. In the present book the term is employed restrictively, to denote mutualistic associations where both partners benefit. Many types of symbiosis are so specialized that the organisms involved are no longer able to live separately. Taxon, pl. taxa Taxonomic entity of undefined rank. Theory of mind The ability to attribute mental states such as beliefs, intentions, desires, emotions to oneself and to others, and to understand that others have mental states that are different from one’s own. Transfer RNA (tRNA) Small RNA molecules that are enzymatically linked to a specific amino acid to form aminoacyl~tRNA complexes. Each tRNA contains a nucleotide triplet known as the anticodon, which binds to the complementary codon in messenger RNA. Cells express at least 1 tRNA for each of the 20 amino acids employed in protein synthesis.

Index

A Abnormal spindle-like microcephaly associated (ASPM), 457, 458 Accessory pigments, 102, 112 Accretion, 28, 29, 31, 32, 40, 45, 46, 54 Acetyl-CoA pathway, 84, 85, 90 Acheulean technology, 414, 417, 439 Actin, 161–163, 175, 188, 189, 202, 325 Aerobic respiration, 9, 78, 133, 134, 150, 151, 165, 222 Aggregative multicellularity, 252 Agriculture, 370, 424, 447–451, 453–455, 460 Alkaline vents, 59–62, 64, 68, 70, 74, 82, 103 Alloparenting, 432, 435 Alternative splicing, 218 Alveolata, 112, 192–194, 210, 221, 271, 282, 285, 292, 293 Amniotes, 10, 341 Amoebozoa, 186, 192, 193, 201, 204, 207, 213, 294 Amorphea, 186, 191–193, 202–205, 208, 218, 263, 271, 294, 303, 316 Amphimedon queenslandica, 318, 322 Amylopectin, 283, 303 Amylose, 303, 304 Anabaena, 256 Angiosperms, 9, 184, 201, 281, 283, 286, 342, 356, 359, 369–373, 382, 387, 389–391, 403 Anoxygenic photosynthesis, 101–103, 106, 120 Anthropocene, 461 Apes, 400, 402, 407–409, 432, 434, 435, 437 Apicomplexa, 184, 192, 208, 209, 221, 280, 285, 286, 290, 292–296, 299, 304, 331

Apomorphies, 9, 189, 192, 284, 315, 338, 401, 411 Archaeal flagellum/archaellum, 196, 199 Archaean genetic expansion, 106 Archaeplastida, 192–194, 203, 271, 276, 281–285, 287, 288, 303–305, 366 Archaezoan model, 164, 166 Ardipithecus ramidus, 406, 432 Asgard superphylum, 175 ATP synthases, 75, 79, 82, 110, 118, 180, 182, 186 Aurignacian culture, 419 Australopithecus afarensis, 407–410 Australopithecus anamensis, 403, 408 Australopithecus sediba, 410 Axoneme, 200, 201, 205, 208, 284, 319

B Bacterial endosymbiosis, 270 Bacterial flagellum, 195, 196, 198 Baculum, 401, 434 Banded iron formations (BIFs), 54, 103, 104, 131, 136, 140, 144, 146 Bangiomorpha pubescens, 211, 281 Basal body, 191, 193, 200, 201, 205, 208, 284 Basalt, 34, 47, 59, 412 Big Bang, 24, 25 Bigellowiella natans, 289 Bikonta, 191, 192 Bilateria, 317, 318, 320, 322, 323, 328–330, 332, 334 Bioelements, 2, 39–40 Bipedality, 401, 406–408, 415, 428, 444

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484 Black smokers, 58–61, 102 Blepharoplast, 200 Body spatial symmetry, 318 Brain mass, 435 Bryophytes, 247, 349, 351, 353–356, 359–361, 363, 365–367, 373, 375, 386, 388 Burgess Shale formation, 312

