Phylogeny, molecular population genetics, evolutionary biology and conservation of the neotropical primates 9781634851657, 163485165X

Table of contents :
PHYLOGENY, MOLECULAR POPULATION GENETICS, EVOLUTIONARY BIOLOGY AND CONSERVATION OF THENEOTROPICAL PRIMATES
PHYLOGENY, MOLECULAR POPULATION GENETICS, EVOLUTIONARY BIOLOGY AND CONSERVATION OF THE NEOTROPICAL PRIMATES
Library of Congress Cataloging-in-Publication Data
CONTENTS
INTRODUCTION
Chapter 1: AN INTRODUCTION TO NEOTROPICAL PRIMATES
ABSTRACT
GENERAL INTRODUCTION TO NEOTROPICAL PRIMATES
STATUS OF NEOTROPICAL PRIMATES
ORGANIZATION OF BOOK
INTRODUCTION OF NEOTROPICAL PRIMATE FAMILIES
Callitrichidae
Cebidae
Atelidae
Ateles
Brachyteles
Alouatta
Lagothrix
Pitheciidae
Cacajao
Callicebus
Chiropotes
Pithecia
Aotidae
ACKNOWLEDGMENTS
REFERENCES
PHYLOGENY, MOLECULAR POPULATION GENETICS
AND PALEOPRIMATOLOGY
Chapter 2: THE PALEOBIOLOGY OF THE RECENTLY-EXTINCT PLATYRRHINES OF BRAZIL AND THE CARIBBEAN
ABSTRACT
INTRODUCTION
The “Giant” Subfossils of Brazil
The Endemic Caribbean Primates
LOCOMOTOR AND POSTCRANIAL ADAPTATIONS
The Brazilian Primates
The Caribbean Primates
CRANIODENTAL MORPHOLOGY AND DIET
The Brazilian Primates
The Caribbean Primates
BODY SIZE
EXTINCTION
The Caribbean
Brazil
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 3: GENETIC HETEROGENEITY AND EVOLUTIONARY DEMOGRAPHIC HISTORY OF THE ENDEMIC COLOMBIAN SAGUINUS LEUCOPUS (PRIMATES) BY MEANS OF DNA MICROSATELLITES
AND COALESCENCE METHODS
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Molecular Analyses
Population Genetics Analyses
RESULTS
Microsatellite Mutation Models
Effective Number Sizes
Gene Drift and Gene Flow in Populations of S. leucopus
Population Expansions in S. leucopus
When Did the Antioquia and Tolima S. leucopus Populations Diverge?
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 4: DIVERSITY OF CEBUS SPECIES FROM
THE SOUTHERN DISTRIBUTION OF THE GENUS
ABSTRACT
INTRODUCTION
THE SOUTHERNMOST CEBUS SPECIES
PHENOTYPIC AND GENETIC VARIABILITY
RESULTS AND DISCUSSION
AN INTEGRATIVE ANALYSIS OF CEBUS CAY
AND C. NIGRITUS DIVERSITY
ACKNOWLEDGMENTS
REFERENCES
Chapter 5:
GENETIC STRUCTURE, SPATIAL PATTERNS AND HISTORICAL DEMOGRAPHIC EVOLUTION OF WHITE-THROATED CAPUCHIN (CEBUS CAPUCINUS, CEBIDAE, PRIMATES) POPULATIONS OF COLOMBIA AND CENTRAL AMERICA BY MEANS OF DNA MICROSATELLITES
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Molecular Procedures
Population Genetics Molecular Analyses
Genetic Diversity and Natural Selection
Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow
Assignation and Genetic Structure Tests
Possible Historical Demographic Changes
Spatial Genetic Analyses
RESULTS
Gene Diversity and Natural Selection
Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow
Genetic Assignment and Genetic Structure
Historical Demographic Changes
Spatial Genetic Structure
DISCUSSION
Population Genetics of C. Capucinus
Molecular Systematics within C. Capucinus
ACKNOWLEDGMENTS
REFERENCES
Chapter 6: INVALIDATION OF THREE ROBUST CAPUCHIN SPECIES (CEBUS LIBIDINOSUS PALLIDUS, C.MACROCEPHALUS AND C. FATUELLUS; CEBIDAE, PRIMATES) IN THE WESTERN AMAZON AND ORINOCO BY ANALYZING DNA MICRO
SATELLITES
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Molecular Procedures
Population Genetics Molecular Analyses
Genetic Diversity and Natural Selection
Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow
Assignation and Genetic Structure Tests
Possible Historical Demographic Changes
Spatial Genetic Analyses
RESULTS
Gene Diversity and Natural Selection
Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow
Genetic Assignment and Genetic Structure
Historical Demographic Changes
Spatial Genetic Structure
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 7: IT IS MISLEADING TO USE SAPAJUS (ROBUST CAPUCHINS) AS A GENUS? A REVIEW OF THE EVOLUTION OF THE CAPUCHINS
AND SUGGESTIONS ON THEIR SYSTEMATICS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Molecular Procedures
Data Analyses
RESULTS
DISCUSSION
Why the Cebus Genus Should Not Substituted by the Sapajus Genus for the Robust Capuchins
The Evolution of the Cebus Genus
The Evolution of the Gracile Capuchins: They Began the History of the Current Capuchins
The Necessity of a New Systematics for the Cebus Genus
ACKNOWLEDGMENTS
REFERENCES
Chapter 8: SEX CHROMOSOMES AND SEX DETERMINATION
IN PLATYRRHINI
ABSTRACT
SEX DETERMINATION
SEX DETERMINING MECHANISMS
SEX CHROMOSOMES IN PRIMATES
PLATYRRHINI SEX CHROMOSOMES
GENES INVOLVED IN SEX DETERMINATION IN PRIMATES
REFERENCES
Chapter 9: CAN MITOCHONDRIAL DNA, NUCLEAR MICROSATELLITE DNA AND CRANIAL MORPHOMETRICS ACCURATELY DISCRIMINATE DIFFERENT AOTUS SPECIES (CEBIDAE)? SOME INSIGHTS ON POPULATION GENETICS PARAMETERS AND THE PHYLOGENY OF THE NIGHT MONKEYS
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Morphological Samples and Procedures
Molecular and Sample Procedures
Mitochondrial Sequences
Microsatellite Markers
Mitochondrial Phylogenetics Procedures
Microsatellite Statistical Analyses
RESULTS
Morphological Results
Mitochondrial Analyses
Microsatellite Analyses
DISCUSSION
Morphometrics, Molecular Population Genetics and Discrimination of Aotus Taxa
Phylogenetics and Systematics of Aotus
ACKNOWLEDGMENTS
REFERENCES
Chapter 10: PHYLOGENETIC RELATIONSHIPS OF PITHECIDAE AND TEMPORAL SPLITS IN REFERENCE TO CEBIDAE AND ATELIDAE BY MEANS OF MITOGENOMICS
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Molecular Procedures
Phylogenetics Procedures
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 11: MICROSATELLITE DNA ANALYSES OF FOUR ALOUATTA SPECIES (ATELIDAE, PRIMATES):
EVOLUTIONARY MICROSATELLITE DYNAMICS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Samples and Molecular Procedures
Population Genetics Analyses
Microsatellite Central Moments
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 12: WHICH HOWLER MONKEY (ALOUATTA, ATELIDAE, PRIMATES) TAXON IS LIVING IN THE PERUVIAN MADRE DE DIOS RIVER BASIN (SOUTHERN PERU)? RESULTS FROM MITOCHONDRIAL GENE ANALYSES AND SOME INSIGHTS IN THE PHYLOGENY OF ALOUATTA
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Molecular Procedures
Data Analysis
Molecular Population Analyses
Phylogenetic Analyses
RESULTS
Molecular Population Genetics
Phylogenetic Inferences
DISCUSSION
How Many Taxa of Red Howler Monkeys are in Peru?
Molecular Phylogenetic Insights into the Systematics of Alouatta
ACKNOWLEDGMENTS
REFERENCES
Chapter 13: HISTORICAL GENETIC DEMOGRAPHY AND SOME INSIGHTS INTO THE SYSTEMATICS OF ATELES (ATELIDAE, PRIMATES) BY MEANS OF DIVERSE
MITOCHONDRIAL GENES
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Molecular Procedures
Data Analysis
Molecular Population Analyses
Phylogenetic Analyses
RESULTS
Gene Diversity and Historical Demographic Evolutionin Ateles Taxa
Molecular Phylogenetic Inferences
DISCUSSION
Current IUCN Classifications and the Evolutionary Demographics of Ateles
New Insights on the Systematics of Ateles
ACKNOWLEDGMENTS
REFERENCES
CONSERVATION BIOLOGY
Chapter 14:
CAPUCHIN MONKEYS IN AMAZONIAN MANGROVE AREAS
ABSTRACT
INTRODUCTION
METHODS
Study Area
Survey
RESULTS
Mangrove Use by Capuchin Monkeys
Localities in Mangrove Suggested for Shelter Residents Groupsof Sapajus apella
Mangrove Areas with Resident Groups
Sapajus apella
Sapajus libidinosus
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 15: HOW DOES THE COLOMBIAN SQUIRREL MONKEY COPE WITH HABITAT FRAGMENTATION? STRATEGIES TO SURVIVE IN SMALL FRAGME
NTS
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
STUDY SITES DESCRIPTION
Tinigua National Park: Continuous Area
San Martin Area: Fragmented Area
PRIMATE DATA COLLECTION
Tinigua National Park: Continuous Area
San Martin Area: Fragmented Area
Data Analysis
RESULTS
Diet
Home Range and Daily Distance
Activity Budget
Group Size and Composition
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 16: CALLICEBUS ORNATUS, AN ENDEMIC COLOMBIAN SPECIES: DEMOGRAPHY, BEHAVIOR AND CONSERVATION
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Study Area
Primate Surveys
Data Analysis
Vegetation Analysis
RESULTS
Forest Fragment Edge Versus Interior Use by Groups of Callicebus ornatus
Fragment Size Effects on Group Size and Density Index
Diversity of Hectare Plots
Additional Observations
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 17: BIOGEOGRAPHY AND CONSERVATION OF THE PITHECIINES: SAKIS, BEARDED SAKIS AND
UACARIS (PITHECIA, CHIROPOTES AND CACAJAO)
ABSTRACT
1. INTRODUCTION
1. The Pitheciinae – Overview
2. RECENT TAXONOMIC REVISIONS
a. Chiropotes
b. Pithecia
c. Cacajao
3. GEOGRAPHIC DISTRIBUTIONS
a. River Basins and Elevation
b. Precipitation and Temperature
c. Forest Canopy Height and Net Primary Productivity
4. PITHECIINE DEMOGRAPHY
5. PITHECIINE ECOLOGY
a. Food Choice
b. Seasonality in Resource Use
c. Ecological Effects of Sympatry among Pitheciines
PITHECIINE CONSERVATION ISSUES
a. Forest Loss and Fragmentation
b. Hunting, Climate Change, and Disease
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
Chapter 18: STATE OF PRIMATES IN NORTHEASTERN PERU: THE CASE OF THE PACAYA-SAMIRIA
NATIONAL RESERVE
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
Sites Used for Monitoring of Primates in the Samiria River
Major Habitat Types and Flood Levels
METHODOLOGY USED
Terrestrial Transects
RESULTS
Population Density of Primates
Population Trends of Primates in the Samiria River
Density and Population Trends by Species
Saguinus Illigeri
Saimiri Boliviensis
Pithecia Isabella
Sapajus Macrocephalus
Cebus Yuracus
Alouatta Seniculus
Lagothrix Lagothricha Poeppigii
Interspecific (Lotka Volterra) Competition
DISCUSSION
Population Density of Primates in the Samiria River
Population Trends of Primates in the Samiria River
ACKNOWLEDGMENTS
REFERENCES
Chapter 19: CURRENT KNOWLEDGE ON PRIMATE DISTRIBUTION
AND CONSERVATION IN BOLIVIA
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
CALLITRICHIDAE
CEBIDAE
AOTIDAE
PITHECIIDAE
ATELIDAE
CONSERVATION OF BOLIVIAN PRIMATES
CONCLUSION
ANNEX 1. GAZETTEER OF PRIMATE DISTRIBUTION LOCALITIESIN BOLIVIA
ACKNOWLEDGMENTS
REFERENCES
ABOUT THE CONTRIBUTORS
INDEX
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ANIMAL SCIENCE, ISSUES AND RESEARCH

PHYLOGENY, MOLECULAR POPULATION GENETICS, EVOLUTIONARY BIOLOGY AND CONSERVATION OF THE NEOTROPICAL PRIMATES

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ANIMAL SCIENCE, ISSUES AND RESEARCH

PHYLOGENY, MOLECULAR POPULATION GENETICS, EVOLUTIONARY BIOLOGY AND CONSERVATION OF THE NEOTROPICAL PRIMATES

MANUEL RUIZ-GARCIA AND

JOSEPH MARK SHOSTELL EDITORS

New York

Copyright © 2016 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected]. NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Names: Ruiz-Garcia, Manuel, editor. | Shostell, Joseph Mark, editor. Title: Phylogeny, molecular population genetics, evolutionary biology, and conservation of the neotropical primates / editors, Manuel Ruiz-Garcia and Joseph Mark Shostell (Professor Titular-Catedrático, Coordinador Unidad de Genética, Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá-Colombia, and others). Description: Hauppauge, New York : Nova Science Publishers, 2016. | Series: Animal science, issues and research | Includes index. Identifiers: LCCN 2016014507 (print) | LCCN 2016024447 (ebook) | ISBN 9781634851657 (hardcover) | ISBN 9781634852043 (H%RRN) Subjects: LCSH: Primates--Latin America--Genetics. | Primates--Evolution--Latin America. | Primates--Latin America--Phylogeny. Classification: LCC QL737.P9 P447 2016 (print) | LCC QL737.P9 (ebook) | DDC 599.8098--dc23 LC record available at https://lccn.loc.gov/2016014507

Published by Nova Science Publishers, Inc. † New York

CONTENTS

Introduction Chapter 1

1 An Introduction to Neotropical Primates Joseph M. Shostell and Manuel Ruiz-Garcia

Phylogeny, Molecular Population Genetics and Paleoprimatology Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