C Calvin-Benson-Basham (CBB) cycle, 85 Capillarity, 364, 373, 375, 379 Carbonaceous deposits, 105, 142 Carbonate deposits, 105, 135 Carboxysomes, 124, 125, 282, 283 Caryoplasmic ratio, 218, 220 Cavitation/gaseous embolism, 364, 372, 377, 378 C-DNA paradox, 214 Cellular adhesion, 255, 260, 261 Cellular junctions, 260, 324–331 Cellulose, 43, 281, 332, 334, 335, 378, 379 Cenozoic global cooling, 389 Centrioles, 201, 207–210, 246, 282 Centrosome, 201, 207–211, 246 Cephalochordates, 334–336 Charophyta/charophytes, 255, 276, 285, 349, 351, 352, 354, 355, 363, 366, 386 Chemiosmosis, 75–81, 150 Chemiosmotic machinery, 170, 180, 181, 186, 187 Chemotrophy, 100, 103 Chengjiang Fossil Site, 312, 337 Chimpanzees, 400–402, 406, 407, 410, 414, 426, 429–432, 441, 443, 446, 452, 453, 456, 457 Chimpanzee Sequencing and Analysis Consortium, 456 Chlamydia, 185, 274, 287 Chlamydomonas, 125, 184, 202–204, 207, 210, 240, 245, 247, 275, 283, 303 Chloroflexibacteria, 109, 111–113, 116, 122 Chlorophyta/chlorophytes, 286, 350, 353 Chlorosomes, 112, 113, 116 Choanocytes, 254, 315, 318, 323 Choanoflagellates, 192, 202, 203, 208, 209, 253–255, 258, 263, 315, 316, 323, 326 Choanozoa, 324 Cholesterol, 151, 160, 212 Chondrites, 29, 40 Chordates, 333–341 Chromoalveolate hypothesis, 292, 295 Chrompodellida, 285, 286, 288, 290, 294–296, 299

Index Ciliates, 173, 184, 186, 199, 201, 202, 204, 206, 208, 213, 221, 222, 237, 245, 292–294, 296–300, 318, 330, 334, 337 Cilium, 199, 207 Clades, 9, 14, 157, 158, 169, 171, 177, 192– 194, 204, 212, 257, 258, 260, 262, 264, 271, 281, 282, 285–287, 292–295, 299, 312, 315, 316, 318, 320–323, 328, 332, 334, 357, 366, 371, 402, 414 Cladistic systematics/cladistics, 9 Clonal multicells, 252 Closed-form organisms, 264 Clothing, 428, 430 Cognitive revolution, 439–443, 453 Cohesion-tension theory, 376 Complex multicellular organization, 169, 258 Conjugation, 234, 235, 245 Constructive neutral evolution, 4 Convective polymerase chain reaction, 62 Convergence, 6, 7, 252 Cooperative breeding, 435 CORR hypothesis, 187, 280 Corticata, 191, 193 C4 photosynthesis, 124, 400, 402–404 Creole languages, 438 Cro‐magnon man, 419 Crown eukaryotes, 211 Cryptista, 194, 205, 271, 287, 288, 292, 294, 295, 298 Cryptophyta, 193, 207, 285, 287, 288, 290–293 Cultural group selection model, 444 Cultural intelligence, 441 Cultural niche construction, 16, 446, 447, 453 Culture, 16, 112, 176, 265, 330, 419, 439–443, 445, 447, 450 Cumulative cultural evolution, 446 Cuticles, 257, 322, 349, 356, 359, 361–364 Cutin, 151, 362 C-value enigma, 218 Cyanidioschyzon merolae, 157, 178, 184, 284 Cyanophora paradoxa, 275 Cysts, 190, 237–239, 241, 353

D Death, 54, 188, 252, 261, 265, 371, 378, 425, 427, 442, 445, 446, 448, 453 Demographic shift, 447 Denisovans, 417, 422, 424, 457, 458 Desiccation tolerance (DT), 241, 348, 355, 357, 365, 369 Deuterostomia, 320, 332 Diaphoretika, 191–193 Dickinsonia, 312, 314

Index Dimethylsulphide, 272 Dinoflagellates, 124, 192, 210, 280, 281, 290, 292, 294, 295, 299, 300, 304, 353 Dinophysis, 300 Diploblastic animals, 317 Discoba, 189, 192, 193 Disproportion, 137, 410 DNA barcoding, 9 Domain shuffling, 235 Donated culture, 446 Doushantuo Formation, 148 Dunbar number, 441 Dynamins, 176, 184, 302 Dyneins, 161, 164, 188, 200, 205, 206