The Paleobiology of the Recently-Extinct Platyrrhines of Brazil and the Caribbean Siobhán B. Cooke, Justin T. Gladman, Lauren B. Halenar, Zachary S. Klukkert and Alfred L. Rosenberger Genetic Heterogeneity and Evolutionary Demographic History of the Endemic Colombian Saguinus leucopus (Primates) by Means of DNA Microsatellites and Coalescence Methods Manuel Ruiz-García, Pablo Escobar-Armel, Norberto Leguizamón and Joseph Mark Shostell Diversity of Cebus Species from the Southern Distribution of the Genus Mariela Nieves and Marta Dolores Mudry Genetic Structure, Spatial Patterns and Historical Demographic Evolution of White-Throated Capuchin (Cebus Capucinus, Cebidae, Primates) Populations of Colombia and Central America by Means of DNA Microsatellites Manuel Ruiz-García and María Ignacia Castillo Invalidation of Three Robust Capuchin Species (Cebus libidinosus Pallidus, C. macrocephalus and C. fatuellus; Cebidae, Primates) in the Western Amazon and Orinoco by Analyzing DNA Microsatellites Manuel Ruiz-García, María Ignacia Castillo, Kelly Luengas-Villamil and Norberto Leguizamón

3 39 41

91

115

135

173

vi Chapter 7

Contents It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus? A Review of the Evolution of the Capuchins and Suggestions on Their Systematics Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil

Chapter 8

Sex Chromosomes and Sex Determination in Platyrrhini Eliana R. Steinberg and Marta D. Mudry

Chapter 9

Can Mitochondrial DNA, Nuclear Microsatellite DNA and Cranial Morphometrics Accurately Discriminate Different Aotus Species (Cebidae)? Some Insights on Population Genetics Parameters and the Phylogeny of the Night Monkeys Manuel Ruiz-García, Adriana Vallejo, Emily Camargo, Diana Alvarez, Norberto Leguizamon and Hugo Gálvez

Chapter 10

Chapter 11

Chapter 12

Chapter 13

Phylogenetic Relationships of Pithecidae and Temporal Splits in Reference to Cebidae and Atelidae by Means of Mitogenomics Manuel Ruiz-García, Geven Rodríguez, Myreya Pinedo-Castro and Joseph Mark Shostell Microsatellite DNA Analyses of four Alouatta Species (Atelidae, Primates): Evolutionary Microsatellite Dynamics Manuel Ruiz-García, Pablo Escobar-Armel, Marta Mudry, Marina Ascunce, Gustavo Gutierrez-Espeleta and Joseph Mark Shostell Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon is Living in the Peruvian Madre de Dios River Basin (Southern Peru)? Results from Mitochondrial Gene Analyses and Some Insights in the Phylogeny of Alouatta Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro and Gustavo Gutierrez-Espeleta Historical Genetic Demography and Some Insights into the Systematics of Ateles (Atelidae, Primates) by Means of Diverse Mitochondrial Genes Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel, Geven Rodríguez and Gustavo Gutiérrez-Espeleta

209

269

287

345

369

395

435

Conservation Biology

477

Chapter 14

Capuchin Monkeys in Amazonian Mangrove Areas R. R. Santos, R. G. Ferreira and A. Araujo

479

Chapter 15

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? Strategies to Survive in Small Fragments Xyomara Carretero-Pinzón, Thomas R Defler and Manuel Ruiz-Garcia

491

Contents Chapter 16

Chapter 17

Chapter 18

Chapter 19

vii

Callicebus ornatus, an Endemic Colombian Species: Demography, Behavior and Conservation Xyomara Carretero-Pinzon and Thomas R. Defler

507

Biogeography and Conservation of Pitheciines: Sakis, Bearded Sakis and Uacaris (Pithecia, Chiropotes and Cacajao) Marilyn A. Norconk and Sarah A. Boyle

521

State of Primates in Northeastern Peru: The Case of the Pacaya-Samiria National Reserve Pablo Puertas, Richard Bodmer and Miguel Antúnez

551

Current Knowledge on Primate Distribution and Conservation in Bolivia Heidy Lopez-Strauss, Robert B. Wallace, Nohelia Mercado and Zulia R. Porcel

575

About the Contributors

641

Index

651

INTRODUCTION

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 1

AN INTRODUCTION TO NEOTROPICAL PRIMATES Joseph M. Shostell1 and Manuel Ruiz-García2 1

Math, Science, and Technology Department, University of Minnesota Crookston, Crookston, MN, US 2 Laboratorio de Genética de Poblaciones Molecular-Biologia Evolutiva, Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC., Colombia

ABSTRACT The Neotropics contains the greatest abundance and diversity of primate species of any bio-region in the world. They make up an impressive and varied assemblage of species, from the small pigmy marmoset weighing one hundred grams, to the woolly spider monkeys (muriquis) tipping the scale at 10-14 kg. Some species in the group, such as the bearded capuchin, show signs of high intelligence evidenced by their use of primitive tools to open nuts and fruit, and many of these species are flagships whose very presence is crucial for the dispersal of seeds and maintenance of primary forests. Unfortunately, a large percentage of Neotropical primate species is threatened or endangered due to various anthropogenic activities including deforestation, illegal hunting and wildlife trade, mining, and road construction. Moreover, there is a general paucity of data pertaining to this group because their habitats can be difficult to access and the sheer expansiveness of the Neotropical area. Here, we present new research findings from thirty-eight of the world’s leading Neotropical primate scientists in order to bridge this informational gap. Specifically we provide up to date biological, molecular, conservation, and phylogenic information on many of these poorly understood, yet amazing creatures. It is our intention that this new information will be used as a resource by the novice and professional alike in order to improve society’s understanding of Neotropical primates and to help protect them long into the future.

Keywords: primates, Neotropics, phylogeny, population genetics, evolutionary biology and conservation

4

Joseph M. Shostell and Manuel Ruiz-Garcia

GENERAL INTRODUCTION TO NEOTROPICAL PRIMATES He looked down at the large seed and then reached for the stone next to the anvil. It was a large stone, one he had selected for the larger seed. Using both hands he raised it high up and then quickly slammed it down on the seed. The capuchin repeated the behavior until the Attalea seed cracked opened...

This book discusses the group of primates referred to as Platyrrhini (flat nosed), also known as Neotropical primates or new world monkeys. Besides having relatively large brains and being quite clever, they have complex social systems, good grasping ability and many possess prehensile tails that support their expert climbing ability. Some, as noted above, are known for their use of tools. Their diversity in form and color is impressive and ranges for example from a small brownish-gold pigmy marmoset of 100 grams to a woolly spider monkey (Brachyteles arachnoides) in excess of 14 kg. The Neotropics is the most taxonomic rich terrestrial system in the world and covers an expansive area including Mexico, the Caribbean, Central America, and South America. Within this area we find roughly 34% of Earth’s primate diversity and some areas of the Neotropics such as the Western Amazon have up to 40% (Mittermeier and Richardson, 2013) of its mammalian biomass (save flying mammals) consisting of primates alone (Janson and Emmons, 1990). Primate diversity, including richness and evenness, varies geographically with precipitation, latitude, forest cover, and anthropogenic factors (Peres and Janson, 1999, Redford, 1992). They are mostly arboreal and, given their frugivorous diets, they are key dispersal agents for the plant communities in which they live (Chapman, 1989). Levey et al. (1994) suggested that approximately 80% of Neotropical plants rely on primates and other frugivorous animals. As a consequence of this diet and mobility, their presence affects forest regeneration and helps forests to maintain a rich community structure (Nunez-Iturri and Howe, 2007). Thus, primates are inexplicably linked to forest ecosystem health and the ability of the forest to maintain important ecosystem services such as carbon sequestration. At a time when atmospheric carbon dioxide emissions are ever increasing and there is a concomitant rise in global temperatures, the preservation of tropical rain forests is paramount. Similar to the canary in the coal mine, the presence of primates indicates good ecosystem health. They are an important part of the food chain as well as evidenced by them being targeted by many carnivores. Moreover, primates have also been used as valued flagships for educational issues regarding the Neotropics. The general public seems to respond more to primates than to a general call for conserving tropical rain forests, perhaps because monkeys have many similar features as to our own and are closely related to us genetically compared to non-primate taxa. Similarities in physiology also make non-human primates ideal models for better understanding human health and designing vaccinations for various tropical diseases such as malaria. Of course this benefit to biomedical research is also a threat to non-human primate survival and will be discussed in more detail later along with hunting, and live capture.

An Introduction to Neotropical Primates

5

STATUS OF NEOTROPICAL PRIMATES Neotropical primates are threatened by many factors, the main categories being habitat loss/degradation, hunting, and live capture. Recent evidence presented by Kim et al. (2015) claims that there is a rise in world deforestation rates, a contradiction to the United Nations Food and Agriculture Organization’s most recent report. The losses of tropical rain forests, where the world has its greatest terrestrial biodiversity and where Neotropical primates live, is staggering with Brazil alone losing about 2.25 million hectares per year (Kim et al., 2015). Both large-scale commercial logging and small-scale slash and burn techniques convert healthy primary forests into agricultural and ranch areas (Mittermeier et al., 1989). Besides denuding an area of trees and plummeting diversity in that area, a once continuous forest becomes fragmented isolating species and disrupting their dispersal routes. Building of roads, expanding of cities, development of mineral mines, construction of hydroelectric dams, are all examples of anthropogenic activities that degrade primate habitat. Hunting poses another major threat to primates. Primates are hunted for food, as agricultural pests, or in some cases just for sport. More often, hunters prefer to target larger species (>2 kg) (Bodmer et al., 1997) and also use the carcasses of primates as bait to lure in and catch jaguars and other predators. Primates are also threatened by live capture. They are kept alive to support biomedical research, such as for the development of new vaccines against malaria and other tropical diseases. Constant demand also comes from zoos and the pet industry. Legal and illegal trafficking occurs to support these demands. Primates are subjected to a number of smaller threats which individually may seem to be inconsequential, but collectively, are dangerous to their constitutions. For instance, disturbance by domestic animals and introduction of invasive species can be detrimental. In the Caribbean for example, the presence of invasive species is thought to have played a significant role in loss of primates. Finally, there are also biological limitations of primates and natural environmental variability. For example, reproduction rates tend to be low in larger species, thus making it difficult for species to adapt to rapid environmental change. Interestingly, environmental changes are hypothesized to be the cause of extinctions of platyrrhines that were in Patagonia (Kay, 2015). These threats, individually or synergistically, are real and have worsened the status of many Neotropical primates as evidenced by 52% of all Neotropical primate species officially recognized as threatened, endangered, or critically endangered on the IUCN Red List (Mittermeier and Richardson, 2013). The latter part of this book (Chapters 13-19) discusses conservation strategies to help protect Neotropical primates.

ORGANIZATION OF BOOK Although there is some overlap, the topics of this book are divided into two general sections. The first one includes chapters two through 13 and focuses on phylogeny, molecular population genetics, and evolutionary biology. The second section includes chapters 14 through 19 addressing topics of conservation. Inclusive of both sections there are several different types of studies. Some for example take a panoramic approach and cover topics linking together most or all of the Platyrrhini. These include a comparison of extinct and extant communities (Chapter 2), a description of sex chromosome systems (Chapter 8), and

6

Joseph M. Shostell and Manuel Ruiz-Garcia

estimations of temporal splits supported be mitogenomic analyses (Chapter 10). Other studies focus on an individual species across one or multiple countries (Chapters 3, 5, 15, and 16) or on multiple species within one or more genera (Chapters 4, 6, 7, 8, 11, 12, 13, and 14) or even on all taxa within a family (Chapter 9). Finally, the highest scale-level of study discusses all Neotropical primates within a significantly large geographical area such as a preserve (Chapter 18) or country (Chapter 19). All 23 primates distributed in Bolivia are mentioned in Chapter 19.

INTRODUCTION OF NEOTROPICAL PRIMATE FAMILIES The systematics of Neotropical primates is complex and controversial among primatologists and consequently there are continuous revisions of classifications in some taxonomic groups. Certainly, the phenotypic similarity among howler monkey species (Alouatta genus) or the high variability within species of capuchins, for instance can make systematics very challenging. We have also seen a steady increase in the number of species with the use of the phylogenic species concept (rather than the biological species concept) and with the discovery of new taxa in museums and the wild (Groves 2001, Mittermeier 1982, Rylands et al., 2012). Based on the work of Mittermeier and Richardson (2013) we currently recognize five Neotropical primate families (Aotidae, Atelidae, Callitrichidae, Cebidae, and Pitheciidae), 20 genera, and 204 species/subspecies (Mittermeier and Richardson, 2013). Therefore, this introduction acknowledges these taxa and the morphological criteria used to validate them. However, molecular data are more consistent with the existence of only three families (Atelidae, Cebidae and Pitheciidae; see chapter 10 of this volume and Schneider and Sampaio, 2015). With the collaborative help of many primatologists, native peoples, and universities, this book covers approximately 60% of Platyrrhini taxa and discusses each of the families and many of their genera in detail. We hope the newly gathered data presented here, including microsatellite and mitochondrial DNA data, will help resolve some of the taxonomic issues regarding Neotropical primates. The application of molecular techniques has greatly enhanced our knowledge of primate systematics and evolution over the last two decades. Today, different than thirty years ago, we can rely on taking small, non-invasive tissue samples from specimens, and more rapidly and less ambiguously compare multiple taxa. Also, the availability of high throughput next generation sequencing has made it easier to process samples, obtain results, and consequently, increase the number of genetic studies focusing on Neotropical primates (Lynch Alfaro, 2015). The next section gives an overview of each of the five Neotropical primate families.

Callitrichidae The Callitrichidae (Chapters 3, 8, 18, and 19) is one of five Platyrrhine families and contains 7 genera, 43 species and 62 taxa. Four of the genera (Callithrix, Cebuella, Callibella, and Mico) represent the marmosets (Figure 1) and the remaining three genera (Saguinus, Leontopithecus, and Callimico) represent the tamarins (Figure 2).