E Early European Modern Humans, 419 Eccrine glands, 428 Ecological inheritance, 16, 91 Ediacaran biota, 142, 211, 213, 312 Electrochemical gradients, 71, 75, 76, 78, 80, 81, 195 Electron bifurcation, 81, 122 Electronvolt, 106 Elysia, 297 Embryophyta, 262, 285, 286 Empathy, 435, 444 Encoded membrane, 75 Endosymbioses, 178, 270 Endurance running, 428, 429 Energy production per gene, 169 Eocytes, 158, 159, 167, 175, 177, 179 Epigenetics, 15, 262, 442 Epithelia, 206, 254, 261, 324–330, 332 Epulopiscium, 160 Equilibrium temperature/equilibrium state, 39, 142 Escherichia, 163, 170, 188, 198, 253 Euchromatin, 220 Euglenozoa, 187, 192, 193, 201, 202, 205, 207, 209, 285 Eukaryote core genome, 188 Eukaryotic flagellum, 199, 200, 205 Eukaryotic-signature genes, 178 Eumetazoa, 317, 318, 320, 322, 329, 331 Eusociality, 443 Euxinic oceans, 141, 147, 272 Evolutionary developmental biology (evo-devo), 15 Excavata, 112, 184, 187, 192, 193, 210, 236, 303 Extended phenotype, 17

485 Extracellular matrix (ECM), 255, 259, 324, 326, 330, 335, 337, 386 Extrinsic mitochondrial proteins, 185

F Fermentation, 85, 137, 139, 150, 172 Fertile Crescent, 424, 448 Flagellar roots, 203 Flagellin, 159, 195, 196 Floridean starch, 283, 303 Food production, 447–452 Foragers, 406, 427, 444, 447, 449–452 Forkhead box Protein2 (FOXP2), 438, 457, 458 Free standard energy of reaction, 76 Fur loss in humans, 427, 430

G Gametes, 187, 208, 234, 240, 243, 245–247, 255, 257, 264, 315, 351, 352, 367, 369 Gaoyuzhuang Formation, 212 Genders, 240, 245–247, 428, 431, 436, 452 Gene polymorphism, 4, 239 Genetic code, 4, 18, 65–71, 76, 90, 100 Genomes, 11, 65, 156, 235, 252, 271, 315, 355, 418, 472 Genotype, 4, 16, 90, 234–236, 240, 244, 256, 458 Geologic time scale, 49, 50 Geosyphon pyriforme, 148, 149 Germ line, 262, 264, 322 Giardia, 164, 204, 236 Glaucophyta/glaucophytes, 113, 271, 275, 281–284, 290, 294, 304, 305 Global glaciations, 39, 48, 49, 133, 141–144, 147 Global thermostat/planetary thermoregulation, 36–38 Gloeobacter, 74, 108 Glycogen, 294, 302–305 Gnatostomes, 336–339 Gorillas, 406, 430, 432, 452, 456 Granites, 34, 45, 47 Great Oxygenation Event (GOE), 85, 105, 130, 342 Green algae, 185, 213, 255, 257, 259, 285, 324, 364 Greenhouse effect, 36, 38, 48, 49, 141, 142 Guillardia theta, 289 Gut microbiome, 332 Gymnosperms, 368–373, 381, 387, 389

486 H Habitable zone, 30, 91, 100 Hagen-Poiseuille law, 374 Halobacteria, 111 Halobacterium salinarum, 198 Haptista, 194, 271, 292, 294, 295 Haptophyta, 193, 194, 207, 285, 288, 290–293, 305 HAR sequences, 457 Hatena arenicola, 298 Heliobacteria, 109, 111, 113, 116 Heterochromatin, 216, 220 Heterocysts, 136, 256 Hidden ovulation, 434 High‐speed throw, 408, 425, 426, 444, 447 Histones, 159, 160, 171, 175, 262 Hohle Fels Flute, 420 Homeohydry, 357, 359–364, 366, 375, 377, 391 Homeotic transcription factors, 328 Hominin taxonomy, 418 Hominoidea, 400, 402 Homo antecessor, 415–417 Homo cepranensis, 415 Homochiral, 56, 89 Homo erectus, 407, 412–414, 418, 424, 426, 429, 432, 433, 437, 439, 459 Homo ergaster, 412, 420, 424 Homo floresiensis, 414 Homo habilis, 403, 410–412, 414, 424 Homo heidelbergensis, 415, 416, 420, 423, 424 Homo neanderthalensis, 416–420, 422, 424, 438 Homoplasies, 6, 7 Homo rhodesiensis, 415 Homo sapiens, 400, 406, 413, 414, 417–420, 423–425, 430, 438, 447, 457, 458 Homo sapiens idàltu, 419 Hopanes, 105 Horizontal gene transfer (HGT), 8, 9, 70, 79, 80, 87, 90, 105, 115, 116, 120, 122, 125, 150, 151, 159, 178, 179, 189, 190, 198, 236, 240, 244, 270, 274, 296, 297, 335, 365 Hox/ParaHox genes, 320, 322, 328–330 Human accelerated region 1 (HAR 1), 456, 457 Human genome, 214, 216–218, 220, 330, 440, 456, 457 Human Genome Project, 456 Human races, 451, 458 Human social organization, 445, 451, 452, 454, 460 Humus, 378–380 Hunther‐gatherers, 423, 436, 447, 460 Huronian global glaciations, 141, 142, 144