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Figure 1. Marmosets photographed by M. R-G: A) Cebuella pygmaea sampled near Nuevo Rocafuerte at the Napo River in the Ecuadorian Amazon in 2013. B) Cebuella pygmaea in the Quistococha Zoo in Iquitos (Peru) in 2010. C) Mico chrysoleuca sampled at the Aripuana River (Brazil) in 2012. D) A Mico argentata that lightly nibbled the neck of M. R-G near the Tapajos River (Brazil) in 2005. E) Mico melanura in Chuchini near the Ibare River (Bolivia) in 2003.

Figure 2. Tamarins photographed by M. R-G: A) An indigenous girl with her pet Saguinus mystax in Contamaná near the Ucayali River in the middle of the Peruvian Amazon in 2010., B) Saguinus fuscicollis weddelli in Santa Cruz de la Sierra (Bolivia) in 2010. C) Saguinus tripartitus sampled near the Napo River in Ecuador in 2013. D) A couple of Saguinus fuscicollis lagonotus at the Belem market in Iquitos in 2002.

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Nevertheless, the genus Callibella seems to not be supported as a full genus by molecular analysis (Schneider et al., 2012). Marmosets and tamarins are much smaller than other Platyrrhines, ranging from approximately 110 to 700 g, even smaller than the squirrel monkey (Cebidae family). Different from other Platyrrhines, they have claws instead of nails at the ends of all their digits save the distal phalanx of the big toe in each foot. The claws are useful for accessing sap, a staple of their diet. Another distinct difference is the number of molars. Callitrichidae have two rather than three molars on each side of the mandibular bones and in the maxillae. Similar to other primates they have longer hind limbs than forelimbs, and unlike many other primates that have non-prehensile tails. The overall distribution range of Callitrichidae extends from Costa Rica, through Bolivia, and into Southern Brazil, but the distributions of marmosets and tamarins rarely overlap. Callitrichidae primates are principally arboreal, diurnal, and omnivorous, subsisting on sap, small animals (frogs, spiders, and lizards), flowers, and fruit. Due to their light weight they can utilize the top layers of the forest canopy staying clear of large predators. Socially this family is complex. They have a flexible mating system exhibiting monogamy, polygyny and polyandry and they engage in cooperative breeding (Rylands, 1996). Most species in this family have twins and gestation is roughly 140-145 days. Their small size, quickness, and presence in the upper canopy make them challenging to study. A total of 14 species are either critically endangered, endangered, or vulnerable. Their threats, are common to those of other Neotropical primates and include deforestation, general habitat degradation, and live collection for biomedical research. In Chapter 3, Ruiz-García et al., discusses the endemic Colombian Saguinus leucopus (Günther, 1877) and provides population genetics findings from blood and hair sampled from 47 individuals. This particular taxon has the smallest distribution range of any known Saguinus species and there is scarcely any molecular data published for it (Ruiz-Garcia et al., 2014). Currently, Saguinus leucopus is classified as endangered by IUCN and is in appendix 1 of CITES. There are no protected areas for this species except for one small preserve. However it is interesting to note that this endemic Colombian species is one of the Neotropical primate species which has managed to adapt and live within the cities. The same has been observed for Aotus lemurinus in Manizales (Colombia), Saguinus bicolor and Saimiri spp in Manaus (Brazil), different species of Callithrix in diverse Atlantic Brazilian cities (such as Vitoria or Rio de Janeiro) and the brown howler monkey (Alouatta clamitans) in the Brazilian city of Porto Alegre. Taxonomy of species in Callitrichidae family (marmosets and tamarins) Kingdom: Phylum: Class: Order: Family: Genera: Species:

Animalia Chordata Mammalia Primate Callitrichidae Callithrix, Cebuella, Callibella, Mico, Saguinus, Leontopithecus, and Callimico Callibella humilis (Van Roosmalen et al., 1998), Callithrix aurita (Geoffroy, 1812), Callithrix flaviceps (Thomas, 1903), Callithrix geoffroyi (Geoffroy, 1812), Callithrix jacchus (Linnaeus, 1758), Callithrix kuhlii (Coimbra-Filho, 1985), Callithrix penicillata (Geoffroy, 1812), Cebuella pygmaea (Spix,

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1823), Leontopithecus caissara (Lorini and Persson, 1990), Leontopithecus chrysomelas (Kuhl, 1820), Leontopithecus chrysopygus (Mikan, 1823), Leontopithecus rosalia (Linnaeus, 1766), Mico acariensis (Van Roosmalen et al., 2000), Mico argentatus (Linnaeus, 1771), Mico chrysoleucus (Wagner, 1842), Mico emiliae (Thomas, 1920), Mico humeralifera (Geoffroy, 1812), Mico intermedius (Hershkovitz, 1977), Mico mauesi (Mittermeier, et al, 1992), Mico leucippe (Thomas, 1922), Mico manicorensis (Van Roosmalen et al., 2000) Mico marcai (Alperin, 1993), Mico melanurus (Geoffroy, 1812), Mico nigriceps (Ferrari and Lopes, 1992), Mico rondoni (Ferrari et al., 2010), Mico saterei (Silva Jr. and Noronha, 1998), Saguinus bicolor (Spix, 1823), Saguinus fuscicollis (1823), Saguinus geoffroyi (Pucheran, 1845), Saguinus imperator (Lönnberg, 1940), Saguinus inustus (Schwarz, 1951), Saguinus labiatus (Geoffroy, 1812), Saguinus leucopus (Güunther, 1877), Saguinus martinsi (Thomas, 1912), Saguinus mystax (Spix, 1823), Saguinus melanoleucus (Ribeiro, 1912), Saguinus midas (Linnaeus, 1758), Saguinus niger (Geoffroy, 1803), Saguinus nigricollis (Spix, 1823), Saguinus oedipus (Linnaeus, 1758), Saguinus tripartitus (Milne-Edwards, 1878).

Cebidae The Cebidae family (capuchins and squirrel monkeys) contains 2 genera (Cebus and Saimiri), 21 species, and 35 taxa (chapters 4-7, 10, 14, 15, 18, 19). This is a diverse and complex family that has the distinction of having a rather confusing and somewhat controversial taxonomy. New mitochondrial data collected from over 450 capuchin monkeys (Ruiz-García al., Chapter 7) supports the removal of Sapajus as a genus and thus, as noted above, recommends the existence of two rather than three genera, in disagreement with early studies (Silva 2001 and 2002, Lynch Alfaro et al., 2012). At the taxonomic level of species, the authors recognize the value of the phylogenetic species concept, but also caution against its indiscriminate use leading down the path of taxonomic inflation (Zachos et al., 2013). Together, chapters 4, 5, 6, and 7 offer genetic data on 944 capuchin monkeys. The Cebidae are divided unequally into two groups, capuchins (Cebus) (Figure 3) and the smaller squirrel monkeys (Saimiri) (Figure 4). It is not our intention to discuss nor describe all species in this family in detail, rather here we provide an overview of each group beginning with the capuchins. Capuchins have a wide variety of names tied to their outward appearance or to their behavior. Take for example, the bearded capuchin (Cebus apella libidinosus), the white fronted capuchin (Cebus albifrons), or the large headed capuchin (Cebus apella macrocephalus) which reflect their appearance. Some capuchins have alternative common names that reflect their behavior and food preference. For example, the large headed capuchin is also referred to as maicero or corn eater in Colombia. Species in the genus Cebus are considered medium-sized primates and range in size from approximately 2.5 to 5 kg (Fragaszy et al., 2004). For the tufted capuchin Cebus apella, Leigh (1994) reported males weighing as much as 6.1 kg compared to females of 3.2 kg, a clear sign of sexual dimorphism.

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Figure 3. Capuchins photographed by M. R-G: A) A Cebus albifrons and his friend: a dog. Photo taken near from Puyo in the Ecuadorian Amazon in 2011. B) A Cebus apella macrocephalus sampled in the Napo River (Peru) in 2003. C) A Cebus albifrons sampled in the Ucayali River (Peru) in 2002. D) A pair of Cebus capucinus sampled in Honduras in 2015. E) A Cebus apella cay (= libidinosus) sampled in Puerto Suárez (Santa Cruz Department; Bolivia) near to the frontier with Brazil in 2010.

Figure 4. Squirrel monkeys: A) A little Saimiri boliviensis peruvensis lost by its mother and adopted in an Indigene community (Bretaña; Puhinauva Channel; Ucayali River; Peru) in 2003. This specimen showed a coat pattern typical of S. boliviensis peruvensis but its mitochondrial DNA was typical of S. sciureus macrodon. B) An adult specimen of Saimiri boliviensis peruvensis at the Centro de Primatologia in Iquitos (Peru) in 2002. C) A mother and her baby (Saimiri sciureus macrodon) below to be sampled in the Ecuadorian city of Coca very near to the Napo River in 2013.

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As an example of size, Cebus apella reach on average a height of approximately 440 mm for males and 390 mm for females (Hakeem et al., 1996). Sexual maturity is reached between 5.5-8 years and 6-10 years in males and females respectively and they tend to be long lived (Hakeem et al., 1996). Capuchins have prehensile tails but their grasping ability is not as developed as the tails of spider monkeys and other atelids. Their thick molars assist them in the mastication of their food, which for Cebus apella in a semideciduous forest of Brazil, may consist of fruit pulp (53.9%), seeds (16%), flowers (11.1%), leaves and new shoots (6.3%), roots (1.5%), and corn (13.9%) (Galetti and Pedroni, 1994). Capuchins, widely distributed in the Neotropics, and given their diet, are key seed dispersal agents for the forest communities they inhabit (Wehncke et al., 1994; Wehncke and Domíguez, 2007). They are one of the most common primates of the Neotropics capable of adapting to multiple forest types (Mittermeier and Roosmalen, 1981). Cebus apella, for example, has a distribution that extends from the Eastern Andes Cordillera of Colombia to approximately 27 degrees south in Brazil, a distribution larger than any other Neotropical primate species (Chapter 6). Capuchin group size, density, and home range size vary with habitat. White-throated capuchins (Cebus capucinus) which have the most northern distribution of all capuchin species, live in groups of 10-20 (Chapter 5) and have densities of 18-30 individuals per kilometer. Home ranges for Cebus apella vary from less than 0.2 km2 to up to 9 km2 (Spironello 2001; Terborgh and Janson, 1983). Their group dynamics are sophisticated with evidence of coordinated group movement (Boinski, 1995) and mobbing behavior against some predators (Chapman, 1986). Perry’s work (2011) suggests capuchins also have social traditions and engage in social learning. Furthermore, there are many studies that document their use of tools which is a clear sign of their intelligence. We observed their intelligence in our own study as well. While Dr. Manuel Ruiz-Garcia (M.R-G) was trying to take a hair sample from a capuchin (Cebus albifrons) with a pair of tweezers the monkey snatched the tool and began to pluck at the hairs of M.R. The roles had suddenly reversed and the capuchin had become the researcher! Threats to capuchins includes biomedical research, deforestation, and being targeted as agricultural pests. In support of conservation measures, Rodrigues dos Santos et al., (Chapter 14) evaluates the use of mangrove areas in Northeastern Brazil as refuge sites for capuchins undergoing threats of habitat loss and subjected to hunting (Figure 5). Saimiri (squirrel monkeys) are much lighter and smaller than capuchins with heights about 300 mm and weights roughly 0.90 to 1.0 kg with males slightly heavier than females (Defler, 2010). They have a golden-orange color on their back, and distal parts of arms and legs whereas the rest of their body is mostly grey, brown, or black. One of their distinguishing features is the white mask around their eyes (Figure 6). They are distributed across Central America and parts of South America (Groves, 2004) and can reach some of the highest densities for Neotropical primates. The Pacaya National Reserve can have squirrel monkey densities up to almost 73 ind/km2, significantly higher than any other primate. Squirrel monkeys are shorter lived than capuchins and typically have a life span of 20 years and live in large groups (Boinski, 1987). As Boinski (2005) highlights, squirrel monkeys have a variety of species dispersal patterns, probably greater than found in any other primate species. For example, S. sciureus males spend most of their life alone as peripheral individuals whereas S. oerstedii males of the same cohort tend stay together (Boinski et al., 2005).

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Figure 5. Many medium and large size Neotropical primates are hunted for food by native communities. A smoked Cebus apella to be consumed in Rivera Alta (Beni River; Bolivia) in 2003. The smoking helps the meat to be preserved for many days in the warm climate of the jungle.

Figure 6. Squirrel monkeys: A) A Saimiri sciureus macrodon sampled in an Indigene community in the Napo River (Peru) in 2003. B) A Saimiri sciureus macrodon sampled in the Amazon River, Colombia in 2002. C) A Saimiri boliviensis boliviensis in the Santa Cruz Department (Bolivia) in 2010.

Their smaller size are both a benefit and detriment in regards to predation. Bird predators are a particular risk for them (Boinski, 1987), but the success of avian predators is a function

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of forest cover (Boinski et al., 2003). Also, given their diminutive size humans rarely hunt them for food. What is considered as an anti-predatory strategy, all pregnant squirrel monkeys within a group birth within a single week (Boinski, 1987). Similar to other Neotropical primates, squirrel monkeys are threatened by habitat loss and fragmentation, but they seem to be habitat generalists and have been observed in both disturbed and undisturbed sites. Within Chapter 15, Carretero-Pinzón discusses how the Colombian squirrel monkey copes with habitat fragmentation. In the last years, with the indiscriminate application of the Phylogenetic species concept, many “supposed” species of Saimiri have been defined (from 5 to 12 different taxa). Nevertheless, Ruiz-García et al., (2015) showed that the molecular data only detected 1 to 3 full species. This is one of the Neotropical primate taxa easily sampled from a genetics point of view due to their large groups and because they are easily attracted to human food. For example, in Manuel Antonio National Park (Costa Rica), many groups of Saimiri oerstedii citrinellus daily move to the hotels around five in the afternoon to receive fruit from tourists. This tolerance to humans allowed M.R.-G to quietly approach two squirrel monkeys and capture them while they ate. He gently held them with bare hands by the nape of their necks to avoid being bitten. Hairs were quickly sampled and the animals were liberated without any problems. There was no need for traps, anesthetics or even tweezers. Taxonomy of species in Cebidae family Kingdom: Animalia Phylum: Chordata Class: Mammalia Order: Primate Family: Cebidae Subfamilies: Cebinae, Saimiriinae Genera: Cebus, Saimiri (squirrel monkey) Species: Cebus apella (with a large variety of subspecies, although for some authors are different species), C. albifrons, C. capucinus, C. flavius, C. kaapori, C. nigritus, C. olivaceus, C. robustus, C. xantosthernos, Saimiri boliviensis (with different subspecies), S. cassiquiarensis (with different subspecies), S. oerstedii (with different subspecies), S. vanzolinii, S. sciureus (with different subspecies), S. ustus. This last species is dubious with molecular analyses.