Index Hydrogenosomes, 186, 187 Hydrologic cycle, 390, 391 Hydroxypropionate cycle, 121, 122, 124 Hyperthermophyly, 88, 90, 198

I Intensification, 447 Intestine, 56, 221, 316–319, 330–334, 340, 449 Intrinsic mitochondrial proteins, 185 Introns, 63, 174, 189, 214, 216–218, 221, 239, 456 Iron-sulfur cluster (ISC) machinery, 188 Isotopes, 41–45, 131, 132, 139, 145, 148, 365 Isotopic fractionation, 42, 43, 54, 131 Isua Greenstone Belt, 54, 103

K Kenyanthropus platyops, 410 Kinesins, 161, 164, 188, 189, 205 Kinetids, 203, 204, 207–211 Kinetoplast, 186, 208 Kinetosomes, 200, 201, 203, 204, 207, 208, 210, 350 Kin selection, 252, 443, 444 Kinship-independent cooperation, 435, 437, 443, 445 Kleptoplasts, 296, 300 Knuckle‐walking, 406, 407

L Lactose, 449 Laminarin, 294, 305 Large fission‐fusion society (LFFS), 451, 452 Last eukaryotic common ancestor(LECA), 165, 166, 188, 190, 191, 201, 209, 211, 222, 236, 258, 316 Last universal common ancestor (LUCA), 18, 72, 75, 79, 85, 87, 89, 158, 168 Leaves, 7, 37, 43, 48, 118, 172, 262, 264, 265, 360, 362, 363, 372, 377, 381–385, 389 Lignified cell walls, 380 Lignin, 151, 365, 369, 372, 373, 378–380 Lissencephaly, 457 Lokiarchaeota, 175–177, 179 Lomagundi event, 140 Lycopodium clavatum, 354

M Macroelements, 2 Macrotrophy, 331

Index Malawimonada, 192, 193 Mass‐independent fractionation of sulfur (MIFS), 131 Mastigont, 203 Mating types, 240, 245, 247 Matrotrophy, 350, 369 Meiosis, 189, 222, 234, 236–241, 243, 244, 247, 350, 351, 353 Meiosis block, 243 Meiospores, 241, 352 Meiotic sex, 19, 234, 236, 242, 245, 247, 262, 280 Membrane stereochemistry, 88 Memes, 442, 443 Menopause, 436 Metabarcoding, 9 Metal-enzymes, 82, 118 Metamonada, 164, 173, 187, 192, 193, 210, 294, 303 Meta‐phylogenomics, 9 Metazoa, 315, 316, 329 Methane photolysis, 139, 140 Methane photo-oxidation, 138, 141 Methanogenesis, 81, 106, 137, 167, 222 Methanotrophy, 59, 84, 85, 141, 222 Microbiomes, 332, 333 Microbiota, 17 Microcephalin (MCPH1), 457 Microcephaly, 457 Microelements, 2, 146 Microfilaments, 161, 188, 189, 191 Microtubule-organizing centers (MTOCTs), 203, 206, 207, 211 Microtubules, 161, 162, 164, 174, 188, 189, 200, 203–208, 210 Miller experiment, 55–57 Mitochondrial contact site and cristae organizing system (MICOS), 182, 183 Mitochondrial genome, 184–186, 315, 422 Mitochondrial Import and Assembly (MIA) machinery, 183, 185 Mitosis variants, 208, 210 Mitosomes, 186–188 Mnemiopsis leidyi, 322 Molecular clocks, 4, 13–14, 105, 147, 211–213, 272, 281, 314, 365, 370 Molecular paleontology, 421 Molecular phylogeny, 9, 18, 125, 164, 190, 273, 315, 318, 323, 336, 354, 357, 421 Monogamy, 431–433, 435, 452 Monophyletic groups, 9, 158, 192, 243, 365 Mousterian, 417, 419, 439, 442 mtDNA haplogroup L3, 422

487 Muller’s ratchet, 188, 235 Multi‐level society (MLS), 451, 452 Multiregional theory, 420 Mycorrhizas, 385–387 Myrionecta rubra, 298, 300 Myths, 441, 446, 458, 461 Myxozoa, 261, 263, 331 Myzocytosis, 292 Myzozoa, 294, 295

N Naegleria gruberi, 184, 189, 190 Native Americans, 425 Neanderthals, 400, 417, 419, 422, 430, 438–440, 457–459 Negentropy, 54 Nematostella, 322, 329 Neodarwinism, 4 Neomuran model, 170–173, 179, 180, 189 Neoproterozoic glaciations, 142, 143, 213 Nephrozoa, 320 Neutral theory of molecular evolution, 4 Niche construction, 16, 17, 91, 223, 253, 446, 447, 453, 460 Non-coding DNA, 215–217, 220, 221 Nostoc, 256, 260 Nucleation factors (NF), 161 Nucleomorph, 221, 288–290, 300 Nucleosynthesis, 24, 25, 41

O Obazoa, 194 Oldowan technology, 412, 415, 439 Olivine, 46 Open-form organisms, 264, 265 Operational Taxonomic Units (OTUs), 9 Opistokonta, 192–194, 202, 204, 209, 294, 315, 316 Orririn tugenensis, 406 Osmotic potential, 376 Out of Africa (OOA) theory, 420, 422 Oxygenic photosynthesis, 18, 101–103, 105, 116–120, 124, 134, 135, 137, 138, 140, 141, 146, 150, 272, 281

P Paleosoils, 106, 131 Paleozoic fluctuations of carbon dioxide, 384, 388 Pangenome, 156, 173, 189, 244, 315

488 Panspermia, 55 Parahoxozoa, 320, 328 Parallel evolution, 6, 459 Paramecium bursaria, 297 Paramylon, 294, 304 Paranthropus boisei, 403, 409 Paraphyletic groups, 9, 10, 158, 159, 314, 337, 366 Parsimony, 9 Parthenogenetic rotifers, 241, 242 Paulinella chromatophore, 273, 274, 276 P450 cytochromes, 151 Pediculus humanus, 429 Pee Dee Belemnite (PDB), 44 Peptidoglycan, 56, 159, 170, 196, 256, 260, 271, 273, 281, 282, 301 Peptidyl transferase, 63, 67, 70, 159 Peridinin, 290, 299 Peridotite, 46 Permo-Carboniferous coal accumulation, 380 Phagotrophy, 172, 173, 218, 222, 281, 331 Phanerozoic eon, 313 Phenetic systematics/phonetics, 5, 6 Phonemic diversity, 438 Phosphatidylinositol (PI), 179, 189 Phosphoenolpyruvate carboxylase (PEPC), 43, 402, 404 Photons, 106–108, 117, 118 Photosynthetic eukaryotes, 19, 87, 117, 121, 124, 144, 147, 148, 156, 180, 247, 348 Photosystems, 81, 106–120, 256, 285 Phototrophy, 100, 107, 110 Phragmoplast, 349 Phycobiliproteins, 112, 113, 272, 282, 284, 288, 290 Phycobilisomes, 112, 113, 119, 282–284, 288, 290, 304, 305 Phyletic tree of the human lineage, 424 Phylogenetic systematics/phylogenetics, 5–7, 9, 84, 89, 105, 115, 160, 165, 168, 169, 172, 175, 177, 178, 188, 190, 192, 193, 204, 221, 258, 287, 289, 322, 326, 328, 332, 336, 350, 365, 367, 371, 401, 414, 457, 458 Phylogenomics, 9, 81, 85, 87, 106, 116, 166, 172, 175, 177, 179, 180, 191–193, 271, 272, 287, 288, 292, 293, 295, 296, 320–322, 328, 350, 357, 365, 366, 370, 419, 421, 425 Physcomitrella, 281, 357, 360, 364 Pidgins, 438 Pilbara Craton, 54, 103, 212 Plantae, 8, 112, 192, 193