Atelidae The Atelidae family (chapters 8, 10, 11, 12, 13, 18, 19) consists of four genera (Ateles, Brachyteles, Alouatta, and Lagothrix) 28 species and 42 taxa (Mittermeier and Richardson, 2013). These are the largest primates of the Neotropics and some are the most threatened. Here we provide an overview of each genus considering its morphology, distribution, threats, and conservation.

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Ateles (Spider Monkeys) Similar to the other Atelidae genera, Ateles or spider monkeys are large bodied and mostly arboreal (Figure 7). Primatologists studying this genus commonly disagree about the classification of its species in part due to the intraspecific and interspecific variation in pelage color (Hines, 2005). Their colors range from red to dark gray in Mesoamerica to black in South America. Spider monkeys have long, thin limbs giving credit to their name. They also have prehensile tails but lack thumbs. As an example of weight, studies of Ateles geoffroyi (Figure 8) have provided a range of 3.2 to 9.0 kg (Ford and Davis, 1992, Emmons and Feer, 1997). As Hines (2005) points out in his doctoral thesis, this relatively large range of weights may be due to including immature adults. Head and body lengths for this species are 305-630 mm plus a tail length of 520-855 mm (Emmons and Feer 1997, Reid, 1997). To support their primarily frugivorous diet (Van Roosmalen, 1980) spider monkeys have a fairly large territory size. The distribution range of spider monkeys includes Southern Mexico and extends through the Amazon of Brazil. All of the species of the Ateles genus are endangered or critically endangered except for Ateles paniscus (Linnaeus, 1758; Red faced spider monkey) that is listed as vulnerable (Figure 9). However, its current listing is more serious than its former listing as a species of least concern. Their plight along with the other species within the Ateles genus has worsened due to hunting, live capture for zoos and pets, and habitat loss. Spider monkeys also have low reproductive rates and are very sensitive to environmental disturbances (Rosenberger and Strier, 1989). In Chapter 13, Ruiz-Garcia et al. discuss data collected from 283 spider monkeys, the largest molecular genetics sample set of Ateles ever analyzed.

Figure 7. Photographs of spider monkeys taken by authors: A) Some individuals of Ateles belzebuth in the Quistococha zoo near to Iquitos (Peruvian Amazon) in 2010. B) An Ateles chamek sampled in the Yavarí River in the Brazilian Amazon near the border of Peru. C) An Ateles chamek sampled in the Santa Cruz Department (Bolivia) in 2010. D) M. R-G (second author) sampling an Ateles fusciceps fusciceps near Santo Domingo de Tsáchila (Ecuador) in 2013.

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Figure 8. Photographs of spider monkeys taken by M.R-G: A) An Ateles geoffroyi yucatanensis sampled in the Mexican Quintana Roo state in 2015. Note the blue eyes. Traditionally, only individuals of A. hybridus were considered to have blue eyes. B) An Ateles geoffroyi yucatenensis sampled in Belize in 2016.

Figure 9. An Ateles paniscus at the Manaus Zoo (Brazilian Amazon) in 2011.

Brachyteles Brachyteles (muriquis) contains the largest individuals of the Atelidae family (Fonseca et al., 1994). The southern woolly spider monkey (Brachyteles arachnoides) males can reach up to 15 kg in weight with lengths (head plus body) of 595 mm (Aguire, 1971). The pelage of Brachyteles is gray to yellow-brown and the two species within the genus vary in face color (complete black or with mottled pink) and presence of opposable thumbs (Rosenberger and Strier, 1989). They subsist on a diet of fruit, leaves, flowers, and to a less extent on twigs, stem, and bark (de Carvalho et al., 2004). Data from Aguirre (1971) suggest that Brachyteles reside in groups of 7 to 8 although Melo et al., (2005) reported group sizes of up to 18. Both have prehensile tails and spend time social grooming (Strier, 1992). The distribution of the endemic Brackyteles in Brazil is very limited due to habitat destruction through deforestation (Bergallo et al., 2000), hunting (Talebi and Soares, 2005), and other anthopogenic pressures. Strier (2000) estimates that there are less than 1,200 Brachyteles individuals left within the Atlantic forest of Southeastern Brazil. Much of the southern species (B. arachnoides) is protected within undisturbed forests of Sāo Paulo State

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Park whereas a large percentage of the northern species (B. hypoxanthos) inhabits private lands (Strier and Fonseca, 1996). The combined total of northern and southern populations of muriquis today ( 1 (ln  > 0). This last value will be present for a long time (several thousand generations) before showing the signature of a population expansion (< 1 or ln  < 0). There is an exception to this general rule, when a bottleneck is so intense the population becomes monomorphic before the demographic expansion, in which case,  < 1 (ln  < 0) all the time. All these  values are consistent in stepwise, logistic or exponential population growth and are not especially affected in diverse mutation models (Kimmel et al., 1998). We used empirical distributions of ln  from 500 coalescence simulations with a  = 5 to determine the statistical significance of  (ln ). In this case, a 95% confidence interval was determined (- 0.25, 0.28). The second test we used was that from Zivothovsky et al., (2000), which calculates an expansion index: Sk = 1 – (K – (RkV/2)/5V2). K and V are the unnormalized kurtosis (fourth central moment) and the allele size variance is estimated from a sample and corrected for sampling bias, respectively, whereas Rk = km/2m (they are the kurtosis and the variance in the repeated number of mutational changes, respectively). The expressions used to estimate V and K are: V = i = 1…n pi (Xi – X)2 and K =  i = 1…n pi (Xi – X)4, where X = i = 1…k pi k, and k represents the alleles in a locus given and pi, the allele frequencies. All the other terms were defined in the previous analysis. We used 6.3 for Rk because it was the value obtained for

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dinucleotide microsatellites by Dib et al., (1996), and because dinucleotide microsatellites were used in the current study. Feldman et al., (1999), used the same data and a geometrical distribution of mutational events, and obtained an estimated 2m of 2.5, which is basically the same as what was obtained by using a truncated Poisson distribution (Zhivothovsky et al., 2000) and by ourselves in the present work (2m = 2.39). The value of Sk is expected to be 0 in a general symmetric stepwise mutation model for a population in equilibrium and of constant size (this was derived by Zhivotovsky and Feldman, 1995). The Sk is positive if an expansion affected the population and it is negative if a bottleneck affected the population. To obtain demographic conclusions of this analysis, the within-population variance and the expansion index are averaged for all of the microsatellites studied within each population and their dynamics are compared. Zhivotovsky et al., (2000) showed that a significant correlation existed between V and Sk (r = 0.58) for a human data set, but this correlation was moderate and, in fact, both statistics could react differently to the changes in population size and have different patterns in different populations. To measure the statistical significance of the Sk values, a jackknife procedure was performed to obtain the variance of Sk and, with this variance, a Student’s t test and a 95% confidence interval were estimated. Finally, the method of O’Ryan et al., (1998) was applied by means of the dlik program with coalescence theory and the Metropolis-Hastings sampling algorithm. This method assumes that some current populations derived from a unique population at some known time earlier. This brief time period permits a negligible number of mutations to affect the observed gene frequencies since the initial split among the populations. Therefore, it is possible to estimate the effective numbers (Ne) of each population. These estimates can be regarded as random variables from the posterior distribution of the Ne. A frequency histogram built from these variables gives an estimated likelihood curve for Ne. We analyzed three hypothetical scenarios. The splits between the northern and southern subpopulations of S. leucopus occurred 100 YA (during the beginning of the twentieth century with the agricultural expansion, habitat degradation, hunting, road building and illegal trade), 500 YA (with the arrival of the Spaniard Conquerors and the beginning of cattle ranching) and 2,000-4,000 YA (with some subtle climatic changes during the Holocene). The form of the histograms indicated which agreed better with the scenarios. Ne was estimated from the most realistic scenarios. Two independent simulations were carried out with 50,000 replicates in each hypothetical scenario. Older scenarios were not investigated because this procedure doesn’t permit the impact of mutations on the gene samples analyzed.

RESULTS Microsatellite Mutation Models The maximum likelihood procedure of Nielsen (1997) showed a very similar  value for a uni-step or for a multi-step mutation model ( = 16.933 ± 21.999 vs.  = 19.878 ± 38.369) (Table 1). The marker D14S51 showed the highest level of genetic diversity, while AP68 and PEPC3 presented the lowest levels of genetic diversity (with AP74, which presented null variability in this species). The overall percentage of multiple step mutations was 5% (which means that 95% of mutations are uni-step) and only two loci showed significant multiple

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mutations (D5S111-20% and D14S51-10%). When the Bonferroni’s correction was applied, only D14S51 showed a significant multi-step mutation pattern. Taking into account all the markers analyzed, 5% of multiple-step mutations were significant (2 = 26.919, 8 df, P < 0.001). Nevertheless, when D14S51 was deleted from the analysis, no significant multiple mutation pattern was found (2 = 9.342, 7 df, NS). Thus for other analyses, it could be considered that the STRPs used are basically uni-step. Table 2 shows the microsatellite pairs which presented significantly different mutation rates. For the uni-step model, 80.93% (17/21) of microsatellite comparison pairs were significant, while for the multi-step model, 76.19% (16/21) were significant. This means that the mutation rates of the STRPs used were basically different among these markers.

Effective Number Sizes With the procedure of Nielsen (1997) considering a typical average mutation rate for STRPs (5.6 x 10-4), the uni-step mutation model offered an effective number of 7,559 individuals whereas the multi-step mutation model showed a value of 8,874 individuals for the overall sample. Additionally, the simulations of Griffiths and Tavaré (1994) (Figure 1) showed the following effective numbers for the overall sample and for both the Antioquia and the Tolima samples. For the overall sample, the effective number was 5,574 ± 3,809, while the Antioquia sample showed an effective number of 3,605 ± 2,770 and the Tolima sample yielded an effective number of 7,285 ± 5,825. Thus, similar effective numbers were obtained with this procedure to those obtained directly from the  statistic calculated through the method of Nielsen (1997). Another interesting result is that the Tolima subpopulation showed higher historical effective numbers than the Antioquia subpopulation, because the first subpopulation presented higher genetic diversity than the second one for some statistics such as . Table 1. Maximum likelihood estimates of  (= 4Ne) with the procedure of Nielsen (1997) and percentages of multi-step mutations for the overall Saguinus leucopus sample analyzed. *SG, Significant values of the chi-square tests indicating that the multi-step mutation model is significantly better than the uni-step mutation model UNI-STEP Markers



log-likelihood

MULTI-STEP 

log-likelihood %multi-step

AP68 AP74 D5S111 D5S117 D6S260 D14S51 D17S804

0.408 0 20.516 8.863 18.531 66.600 20.065

Mean

16.939 + 21.998

20.796 + 37.911

Effective Numbers

7,559 individuals

8,874 individuals

-4.12249 0 -27.82252 -22.40753 -21.47175 -24.03704 -24.43254

0.408 0 6.898 5.934 14.575 113.466 17.220

-4.13346 0 -25.12429 -22.57999 -22.72971 -32.82591 -23.90754

0 0 20 0 0 10 5

2

0.02 NS 0 NS 5.39 SG 0.34 NS 2.51 NS 17.57 SG 1.05 NS

5 + 7.368 26.92 SG 7 df p < 0.001

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Saguinus total 0,6

207 0,5

0,4

Likelihood

AP68

D55111 0,3

D55117

7939

D65260 D14551

7939 9618

0,2

Pepc3 D175804

6553 0,1

0

0,46416

0

10

20

30

17,78279 14,67799

40

50

60

70

80

Título del eje

21,54435

(A) 0,5

0,45

ANTIOQUIA

141 207

0,4

0,35

2072

Likelihood

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Figure 1. (Continued).

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TOLIMA

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Figure 1. Simulations using the coalescence theory and procedure of Griffith and Tavaré (1994) to estimate the parameter  = 4Ne (Ne = Effective number;  = mutation rate per generation). A mutation rate of 5.6 x 10-4 was assumed and the effective numbers were determined with the highest likelihood values for each one of the markers used. Specific samples are indicated as (A) total Colombian S. leucopus, (B) Antioquia S. leucopus, and (C) Tolima S. leucopus.

Table 2. Differences among the diverse mutation rates for the Saguinus leucopus microsatellites analyzed. Assuming a multi-step mutation model (A) and assuming a uni-step mutation model (B). ** Significant probability (the probability level was ’ = 0.05/21 = 0.0023 with the Bonferroni’s correction), * Significant probability at  = 0.05 B AP68 A AP68 D5S111 D5S117 D6S260 D14S51 D17S804 PEPC3

41.98** 36.89** 37.19** 57.38** 39.55** 0.020

D5S111 47.40** 5.09* 4.78* 15.40** 2.43 41.95**

D5S117 36.57** 10.83** 0.30 20.49** 2.66 36.87**

D6S260 34.69** 12.70** 1.87 20.19** 2.36 37.16**

D14S51 39.83** 7.57* 3.25 5.13* 17.84** 57.36**

D17S804 40.62** 6.78* 4.05* 5.92* 0.79

PEPC3 3.10 47.34** 36.51** 34.64** 39.77** 40.56**

39.52**

Gene Drift and Gene Flow in Populations of S. leucopus The method of Ciofi et al., (1999) showed that the gene drift model between the two detected subpopulations is more probable than the gene flow model with a Bayesian factor of around 1.75 (Figure 2). The log-likelihood values showed a normal and symmetrical

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distribution. Also, the two independently carried out runs showed the exact same results. The marginal F posterior distributions for both subpopulations seemed to be correct. Therefore, the analysis seems to be valid. This result does not completely agree with that determined by Ruiz-García et al., (2014a) with Migrate 3.6 Software, where the gene flow was estimated to be high between the two populations of S. leucopus.