Index Plasmids, 174, 188, 235 Plasmodesmata, 259, 350, 404 Plasmodium, 184, 209, 217, 221, 253, 255, 296 Plastid divisome, 301, 302 Plastidial Phosphate Translocators, 275 Plastome, 271, 272, 295 Plesiomorphic, 9 Poaceae, 400, 402, 404 Podiates, 191, 193 Poikilohydry, 355, 357 Polar lipids, 73, 87 Polyphyletic groups, 9 Polysporangiophytes, 356, 357, 359, 361, 364, 365, 381, 388 Porphyra, 164, 283 Precision grip, 410, 411 Primary chloroplast, 271–282, 287, 291, 304 Primary cilium, 207 Primate phylogenetic tree, 401 Primates, 5, 9, 220, 400–402, 408, 409, 411, 425, 427–429, 431–435, 437, 441, 444, 446, 453, 456, 457 Prochlorophytes, 113 Projected coercive threat, 453 Protein import in secondary chloroplasts, 291 Protists, 9, 12, 17, 164, 173, 186, 190, 192, 193, 199, 201, 202, 205, 207, 208, 212, 214, 221, 237, 253, 315, 331, 353 Proton motive force (PMF), 77–83, 85, 108, 110, 111, 114, 115, 118, 119, 182, 195 Proton pumps, 68, 78, 80, 81 Protoribosomes, 70, 71, 87 Protostomia, 320, 332 Protoviruses, 70, 71, 87 Pteridophytes, 367, 368, 373, 388 Pthirus pubis, 429 Purple bacteria, 109, 111, 113–116, 121 Pyrenoids, 124, 125, 289 Pyrite, 39, 131, 132, 134, 135

Q Quorum sensing, 253

R Radiometric dating/radiometry, 42, 44 Reaction centre (RC), 107–109, 111–118, 120 Reactive oxygen species (ROS), 150, 237 Reciprocal cooperation, 443, 444, 446, 447 Red beds, 131, 132, 136 Reductive acetyl-coenzyme A/Wood– Ljungdahl cycle, 121, 122

Index Reductive citric acid cycle, 121–123 Reproductive cycle in land plants, 350, 351 Reproductive cycle/life cycle, 4, 24, 190, 201, 209, 221, 234, 238, 240, 242, 247, 252, 262, 275, 328, 335, 350–355, 359, 361, 364, 378, 387 Resurrection plants, 357 Reverse electron transport, 81 Rhizaria, 112, 192–194, 205, 271, 273, 285, 289, 293, 294 Rhodophyta/red algae, 113, 148, 164, 201, 208, 213, 260, 281–285, 288, 290, 292, 301, 304, 364 Rhodopsins, 100, 107, 109, 110 Rhynia gwynne-vaughanii, 356 Rhynie Chert, 356, 359 Riboswitches, 63 Ribozymes, 63, 65, 66, 90 Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO), 43, 122–125, 151, 282, 284, 402, 404 RNA world, 63 Rock weathering, 37, 149, 348, 384, 387–389 Roots, 89, 172, 177, 192, 193, 203, 204, 206, 207, 223, 264, 265, 316, 363, 364, 377, 378, 380–386, 388, 391, 404, 414, 449 Runaway ice‐albedo feedback, 143

S Saccharomyces cerevisiae, 157, 178, 210, 261, 263 Sahelanthropus tchadensis, 406 Savanna, 342, 400, 402, 403, 405–407, 409, 412, 427, 428, 433, 452, 460 Savanna theory, 407 Secondary chloroplasts, 221, 271, 272, 285–296, 300 Sec translocons, 72, 89 Sedimentary rocks, 34, 45, 54, 103–106, 130–132, 213, 312 Seeds, 9, 208, 357, 359, 360, 367, 369, 370, 381, 388, 404, 446, 447, 449 Selfish gene, 16, 235 Serial symbiosis, 294, 295 Serpentinization, 49, 59, 84, 139 Sex, 4, 19, 235, 236, 238, 239, 241, 242, 244–246, 257, 262, 280, 367, 428, 431, 434, 436 Silicate/carbonate cycle, 47, 388 Simons Genome Diversity Project, 422, 440 Singlet oxygen, 150

489 Sister clades/sister groups, 9, 89, 157, 158, 171, 177, 192, 194, 212, 263, 281, 286, 296, 299, 314, 324, 336, 357, 366, 424 Skeletal DNA hypothesis, 219, 221 Social rearing, 431, 435, 453 Sorting and assembly machinery (SAM), 182, 183, 185 Speciation, 243 Species definition, 243 Spindle polar bodies (SPBs), 207–211 Spliceosomal introns, 63, 174, 189, 217 Spliceosomes, 63, 69, 189 Sponge evolution, 253, 254 Sporopollenin, 151, 237, 351–354 Standard reduction potential, 76, 77, 103, 114, 115, 117 Starch, 125, 283, 284, 287, 294, 303–305, 370 Stem cells, 255, 350 Stem eukaryotes, 211 Sterans, 213 Stereoisomers, 56, 67 Sterols, 160, 179, 212 Stomata, 356, 357, 359–364, 375, 378, 382, 389, 391, 402 Storage polysaccharides in eukaryotes, 294, 302 Stramenopila, 186, 192, 204, 207, 209, 271, 285, 288, 290–293, 295, 305 Stramenopila, Alveolata and Rhizaria (SAR), 192–194, 293, 295 Strelley Pool Formation, 54, 103 Streptophyta/streptophytes, 276, 281, 285, 286, 349–351, 366 Stromatolites, 54, 103, 105 Substrate-level phosphorylation (SLP), 76, 81, 150, 186 Sulphur green bacteria, 109, 112–116, 122 Symbiosis, 17, 49, 180, 270, 271, 273–275, 280, 287, 288, 294–296, 298, 385–387 Symplast, 260, 350, 363 Synechocystis, 117, 125 Syngamy, 151, 189, 222, 234, 239, 240, 245, 247, 350, 351, 353, 354 Syntrophic models, 167

T Tag peptides, 276, 277, 291 Technological advance in human history, 440, 446 Tectonic activity, 32, 35, 37, 39–41, 136 Tectonic plates, 35, 36, 47

490 Tectonics, 32–35, 37–39, 41–43, 45–47, 130, 134, 136, 137 Telome theory, 382, 383 Testosterone, 428 Tetraether lipids, 88, 89 Thaumarchaeota, Aigarchaeota,Crenarchaeota and Korarchaeota (TACK), 158, 159, 175–178 Theory of mind, 435 Thermophoresis, 62, 82, 86 Thiomargarita, 160, 170 Thylakoids, 73, 79, 108, 117–119, 151, 180, 276, 277, 282–285, 288, 290, 291, 301 TOC-TIC machinery, 276 Tracheophytes, 365, 366, 369, 371, 373, 379, 384–386, 388–390 Transduction, 57, 100, 234 Transformations, 1, 82, 130, 234, 272 Translocase of the inner mitochondrial membrane (TIM), 183, 186 Translocase of the outer mitochondrial membrane (TOM), 182, 183, 185, 186, 192 Tree of life, 8, 18, 69, 89, 157, 158, 171, 172, 175, 192 Trichoplax, 326, 330, 331 Trilete mark, 351, 354 Triplet oxygen, 149, 150 Triploblastic animals, 317, 318 Tubulin, 161, 162, 164, 175, 188, 189, 201 Tunicates, 281, 334–336 Turkana boy, 414

U Undulipodium, 199

Index Unikonta, 190, 192, 193, 204 Uniparental mitochondrial transmission, 187 Uniparental transmission of plastids, 247 Uraninite, 131, 132 Urbilater, 332, 333

V Vein density, 389, 390 Vertebrates, 9, 10, 214, 234, 264, 325, 326, 328, 329, 332–337, 339, 340, 407, 430, 435, 458 Viridiplantae, 113, 260, 262, 281, 282, 284–286, 290, 303, 305 Volvox, 257, 264

W Warfare, 455 Water‐induced wrinkling, 429 Water oxidizing complex (WOC), 118, 120 Water photolysis, 134, 140 Water potential, 364, 375–378 Weapons, 425, 444, 452, 453 White-rot fungi, 379 White smoker, 59

X Xylem, 259, 359–365, 378, 379, 381, 389, 390

Z Zircons, 41, 44, 45, 47, 54, 130 Zosterophyllum shengfengense, 385