Figure 2. Simulation II graphs (A and B) and F marginal posterior distribution graphs (C and D) of Saguinus leocupus generated using the method of Ciofi et al., (1999). The gene drift model, between the two subpopulations of Saguinus leucopus (Antioquia and Tolima) detected, is more probable than the gene flow model with a Bayesian factor of around 1.75. The log-likelihood values showed a normal and symmetrical distribution and the two runs, independently carried out, showed exactly the same results. Therefore, the marginal F posterior distributions for both subpopulations seem to be correct. Y-axe: number of Markov Chains Monte Carlo simulations (MCMC).

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Population Expansions in S. leucopus The test of Zhivotovsky et al., (2000) had Sk and V values of -0.1193 and 4.144, respectively, with Sk not significantly different from 0 (t = -0.564) and a 95% confidence interval of (-1.411, 0.295). This means that no significant demographic changes were revealed for this species and the genetic variance was moderate compared with other species that we have previously studied (Ruiz-García, 2010; Ruiz-García et al., 2015a,b). Nevertheless, the test of Kimmel et al., (1998) showed an imbalance index of  = 1.457 (ln  = 0.376). This result was significant for 500 coalescence simulations and for a jackknife procedure (t = 2.69, 95% confidence interval [1.125, 1.791]). This means that the overall S. leucopus sample experienced a population expansion after an initial bottleneck and that a relatively short time period had elapsed since the foundation of this population, which agree quite well with the next analysis. Thus, one test does not detect any significant population change but another detected a population expansion as Ruiz-García et al., (2014a) detected for mitochondrial DNA.

When Did the Antioquia and Tolima S. leucopus Populations Diverge? The method of O’Ryan et al., (1998) showed that the best fit histograms (for both subpopulations) were those obtained with a divergence scenario between both subpopulations 2,000-4.000 YA (Figure 3). If this is the most correct scenario (it showed a more complete normal curve distribution than the other two scenarios), the effective numbers for the Antioquia and Tolima subpopulations could be around 7,100 and 6,900 individuals, respectively. In this case, the Antioquia population was slightly larger than the Tolima population, in contrast to that determined with the simulations by Griffiths and Tavaré (1994). However, if we adopted the other two scenarios, 100 YA or 500 YA, the Tolima population yielded higher effective sizes than the Antioquia one (4,300 vs 3,700 individuals and 5,700 vs 5,100 individuals, respectively) as in the previous analyses. However, the different procedures used to determine effective number sizes in S. leucopus offered very similar results irrespective of the mathematical properties of each one of the procedures.

DISCUSSION The microsatellite gene diversity estimated for the overall S. leucopus sample and for the Antioquia and Tolima samples (He = 0.541 ± 0.336, 0.488 ± 0.313 and 0.458 ± 0.365, respectively) were smaller than the gene diversity estimates from microsatellites for other Neotropical mammals, such as the jaguar (He = 0.85, Ruiz-García et al., 2006b), the ocelot (He = 0.92, Ruiz-García, 2001), the red brocket deer (He = 0.71, Ruiz-García et al., 2009) or the capybara (He = 0.62, Maldonado-Chaparro et al., 2010). However, these values for S. leucopus are similar to those obtained for the same microsatellite set in other Neotropical primate taxa (see Ruiz-García et al., 2014a). Thus, although the geographical distribution range of this species is very small and highly threatened by human activity, their gene

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diversity levels are not impoverished, based on the mutation models and microsatellites we used. This is relevant information for the conservation purposes.

Figure 3. The best fit histograms (using the method of O’Ryan et al., 1998) showed that the subpopulations of Saguinus leucopus, Antioquia and Tolima, diverged around 2,000-4,000 YA (in light blue). Poorer fit histograms are marked in marine blue (100 YA) and yellow (500 YA). Y-axe: number of Markov Chains Monte Carlo simulations (MCMC).

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The different procedures we used showed relatively similar effective population sizes for the overall population of S. leucopus (Nielsen [1997], 7,600-8,900; Griffiths and Tavaré [1994], 5,600-10,900; O’Ryan et al., [1998], 8,000, 10,800, and 14,000 for divergences from 100, 500 and 2,000-4,000 YA). We do not know the real Ne/N rate for this species but if we speculate that this rate is about 0.6, this could mean the historical censuses values for the overall S. leucopus population could be around 12,600-14,800 animals for the first method used, around 9,300-18,200 individuals for the second method and 23,300 (2,000-4,000 YA), 18,000 (500 YA) and 13,300 (100 YA) specimens for the third method applied. IUCN considered that no more than 10,000 mature specimens of this species remain in Colombia. Our genetics estimations were generally slightly higher than this IUCN census value. This could mean that a certain demographic decrease of the population occurred in relatively recent times, but without an appreciable impact at the genetic level. Indeed, Ruiz-García et al., (2014a) did not detect any bottleneck in this endemic Colombian species. However, both the genetics and the IUCN census population sizes are of the same magnitude, with relative agreement between both. During 1996-2002, IUCN classified this species as A1C (this means that the population declined 20% in the last 10 years- 3 generations-), B1 + B2C (distribution with an extension of less than 2,000 km2, very fragmented and no more than 10 large localities) and C2A (less than 10,000 mature individuals and no subpopulation estimated to have more than 1,000 mature individuals). In 2003-2004, IUCN classified this species as A2C (reduction equal or larger than 30% in the next 10 years). Thus our genetics population size estimations could agree with these estimations provided by IUCN, with the last one the most realistic. In fact, our analysis with the procedure of Ciofi et al., (1999) detected important gene drift differentiating both the Antioquia and Tolima populations. Nevertheless, this does not necessarily mean that both populations are different subspecies as suggested by HernándezCamacho and Cooper (1976) and Defler (2010), especially if the genetics differentiation occurred recently. Indeed, several indirect gene flow estimates were obtained with the ndimensional island model (mean value and 99% confidence interval; Takahata 1983; Crow and Aoki 1984; Ruiz-García and Álvarez 2000) and by using the private allele method (Slatkin 1985; Barton and Slatkin 1986) between the Antioquia and the Tolima populations. These gene flow estimates with the n-dimensional model for the FST statistic and for the private allele method were Nm = 1.58 ± 0.77 and Nm = 0.63, respectively. This supported a limited gene flow between these two subpopulations, but a historical one. It is really important to determine when the populations (Antioquia and Tolima) began to be more affected by gene drift than by gene flow. The procedure of O’Ryan et al., (1998), showed ambiguous results. This procedures only determines population sizes in short periods of time due to the impossibility of incorporating mutations in the model. The normal distribution curve was better for 2,000-4,000 YA than for a split in the last 100 or 500 Y. This could mean that some climatological changes during the Holocene could be responsible for the initial divergence between the S. leucopus populations. Around 2,000-3,300 YA, during the Huascarán cold period in Peru (León-Canales, 2007), there was a drop in the temperature which could have had some influence on S. leucopus. Analyses of the O18 isotopes indicated a very significant decrement of temperature in the Andes and this could have initiated the fragmentation of the Andean forest connecting the current areas of Antioquia and Tolima, which in turn could have negatively influenced the gene flow of S. leucopus between these areas. Van der Hammen (1992) analyzed the Primavera lagoon in the Sumapaz Páramo and

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demonstrated some very cold periods in the Colombian Andes around 3,000 YA and between 6,300 and 4,700 YA. Additionally, Van der Hammen (1992) reported very active and catastrophic volcanic activity in the Colombian Andean Central Cordillera around 7,400, 6,200, 3,600 and 2,600 YA and this could have helped to fragment the forest S. leucopus inhabited. Another possibility is that this initial genetic fragmentation is really older than the Huascarán cold period but we cannot test it due to the limitations of the procedure of O’Ryan et al., (1998). Ruiz-García et al., (2014a) showed that the most recent mitochondrial haplotype diversification was around 16,000–8,000 YA. This agrees quite well with the Upper Pleniglacial period, around 22,000–14,000 YA, concomitant with the major cold and dry periods (20,000–18,000 YA) of the fourth large Pleistocene glaciation (Climap, 1976; Brown, 1982; Haffer, 1997, 2008; Van der Hammen et al., 1991). Clark (2002) showed that the rain level and the humidity in the Amazon basin at that time were extremely low. Even the Atlantic Ocean along the Brazilian coast had its temperature lowered to almost 6°C. Metivier (1998) showed that the central and northern Andes had an ice surface area totaling around 371,306 km2, 18,000 YA. This means an ice cover nearly 100 times greater than today. The last glacial advance in the Andes occurred during that epoch (Younger Dryas or III Dryas; Clapperton, 1993). This last glacial advance is reported to have happened in different parts of the Andean cordilleras (Wright, 1983), such as the Manachaque Valley (Cordillera Blanca), the Upismayo-Jalacocha (Cordillera de Vilcanota), and Puna de Junin in Peru, Choque-Yapu mountain in Bolivia and the Chimborazo volcano in Ecuador. Indeed, Rodbell and Seltzer (2000) showed that the glacial limits in the Cordillera Blanca (San Martin Department in Peru), around 12,000 YA, were around 3,170 and 3,827 masl. Compare this to today’s limit of around 4,600 masl. Therefore, the beginning of the partial genetics isolation between the S. leucopus populations could have initiated earlier than the 2,000-4,000 YA that we detected with the procedure of O’Ryan et al., (1998). Additional data could justify the beginning of the S. leucopus genetics fragmentation in the last 10,000-20,000 YA, which is in line with the Refugia hypothesis proposed by Haffer, (1969, 1997, 2008). Here are some examples that could be relevant. Iriondo and Latrubesse (1994) discovered evidence for a dry Late Glacial climate in the central area of the lower Amazon and found evidence in favor of reduced discharges of the rivers in this area. In this same area, west of the city of Santarém in the Brazilian Amazon, Tricart (1974) determined a drier type of vegetation during the last stage of the Würm (Wisconsin) glaciation that was different to the present phytostabilizing rainforest. Similarly, Freitas et al., (2001) and Van der Hammen and Absy (1994) determined, between 9,000-3,000 YA, that savanna vegetation expanded at the expense of forest in the area of Humaitá and in the region to the southeast of Porto Velho during the Last Glacial Maximum. Based on oxygen isotopic of planktonic foraminifera samples recovered from a marine sediment core in a region of Amazon River discharge, Maslin and Burns (2000) determined that the Amazon Basin was extremely dry during the Younger Dryas period (13,000 - 11,600 YA). The Amazon’s discharge was reduced by at least 40% as compared with that of today. The moisture increased steadily afterwards during the Holocene. This dry period, affected terrestrial organisms and aquatic organisms alike. For example, Farias et al., (2010) investigated the demographic history and population structure of a fish, Colossoma macropomum, sampled in the Amazon basin and the Bolivian sub-basin. They determined an inter-basin divergence to have occurred approximately 17,000 YA, which could be similar to the temporal period when the fragmentation of S. leucopus population began. Very similarly,

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Ledru et al., (1998) and Van der Hammen and Hooghiemstra (2000) reported the absence (or very small amounts), of sediments in many locations of the Amazonia and central Brazil during the Last Glacial Maximum (24,000-17,000 YA). The Last Glacial Maximum was represented by a hiatus of several thousand years or more, indicative of drier climates. On the other hand, the strongest evidence in favor of the genetic fragmentation in very recent times (100-500 YA) is based on procedures from O’Ryan et al., (1998) Nielsen (1997), and Griffiths and Tavaré (1994). Effective population sizes based on a divergence that occurred 2,000-4,000 YA are clearly higher than the values estimated with these other procedures. However, we must not forget that the mitochondrial results (Ruiz-García et al., 2014a) and one of the tests applied to microsatellites revealed a historical population expansion for this species probably during a fraction of the Pleistocene. The mitochondrial data showed a population expansion that was initiated around 0.3 MYA in S. leucopus (Ruiz-García et al., 2014a). This population expansion period coincided with the population expansion of other Neotropical mammals, such as the Humboldt woolly monkeys (Lagothrix lagotricha; RuizGarcía et al., 2014b), the Andean fox (Pseudoalopex culpaeus; Ruiz-García et al., 2013a) and the jaguarundi (Puma yagouaroundi; Ruiz-García et al., 2013b). Even the Pleistocene climatic fluctuations could have had an important influence on fish, such as the piranha Serrasalmus rhombeus. Hubert et al., (2007) showed a population expansion for this species occurring between 800,000-400,000 YA. Farias et al., (2010) detected the beginning of a population expansion approximately 350,000 YA for the Amazon population of C. macropomum (previously cited). This demographic growth was greatly accelerated in the last 125,000 YA. Therefore, it seems clear that palynological analyses support the idea that lowland Neotropical humid forests were once more restricted (Absy et al., 1991; Burnham and Graham, 1999). Recent isotopic evidence suggests that precipitation was lower in the eastern Amazon than in the western Amazon during the last glaciation (Cheng et al., 2013). However, the Refugia hypothesis has been widely criticized on both theoretical and empirical grounds in the last decades (Endler, 1982; Weitzman and Weitzman, 1982; Lundberg, 1998; Colinvaux et al., 2000). For instance, Hubert et al., (2007) determined that the Aripuana Refuge was important for S. rhombeus. In contrast, the palaeovegetation study of Amazonia by Anhuf et al., (2007) did not indicate forest refugia during the Pleistocene in the area hypothesized by Hubert et al., (2007). Thus, the role of the Refugia hypothesis in limiting population sizes and dispersal remains unclear for some authors (Antonelli et al., 2010; Beling et al., 2010; Jaramillo et al., 2010). Nevertheless, the case of S. leucopus could offer interesting insights in favor of the influence of Pleistocene climatic changes on the generation of genetic diversification, which is a prelude to speciation phenomena.

ACKNOWLEDGMENTS Thanks go to the SDA (Secretaria Distrital Ambiental de Bogotá DC, Colombia) for the project entitled “Fortalecimiento del control y prevención del tráfico ilegal de fauna silvestre, especialmente de Primates, Perezosos y Ardillas, a través de la determinación de zonas sometidas a extracción ilegal utilizando pruebas de genética molecular de poblaciones,” which allowed us to obtain the necessary financial resources to carry out the current study. In

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addition, thanks to Amador Ávila, Claudia Brieva and Carlos del Valle (URRAS, Universidad Nacional de Colombia) as well as to the Natura Foundation for providing S. leucopus samples and financial resources (Natura Foundation). Also, thanks to L.M. Borrero and D.M. Ramírez from the Medellín Zoo, Tinka Plese (UNAU, Medellín) and Cornare (Medellín), for providing monkey samples.

REFERENCES Absy, M. L., Cleef, A., Fournier, M., Martin, L., Servant, M., Siffedine, A., Ferreira da Silva, M., Soubies, F., Suguio, K., Turcq, B., Van Der Hammen, T. (1991). Mise en évidence de quatre phases d’ouverture de la foret dense dans le sud-est de l’Amazonie au cours des 60 000 dernières années. Première comparaison avec d’autres régions tropicales. C. R. Acad. Sci. Paris. 312: 673 - 678. Ackermann, R. R., Cheverud, J. M. (2000). Phenotypic covariance structure in tamarins (genus Saguinus): A comparison of variation patterns using matrix correlation and common principal component analysis. American Journal of Physical Anthropology 111: 489-501. Ackermann, R. R., Cheverud, J. M. (2002). Discerning evolutionary processes in patterns of tamarin (genus Saguinus) craniofacial variation. American Journal of Physical Anthropology 117: 260-271. Anhuf, D., Ledru, M.P., Behling, H., Da Cruz, J.F.W., Cordeiro, R.C., Van der Hammen, T. et al., (2006). Paleo-environmental change in Amazonian and African rainforest during the LGM. Palaeogeography Palaeoclimatology 239: 510–527. Antonelli, A., Quijada-Mascarenas, A., Crawford, A.J., Bates, J.M., Velazco, P.M., Wuster, W. (2010). Molecular studies and phylogeography of Amazonian tetrapods and their relation to geological and climatic models. In Hoorn, and C., Wesselingh, F. (Eds.), Amazonia, Landscape and Species Evolution: A Look into the Past. Wiley-Blackwell, Oxford, Pp. 386–404. Barton, N.H., Slatkin, M. (1986). A quasi-equilibrium theory of the distribution of rare alleles in a subdivided population. Heredity 56: 409-416. Behling, H., Bush, M., Hooghiemstra, H. (2010). Biotic development of Quaternary Amazonia: a palynological perspective. In Hoorn, C., Wesselingh, F. (Eds.), Amazonia, Landscape and Species Evolution: A Look into the Past. Wiley-Blackwell, Oxford, Pp. 335-348. Bennett, S. E. (2003). Los micos de Colombia. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt y Fundación Tropenbos. Pp. 1-260. Bernstein, I. S., Balcaen, P., Dresdale, L., Gouzoules, H., Kavanagh, M., Patterson, T., Newman-Warner, P. (1976). Differential effects of forest degradation on primate populations. Primates 17: 401-411. Brown, K. S. (1982). Paleoecology and regional patterns of evolution in Neotropical forest butterflies. In: Prance GT (Ed.) Biological diversification in the tropics. Columbia University Press. Bruford, M. W., Wayne, R. K. (1993). Microsatellites and their application to population genetics studies. Current Opinion in Genetics and Development 3: 939-943.

108

Manuel Ruiz-García, Pablo Escobar-Armel, Norberto Leguizamón et al.

Burnham, R. J., Graham, A. (1999). The history of Neotropical vegetation: new developments and status. Annals of the Missouri Botanical Garden 86: 546-589. Cabrera, A., Yepes, J. (1940). Historia Natural Ediar. Mamíferos Sud-Americanos. Compañía Argentina de Editores, Buenos Aires, Argentina. Calle, Z. (1992). Informe de actividades y resultados: Censo preliminar y recomendaciones para el manejo de una población natural de Saguinus leucopus en la zona de influencia del Proyecto Hidroeléctrico La Miel II. Unpublished. Cheng, H., Sinha, A., Cruz, F. W., et al., (2013). Climate change patterns in Amazonia and biodiversity. Nature Communications 4: 1411. Ciofi, C., Beaumont, M. A., Swingland, E.O., Bruford, M.W. (1999). Genetic divergence and units for conservation in the Komodo dragon Varanus komodoensis. Proceedings of the Royal Society London B 266: 2269-2274. Clapperton, C. (1993). Quaternary Geology and Geomorphology of South America. Ed. Elsevier. Amsterdam, The Netherlands. Clark, P.U. (2002). Early deglaciation in the Tropical Andes. Science 298: 7a. Climap, (1976). The surface of the ice-age Herat. Science 191: 1131-1137. Colinvaux, P.A., Oliveira, P.E., Bush, M.B. (2000). Amazonian and Neotropical plant communities on glacial time-scales: The failure of the aridity and refuge hypotheses. Quaternary Sci. Rev. 19: 141-169. Crow, J.F., Aoki, K. (1984). Group selection for a polygenic behavioral trait: estimating the degree of population subdivision. Proceedings of the National Academy of Sciences USA 81: 6073-6077. Cuartas-Calle, C. A. (2001). Partial distribution of tamarins (Saguinus leucopus, Callitrichidae) in Departamento de Antioquia, Colombia. Neotropical Primates 9: 107111. Defler, T. R. (2003). Primates de Colombia. Colombia, Bogotá: Conservación Internacional. Defler, T. R. (2010). Historia natural de los primates colombianos. Editorial Universidad Nacional de Colombia. Pp. 1-609. Dib, C., Faure, S., Fizames, C. (1996). A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature 380: 152-154. Ellesworth, J. A., Hoelzer, G. A. (1998). Characterization of microsatellite loci in a new world primate, the mantled howler monkey (Alouatta palliata). Molecular Ecology 7: 657–658. Endler, J. (1982). Pleistocene forest refuges: fact or fancy? In Biological Diversification in the Tropics. Prance, G. T. (Ed.). Pp. 179-200. New York: Columbia University Press. Farias, I. P., Torrico, J. P., García-Dávila, C., Freitas Santos, M. C., Hrbek, T., Renno, J. F. (2010). Are rapids a barrier for floodplain fishes of the Amazon basin? A demographic study of the keystone floodplain species Colossoma macropomum (Teleostei: Characiformes). Molecular Phylogenetics and Evolution 56: 1129–1135. Feldman, M. W., Kumm, J., Pritchard, J. K. (1999). Mutation and migration in models of microsatellite evolution. In Goldstein, D. G., and Schlötterer, C. (Eds.), Microsatellites: evolution and applications. Oxford, United Kingdom: Oxford University Press. Freitas, H. A., Pessenda, L. C. R., Aravena, R., Gouveia, S. E. M., Ribeiro, A.S., Boulet, R. (2001). Late Quaternary vegetation dynamics in the southern Amazon basin inferred from Carbon isotopes in soil organic matter. Quaternary Rese. 55: 39-46.

Genetic Heterogeneity and Evolutionary Demographic History …

109

Griffiths, R. C., Tavaré, S. (1994). Simulating probability distributions in the coalescent. Theoretical Population Biology, 46 131-159. Haffer, J. (1969). Speciation in Amazonian forest birds. Science 165: 131-137. Haffer, J. (1997). Alternative models of vertebrate speciation in Amazonia: an overview. Biodiversity Conservation 6: 451-476. Haffer, J. (2008). Hypotheses to explain the origin of species in Amazonia. Brazilian Journal of Biology 68: 917-947. Hernández-Camacho, J., Cooper, R.W. (1976). The nonhuman primates of Colombia. In Thorington Jr RW, Heltne PG (Eds.), Neotropical Primates: Field Studies and Conservation (pp. 35-69). Washington D.C.: National Academy of Sciences. Hershkovitz, P. (1977). Living new world monkeys (Platyrrhini). Chicago: University of Chicago Press. Hubert, N., Duponchelle, F., Nunez, J., Garcia-Davila, C., Paugy, D., Renno, J.F. (2007). Phylogeography of the piranha genera Serrasalmus and Pygocentrus: implications for the diversification of the Neotropical ichthyofauna. Molecular Ecology 16: 2115–2136. Iriondo, M., Latrubesse, E. M. (1994). A probable scenario for a dry climate in central Amazonia during the late Quaternary. Quaternary International 21: 121-128. Jacobs, S.C., Larson, A., Cheverud, J.M. (1995). Phylogenetics relationships and orthogenetic evolution of coat color among tamarins (genus Saguinus). Systematic Biology 44: 512532. Jacobs-Cropp, S., Larson, A., Cheverud, J. M. (1999). Historical biogeography of tamarins, genus Saguinus: The molecular phylogenetic evidence. American Journal of Physical Anthropology 108: 65-89. Jaramillo, C., Hoorn, C., Silva, S.A.F., Leite, F., Herrera, F., Quiroz, L., Dino, R., Antonioli, L. (2010). The origin of the modern Amazon rainforest: implications of the palynological and palaeobotanical record. In Hoorn, C., Wesselingh, F. (Eds.), Amazonia, Landscape and Species Evolution: A Look into the Past. (pp. 317-334). Wiley-Blackwell, Oxford. Kimmel, M., Chakraborty, R., King, J. P., Bamshad, M., Watkins, W. S., Jorde, L. B. (1998). Signatures of population expansion in microsatellite repeat data. Genetics 148, 19211930. Ledru, M.P., Bertaux, J., Sifeddine, A., Suguio, K. (1998). Absence of Last Glacial Maximum records in lowland tropical forests. Quaternary Research 49: 233-237. León-Canales, E. (2007). Orígenes humanos en los Andes peruanos. Universidad de San Martín de Porres. Lima, Perú. Pp. 1-328. Lundberg, J.G., Marshall, L., Guerrero, J., Horton, B., Malabarba, C., Wesselingh, F. (1998). The stage for Neotropical fish diversification: history of a tropical South American river. In Malabarba, L.R., Reis, R., Vari, R., Lucena, Z., and Lucena, C.A., (Eds.). Phylogeny and Classification of Neotropical Fishes. (pp. 13-48). Porto Alegre, Brazil: Pontificia Universidade Católica Do Rio Grande Do Sul. Maldonado-Chaparro, A., Bernal-Parra. L. M., Forero-Acosta, G., and Ruiz-García, M. (2011). Estructura genética de un grupo de capibaras (Hydrochoerus hydrochoeris, Hydrocheridae, Rodentia) mediante marcadores microsatélites en los Llanos Orientales colombianos. Revista de Biología Tropical (International Journal of Tropical Biology) 59: 1777-1793. Maslin, M.A., Burns, SJ. (2000). Reconstruction of the Amazon Basin effective moisture availability over the past 14,000 years. Science 290: 2285-2287.

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Metivier, S. P. (1998). A reconstruction of glacial extent, temperature and precipitation in South America at the time of the last glacial maximum. PhD Thesis. Syracuse University. Mittermeier, R. A., Coimbra-Filho, A. F. (1981). Systematics: species and subspecies. In Coimbra-Filho, A. F., Mittermeier, R. A. (Eds.). Ecology and behavior of Neotropical Primates. Vol 1. (pp. 29-109). Mittermeier, R. A., Rylands, A. B., Coimbra-Filho, A. F., da Fonseca, A. B. (1988). Ecology and behavior of Neotropical Primates. Vol.2. World Wildlife Fund, Washington, D.C. Pp. 1-610. Moore, A. J., Cheverud, J. M. (1992). Systematics of the Saguinus oedipus group of the bareface tamarins: Evidence from facial morphology. American Journal of Physcial Anthropology 89: 73-84. Nielsen, R. (1997). A likelihood approach to population samples of microsatellite alleles. Genetics 146: 711-716. O’Ryan, C. E., Harley, H., Bruford, M. W., Beaumont, M., Wayne, R. K., Cherry, M. I. (1998). Microsatellite analysis of genetic diversity in fragmented South African buffalo populations. Animal Conservation 1: 85-94. Poveda, K. (2000). Uso de hábitat de dos grupos de tití de pies blancos (Saguinus leucopus) en Mariquita, Colombia. Bachelor Thesis, Universidad Nacional de Colombia, Bogotá DC. Poveda, K., Sánchez-Palomino, P. (2004). Habitat use by the White-footed tamarin, Saguinus leucopus: a comparison between a forest-dwelling group and an urban group in Mariquita, Colombia. Neotropical Primates 12: 6-9. Rodbell, D.T., Seltzer, G.O. (2000) Rapid ice margin fluctuations during the Younger Dryas in the tropical Andes. Quaternary Research 54: 328-338. Rogers, J., Witte, S.M., Slifer, M.A. (1995). Five new Microsatellite DNA polymorphisms in the squirrel monkey (Saimiri boliviensis). American Journal of Primatology 36: 151. Roncancio, N. (2008). Densidad poblacional de Saguinus leucopus en áreas alteradas con diferentes características físicas y biológicas en el departamento de Caldas. MS. Thesis. Universidad Nacional de Colombia, Bogotá, Colombia. Ruivo, E.B., Carroll, J.B., Morales-Jiménez, A.L. (2005) The Silvery-Brown Tamarin (Saguinus leucopus) Conservation Project. Neotropical Primates 13: 36-39. Ruiz-García, M. (2001). Diversidad genética como herramienta de zonificación ambiental: Estudios moleculares (microsatélites) en el caso de Primates y Félidos neotropicales comportan una nueva perspectiva. In Defler, T., and Palacios, P. A., (Eds.), Zonificación Ambiental para el Ordenamiento Territorial en la Amazonía Colombiana, (pp 85-108). Bogotá DC: Instituto Amazónico de Investigaciones, Imani and Instituto de Ciencias Naturales, Universidad Nacional de Colombia. Ruiz-García, M. (2005). The use of several microsatellite loci applied to 13 Neotropical primates revealed a strong recent bottleneck event in the woolly monkey (Lagothrix lagotricha) in Colombia. Primate Report 71: 27–55. Ruiz-García, M. (2010). Changes in the demographic trends of pink river dolphins (Inia) at the micro-geographical level in Peruvian and Bolivian rivers and within the Upper Amazon: Microsatellites and mtDNA analyses and insights into Inia’s origin. In RuizGarcía M., Shostell, J. (Eds.). Biology, Evolution, and Conservation of River Dolphins Within South America and Asia. Nova Science Publishers., Inc.. New York, USA. Pp. 161-192.

Genetic Heterogeneity and Evolutionary Demographic History …

111

Ruiz-García, M., Alvarez, D. (2000). Genetic microstructure in two Spanish cat populations I: Genic diversity, gene flow and selection. Genes and Genetic Systems 75: 269-280. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P., Shostell, J. M. (2006a). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report 73: 3–47. Ruiz-García, M., Payán, E., Murillo, A., Alvarez D. (2006b). DNA Microsatellite characterization of the Jaguar (Panthera onca) in Colombia. Genes and Genetics Systems 81: 115-127. Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., GutierrezEspeleta, G., Shostell, J. M. (2007a). Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: Genetic structure and recent bottlenecks. Folia Primatologica 78: 73–87. Ruiz-García, M., Castillo, M. I., Álvarez, D., Gardeazabal, J., Borrero, L. M., Ramirez, D. M., Carrillo, L., Nassar F., Gálvez, H. (2007b). Estudio de 14 especies de Primates Platirrinos (Géneros Cebus, Saimiri, Aotus, Saguinus, Cebuella, Callithrix, Lagothrix, Alouatta, y Ateles), utilizando 10 loci microsatelites: Análisis de la diversidad génica y de la detección de cuellos de botella con propósitos conservacionistas. Orinoquia 11: 19-37. Ruiz-García, M., Martínez-Aguero, M., Alvarez, D., Goodman S. (2009). Variabilidad genética en géneros de Ciervos neotropicales (Mammalia: Cervidae) según loci microsatélitales. Revista de Biología Tropical. (International Journal of Tropical Conservation and Biology) 57: 879-904. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L.F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). Internation Journal of Primatology 32: 1218–1241. Ruiz-García, M., Rivas-Sánchez, D., Lichilín-Ortiz, N. (2013a). Phylogenetics relationships among four putative taxa of foxes of the Pseudoalopex genus (Canidae, Carnivora) and molecular population genetics of Ps. culpaeus and Ps. sechurae. In: Ruiz-García, M., and Shostell, J. (Eds). Molecular Population Genetics, Phylogenetics, Evolutionary Biology and Conservation of the Neotropical Carnivores. (pp. 97–128). Nova Science Publishers, New York. Ruiz-García, M., Pinedo-Castro, M. (2013b). Population genetics and phylogeographic analyses of the jaguarundi (Puma yagouaroundi) by means of three mitochondrial markers: the first molecular population study of this species. In: Ruiz-García, M., and Shostell, J. (Eds). Molecular Population Genetics, Phylogenetics, Evolutionary Biology and Conservation of the Neotropical Carnivores. (pp. 245-288). Nova Science Publishers, New York. Ruiz-García, M., Escobar-Armel, P., Leguizamón, N., Manzur, P., Pinedo-Castro, M., Shostell, J.M. (2014a). Genetic characterization and structure of the endemic Colombian silvery brown bare-face tamarin, Saguinus leocopus (Callitrichinae, Cebidae, Primates). Primates 55: 415-435. Ruiz-García, M., Pinedo-Castro, M., Shostell, J.M. (2014b). How many genera and species of wolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular analysis of Lagothrix flavicauda, an endemic Peruvian primate species. Molecular Phylogenetics and Evolution 79: 179–198.

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Ruiz-García, M., Castillo, M. I. (2016a). Genetic structure, spatial patterns and historical demographic evolution of white-throated capuchin (Cebus capucinus, Cebidae, Primates) populations of Colombia and Central America by means of DNA microsatellites. In Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Ruiz-García, M., and Shostell, J. M. (Eds.). Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I., Luengas, K., Leguizamon, N. (2016b). Invalidation of three robust capuchin species (Cebus libidinosus pallidus, C. macrocephalus and C. fatuellus; Cebidae, Primates) in the Western Amazon and Orinoco by analyzing DNA microsatellites. In Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Ruiz-García, M., and Shostell, J. M. (Eds.). Nova Science Publishers, Inc. New York, USA. Slatkin, M (1985). Rare alleles as indicators of gene flow. Evolution 39: 53-65. Snow, C. A. (1986). A life history study of the Geoffroy’s tamarin, Saguinus geoffroyi, with emphasis on male-female relationships in captive animals. PhD Thesis, Kent State University. Takahata, N. (1983). Gene identity and genetic differentiation of populations in the finite island model. Genetics 104: 497-512. Tricart, J. (1974). Existence de périodes sèches au Quaternaire en Amazonie et dans les régions voisines. Rev. Géomorph. Dyn. 23: 145-158. Van der Hammen, T. (1992). La paleoecología de Suramérica Tropical. Cuarenta años de investigación de la historia del medio ambiente y de la vegetación. In Historia, Ecología y Vegetación (pp. 1-411). Bogotá, Colombia: Corporación Colombiana para la Amazonía-Araracuara. Van Der Hammen, T., Absy, ML. (1994). Amazonia during the last glacial. Palaeogeography, Palaeoclimatology, Palaeoecology 109: 247-261. Van Der Hammen, T., Hooghiemstra, H. (2000). Neogene and Quaternary history of vegetation, climate, and plant diversity in Amazonia. Quaternary Science Review 19: 725-742. Van der Hammen, T., Duivenvoorden, J. F., Lips, J. M., Urrego, L. E., Espejo, N. (1991). El cuaternario tardío del área del Medio Caquetá (Amazonia colombiana). Colombia Amazónica 5: 63-90. Vargas, T. N., Solano, C. (1996). Evaluación de los problemas en determinar potenciales áreas de conservación para Saguinus leucopus en una sección del medio valle del Magdalena. Neotropical Primates 4: 13-15. Weber, J. L., May, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. The American Journal of Human Genetics 44: 388–396. Weber, J. L., Wong, C. (1993). Mutation of human short tandem repeats. Human Molecular Genetics 2: 1123-1128. Weitzman, S. H., Weitzman, M. J. (1982). Biogeography and evolutionary diversification in Neotropical freshwater fishes with comments on the refuge theory. In Prance, G. T. (Ed.). Biological Diversification in the Tropics. (pp. 403–422). Columbia University Press, New York. Wright Jr, H. E. (1983). Late-Pleistocene glaciation and climate around the Junin Plain, central Peruvian highlands. Geografiska Annaler 65A: 35-43.

Genetic Heterogeneity and Evolutionary Demographic History …

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Zhong, F., Brady, A. G., Shi, J. (1996). Strategy using pooled DNA to identify 56 short tandem repeat polymorphisms for the Bolivian squirrel monkey. BioTechniques 21: 580586. Zhivotovsky, L. A., Feldman, M. W. (1995). Microsatellite variability and genetic distances. Proceedings of the National Academy of Sciences 92: 11549-11552. Zhivotovsky, L. A., Bennett, L., Bowcock, A. M., Feldman, M. W. (2000) Human population expansion and microsatellite variation. Molecular Biology and Evolution 17: 757-767.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 4

DIVERSITY OF CEBUS SPECIES FROM THE SOUTHERN DISTRIBUTION OF THE GENUS Mariela Nieves and Marta Dolores Mudry *

Grupo de Investigación en Biología Evolutiva, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IEGEBA (CONICET-UBA), Ciudad Universitaria, Buenos Aires, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas CONICET

ABSTRACT Taxonomists recognize different species and subspecies within the genus Cebus (capuchin monkeys, caiararas and macaco-prego) but classifications have been controversial and continuously revised. Relatively recently, evolutionary cytogenetic and molecular genetics studies using other molecular markers have suggested alternative classifications. The distinctive cytogenetic feature of Cebus is its conspicuous heterochromatic extracentromeric regions distributed along the whole karyotype. These are considered good markers for taxonomic diagnosis. The detection of specific mitochondrial haplotypes and morphological variation are also useful tools in phylogenetic, evolutionary and taxonomic studies. C. nigritus and C. cay naturally distributed in Northern Argentina, represent the southernmost geographic distribution of Cebus. These species show phenotypic variations mostly regarding the color of the body pelage with a highly homologous karyotype, although easily distinguishable from each other. Our aim in this chapter is to provide a better understanding of the variability within capuchin monkeys through the analysis of evolutionary cytogenetics, genome size estimations, geometric morphometrics and molecular genetics data. We studied 275 individuals (C. cay and C. nigritus) of which 44 were new specimens. The thirty-two new C. cay did not show any of the chromosomal reorganizations previously described. The analysis of C-band variability and frequency of polymorphisms allowed us to identify a species-specific pattern. We also detected connections between the three observed morphotypes and the geographic origin at the intraspecific level. One (P1) was associated with the Paraguayan C. cay with one heterochromatin variation pattern. A second (P2) *

For correspondence: [email protected], [email protected].

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Mariela Nieves and Marta Dolores Mudry was related to C. cay individuals from Salta, Bolivia, Peru with a different heterochromatin pattern. The third (P3) corresponded to C. nigritus specimens with a third heterochromatin pattern. We also detected a significant variability in genome size among the C. cay’s samples in agreement with the molecular genetics and geometric morphometrics analyses. Variations in skull morphologic patterns support the differentiation between C. nigritus and C. cay as well as the differentiation among Northwest Argentina, Southern Bolivia and Paraguay forms. In summary, our findings of Cebus cay and C. nigritus provide a pattern of diversity associated with the limits of this genus’s distribution. We propose a more holistic interpretation for understanding the present day diversity of these species in the context of the evolutionary history of the Cebus genus.

Keywords: Cebus cay, Cebus nigritus, cytogenetics, mtDNA, morphometric geometric, taxonomy

INTRODUCTION The genus Cebus (Erxleben, 1777) has a long evolutionary history. It groups the neotropical primates known as capuchin monkeys, caiararas or macaco-prego and has one of the most confusing taxonomies of neotropical mammals (Rylands, 2000). It is among the widest distributed Platyrrhini, inhabiting almost all forested areas from Honduras and Nicaragua, in Central America, to South America in the Argentinean provinces of Misiones, Salta and Jujuy (Cabrera, 1957; Wilson and Reeder, 2005; Madden et al., 2007). Its wide geographical distribution is accompanied by a huge ecological, morphological, phenotypical (particularly the coat color), behavioral, and genetic diversity at both the inter and intrapopulation levels. This diversity has challenged the taxonomy of the genus and has helped to create a continuous source of controversy and classification revisions (Hershkovitz, 1949; Hill, 1960; Assumpçao, 1983; Silva Jr., 2001; Rylands et al., 2012 among others). Elliot (1913) was the first to establish the basic classification of capuchin monkeys, separating the species of the Cebus genus into two groups: tufted (or crested) and un-tufted (or not crested). However, there is a consensus that this feature is not a good diagnostic for the species, due to phenotypic variations. Hershkovitz (1949), adopting this criterion, recognized four species: C. apella as the only member of the tufted group and C. albifrons, C. capucinus and C. nigrivittatus in the un-tufted group. More recently, Groves (2001) recognized eight species, splitting C. apella into four distinct taxa (C. apella, C. libidinosus, C. xanthosternos and C. nigritus) and adding C. kaapori to the three pre-existing species in the un-tufted group. Silva Jr. (2001) presented an alternative taxonomic arrangement in which he recognized the untufted and tufted groups as subgenera (Cebus and Sapajus respectively) and did not recognize any subspecies. For example, the designation of Cebus apella, is still ubiquitous especially for management purposes and captivity conditions. Even with all the efforts to elucidate the classification of the group including individuals in zoos and reproduction centers, most of their identification is based only on the phenotype (Nieves et al., 2008). Recent biogeography, morphology and molecular genetics studies suggested a new division of the genera as follows: Cebus, the gracile capuchins and Sapajus the robust

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capuchins (Lynch Alfaro et al., 2012a,b). However, numerous studies conducted over the past 30 years in the field of evolutionary cytogenetics and molecular genetics that used other mitochondrial and nuclear markers do not support this division (Matayoshi et al., 1986, 1987; Mudry, 1990; Martinez et al., 2002; Ascunce et al., 2003; Seuánez et al., 2005; Nieves, 2007; Amaral et al., 2008; Nieves et al., 2011; Bi et al., 2012; Ruiz-García et al., 2010, 2012 among others). Cebus is characterized by presenting two chromosomal numbers: 2N = 52 and 2N = 54. Particularly 2N = 52 and 2N = 54 in C. albifrons (Koiffmann and Saldanha, 1974; García et al., 1976); 2N = 52 in C. olivaceus = C. nigrivitattus (Dutrillaux et al., 1978) and C. kaapori (Amaral, unpublished data); and 2N = 54 in C. capucinus (Dutrillaux et al., 1978), C. paraguayanus = C. (Sapajus) cay (Matayoshi et al., 1986), C. (Sapajus) nigritus (Mudry, 1990), C. (Sapajus) xanthosternos (Seuánez et al., 1986) and C. (Sapajus) apella (García et al., 1978). However, to date, the karyotype descriptions of Cebus (Sapajus) robustus and Cebus (Sapajus) macrocephalus have not been published. Only the number and chromosome morphology of Cebus (Sapajus) flavius and C. (Sapajus) libidinosus is known (da Silva et al., 2010). The distinctive cytogenetic feature of the genus is the presence of conspicuous heterochromatic regions corresponding to repeated sequences distributed along the whole karyotype in all the species described up to date. This remarkable genome trait refers not only to the number of heterochromatic blocks but also to the size, frequency and proportion of repeated sequences exhibited by each chromosome pair (Nieves, 2007). In fact, and a peculiarity of Cebus karyotypes, the heterochromatin pattern is a good marker for taxonomic diagnosis particularly when the geographical origin of specimens is unknown. This feature has a great relevance among these neotropical primates who exhibit remarkable phenotypic plasticity (Martinez et al., 2004; Nieves et al., 2008; Penedo et al., 2014). In addition, population and conservation studies use the analysis of the mitochondrial DNA as informative genetic markers. Mainly, the analysis of the non-coding control region or D-loop shows a high substitution rate with polymorphisms concentrated in the hypervariable region, which has been the focus of several studies for recent demographic events in different populations (Hewitt, 2000). This region has been used in population genetic analyses and captivity surveys in a variety of neotropical primates including Cebus (Ascunce et al., 2003; Casado et al., 2010). Then, the detection of specific mitochondrial haplotypes enables us to determine the existence of genetic sub-structuration in a particular species’s geographic distribution. Morphological variation is also of potential interest in phylogenetic, evolutionary and taxonomic studies especially in a group where some complex traits might provide further information. For instance, it is well known that the skull represents a complex structure with numerous biological functions, and therefore subjected to many selective pressures. However, few studies focus on the analysis of skull morphology variation of the above mentioned taxa (Silva Jr., 2001; Avila, 2004; Cáceres et al., 2014; Arístide et al., 2013; Wright et al., 2015). Although numerous forms have been described over the years, the tufted capuchins of the Amazon and Guianas are considered to be black capped capuchins Cebus apella (Sapajus apella) and those in the south to a further five forms with poorly understood distributions [libidinosus, xanthosternos, robustus, nigritus and, variably, cay, paraguayanus and pallidus. However, the most recent revisions, based on morphology, differ (Groves, 2001; Silva Jr., 2001).

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THE SOUTHERNMOST CEBUS SPECIES Particularly regarding the southernmost distribution species, Cebus nigritus and C. cay, Groves (2001) recognized 3 species: C. libidinosus, Cebus, nigritus, and Cebus xanthosternos. C. libidinosus had 4 subspecies: Cebus libidinosus libidinosus, C. l. pallidus (Gray, 1866), C. l. paraguayanus (Fischer, 1829) and C. l. juruanus (Lönnberg, 1939). Cebus nigritus had 3 subspecies: C. n. nigritus, C. n. robustus, and C. n. cucullatus (Spix, 1823). Finally, Cebus xanthosternos had no subspecies (Fragaszy et al., 2004; Rylands et al., 2005). C. libidinosus paraguayanus and C. libidinosus pallidus are both considered junior synonyms of C. cay according to Silva Jr. (2001) and Rylands et al. (2012). They have a joint distribution that comprises a wide range of habitats at the margin of the southern distribution of the Cebus genus. Also, C. nigritus cucullatus (C. nigritus sensu Silva Jr., 2001) inhabits the Atlantic forest of Brazil along the Doce River in Minas Gerais. Its distribution range extends south along the coast to the northern part of Misiones province in Argentina and includes part of Rio Grande do Sul state (the great river of the south), Brazil (Figure 1). Its westernmost limit abuts the Paraná River which acts as a geographic barrier separating it from C. libidinosus paraguayanus/C. cay paraguayanus (IUCN 2012) (Arístide et al., 2013). C. nigritus (CNI) and C. cay, (CCY) are naturally distributed in Northern Argentina and represent the southernmost geographic distribution of the genus along with a population from Rio Grande do Soul in Brazil. These two Argentinian species show phenotypic variations mostly regarding the color of the body pelage which can vary from brown, dark brown, to black. This is particularly evident with the face (Cabrera, 1957). These similar species illustrate an outstanding feature of the Cebus genome. Although they have a highly homologous karyotype, they are easily distinguishable at the chromosomal level by applying G and C banding techniques. This is due to the absence of the extracentromeric heterochromatin block on chromosome 11 in CNI, which in CCY represents 75% of the q arm of the same chromosome (Mudry, 1990). These species have been the subject of several intraspecific variation studies, mainly at the cytogenetic level (e.g., Matayoshi et al., 1986, 1987; Mudry, 1990; Ponsà et al., 1995; Martinez et al., 2002, 2004; Nieves, 2007), as well as at the molecular genetics level where studies on arboreal primates have increased in recent years. Mainly, the analysis of the non-coding control region (CR) of the mitochondrial DNA (mtDNA) or D-loop shows a high substitution rate with polymorphisms concentrated in the hypervariable region, which has been the focus of several studies for recent demographic events in different populations (Hewitt, 2000). This region has been used in population genetic analyses and captivity surveys in a variety of neotropical primates including the southernmost Cebus species (Ascunce, 2002; Ascunce et al., 2003; Casado et al., 2010). Both approaches revealed the existence of a high degree of intraspecific polymorphism and a marked differentiation between these taxa, in agreement with external phenotype (mainly coat coloration) observations (e.g., Mantecón et al., 1984). In contrast, the traditional morphometric methods using the pattern of variation in skull morphology in southern C. libidinosus (C. cay) populations failed to find any significant differences between them (Avila, 2004). With the aim of providing a more comprehensive explanation of the variability of capuchin monkeys we thoroughly analyzed the data in our laboratory that we have accumulated over the last 30 years. We also reviewed the scientific literature for Cebus

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studies covering karyotype characterization, evolutionary cytogenetics, genome size, molecular genetics, and geometric morphometrics. These data were analyzed using methodologies described below.

Figure 1. Map illustrating the proposed natural distributions for the two southernmost species of Cebus following IUCN Red List maps (IUCN 2012) and Arístide et al., (2013). The origins of samples are marked.

PHENOTYPIC AND GENETIC VARIABILITY Phenotypic diagnosis: taking into account the descriptions of Cabrera (1957) and Groves (2001) we analyzed the accuracy of the taxonomic diagnoses performed with the pool of individuals reviewed in this chapter (Table 1, Figure 1). Cebus nigritus -CNI- (black capuchin) has a very dark brown, gray, or even black pelage. If it has a dorsal stripe it is very dull. Its face is white, sideburns poorly marked and its limbs are darker (usually blackish) than its body. It has pointed crown tufts as an adult but they wear away with age. Cebus cay CCY-(black-striped capuchin) has a yellow-white head with black sideburns. Its light-colored body contrasts with the crown and sideburns. Cebus cay also has a prominent dark dorsal stripe and limbs that are mostly dark to blackish. Its underside is yellowish or reddish, and often overlain with black.

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Mariela Nieves and Marta Dolores Mudry Table 1. List of Cebus cay and C. nigritus samples analyzed for this chapter

SPECIES

Institution

Geographic origin

N

Phenotype

Cebus cay

ZBA

Unknown

48

P1

ZBA/ECAS CAPRIM CAPRIM CRIMOP ZBA Argentina IICS CAPRIM PUCCH

Paraguay Paraguay Paraguay Paraguay Unknown Salta Paraguay Paraguay Perú Unknown Captive born Paraguay Bolivia Salta Salta Salta Salta Salta Unknown Salta Misiones PNI Misiones Misiones Unknown Brazil

42 11 48 15 18 3 11 22 4 2 2 20 8

P1 P1 P1

11 1 3 2 1 2

P3 P1 P3 P3 P3 P3

PEEP EFA

Cebus nigritus

PEEP Argentina

ZBA PUCCH

P2 P1 P1 P1 P1 P1 P1 P2

Cytogenetic characterization References Present work Mudry et al., 1984, Mantecon et al., 1984 Mudry et al., 1987 Mudry and Labal 1988 Mudry 1990

Martinez et al., 2004 Nieves et al., 2008

Present work Present work

Present work Steinberg et al., 2014 Mudry et al., 1991 Mudry 1990 Present work Nieves et al., 2008

P1: Cebus cay from Northeastern populations; P2: Cebus cay from Northwestern populations; P3: Cebus nigritus.

Karyological diagnosis: all the specimens analyzed in this chapter or referred to in previous studies (N = 275, Table 2) were characterized following standard techniques and our own recently reviewed modifications. We cultured heparinized blood samples and conducted G- and C-band protocols (Steinberg et al., 2014). To confirm the species status of each specimen studied, metaphases spreads with conventional staining (G and C banding) were analyzed and compared to the accepted Cebus sp.: Cebus albifrons (García et al., 1976); C. apella (García et al., 1978); C. capucinus (Dutrillaux et al., 1978); C. paraguayanus = C. cay (Matayoshi et al., 1986); C. nigritus (Mudry et al., 1991); C. nigrivitattus (Martinez et al., 1999); and C. xanthosternos (Seuánez et al., 1986).

Table 2. Number of Cebus cay and C. nigritus individuals with C-band polymorphisms for each of the seven chromosome pairs with extracentromeric heterochromatin. Each of the geographic origin of the samples are summarized C+ Chromosome Paraguay pair 4 absence 0 polymorphic 1 6 absence 53 polymorphic 83 11 absence 0 polymorphic 1 12 absence 0 polymorphic 75 13 absence 5 C+ Chromosome Paraguay pair polymorphic 5 17 absence 48 polymorphic 113 19 absence 6 polymorphic 74 N of 171 individuals/origin

Salta

Peru

Bolivia

Misiones

Brazil

Brazil

Unknown

Unknown

0 0 5 4 0 0 0 0 0 Salta

0 0 4 0 0 0 0 0 0 Peru

0 0 0 1 0 0 0 0 0 Bolivia

0 0 0 11 17 0 0 0 0 Misiones

0 0 1 0 1 0 0 0 0 Brazil

0 0 2 0 0 0 0 0 2 Brazil

0 0 1 0 1 0 0 1* 0 Unknown

0 1 6 60 0 0 0 0 1 Unknown

1* 1 4 6 3 9

0 4 0 0 0 4

0 0 1 1 0 1

2* 1 11 4 9 17

1* 1 0 0 0 1

0 0 0 0 0 2

0 0 1 1 0 1

4*§ 2 60 5 60 67

N: number of individuals; *: paracentric inversion; §: Absence/presence of the heterochromatic block.

Captive Born 0 0 1 0 0 0 0 0 0 Captive Born 0 0 2* 0 0 2

N Total

N Total

275

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From this characterization we performed an analysis of the variability of C-band patterns and frequencies of polymorphisms. This was done for the seven chromosome pairs described in Cebus with extracentromeric heterochromatic blocks (#4, 6, 11, 12, 13, 17 and 19) for all specimens (Table 2). Genome Size: To assess genome size, a Feulgen image analysis densitometry was used (Hardie et al., 2002). Air-dried blood smears were prepared from 18 individuals of C. cay and C. nigritus and stored in the dark prior to staining. A minimum of 30 lymphocyte nuclei were measured per individual sample. Integrated optical densities were converted to genome size in picograms using erythrocytes of Gallus domesticus (1C = 1.25pg) as the internal standard. They were stained in the same run as the unknowns. For data analysis and comparison, a 2way nested ANOVA was run using the 2013 version of InfoStat (InfoStat Group, FCA National University of Córdoba, Argentina) (Fantini et al., 2011). Molecular analyses: DNA extractions were carried out using whole blood samples anticoagulated with EDTA, following the protocol of Corach et al., (1995). Through the use of universal primers (L15926 and H00651) the D-loop region was amplified and sequenced obtaining a region of ~600 bp in 43 of the 275 individuals from both species listed in Table 2. Afterwards a new primer pair was designed by using two of the CNI sequences. An amplicon of 450 bp was obtained and tested in the remaining CNI and CCY referred specimens (Hassel et al., 2013). Geometric morphology: a total of 41 specimens of Cebus were selected from collections deposited in the Museo Argentino de Ciencias Naturales (MACN) and Museo de La Plata (MLP). Based on the indicated geographic origin and the natural distributions, each specimen was assigned either to C. nigritus (CNI, specimens from Misiones province, Argentina) or to C. cay without initially considering subspecies (CCY, those from Salta and Jujuy provinces, Argentina; and from Paraguay and Southern Bolivia) (Figure 1). Only adult specimens were used in the analyses. The patterns of skull size and shape variation among the southernmost distributed populations of Cebus were studied using three-dimensional geometric morphometrics techniques (Arístide et al., 2013).

RESULTS AND DISCUSSION As we described in the previous sections, Cebus is considered by many specialist primatologists to be one of the most taxonomically complex Platyrrhini. However, it has been among the least studied, with relatively few published works on its systematics and taxonomy. Of all the disciplines, cytogenetics has probably made the greatest contributions to the understanding of this genus’s diversity. Our expertise in this field of genetics enables us to pose a discussion about the variability observed in the Cebus species from the southernmost geographic distribution. We complement this information with our own data which includes geometric morphometrics, the analysis of genome size and the study of certain mitochondrial sequences. In this chapter we propose a more holistic interpretation for understanding the present day diversity of these species in the context of the evolutionary history of the genus.

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Of the 275 animals we studied, 255 were assigned to Cebus cay (CCY) and 20 to Cebus nigritus (CNI). Three well distinguishable morphotypes were observed (P1, P2, and P3) and are described here (Figure 2).

Figure 2. Illustration of the G-band, C-band and phenotype variability observed in the Cebus cay and Cebus nigritus specimens studied. Colored arrows indicate chromosomal identity: Green, chr.4; Yellow, chr.11; Red, chr.12; Black, chr.13; Blue, chr.17; Turquoise, chr.19. (A) Cebus cay, morphotype 1: (i) Illustration of G-banded karyotype; (ii) C- banded metaphase. Arrows indicate the heterochromatic blocks (Band C+). Note the absence of pair 6 bands C+; (iii) Photograph of a representative morphotype 1 individual. (B) Cebus cay, morphotype 2: (i) Illustration of G-banded karyotype; (ii) C- banded metaphase. Arrows indicate the heterochromatic blocks (Band C +). Note the heteromorphism of chromosomes 17 and 19, given the absence of band C + in one of the counterparts; (iii) Photograph of a representative morphotype 2 individual. (C) Cebus nigritus, morphotype 3: (i) Illustration of G-banded karyotype; (ii) C- banded metaphase. Arrows indicate the interstitial heterochromatic blocks (Band C +). Note the absence of pair 11 bands C+ and the heteromorphism of chromosomes 17 and 19, given the absence of band C + in one or both of the counterparts; (iii) Photograph of a representative morphotype 3 individual. C-band metaphase of (Aii) and G-band karyotype (Ci) were taken from Fantini et al., (2011) and Steinberg et al., (2014) respectively where Drs. Mudry and Nieves are co-authors too.

P1 (A, iii): Cebus cay (morphotype 1) is from the northeastern populations. Both sexes have a tuft of short black hair. The tuft can be divided forming two tufts on the sides of the head, although it is a highly polymorphic trait. The shoulders have a lighter coat than the rest of the body. The mantle and ventral areas of the body vary from light brown to dark brown. The tail is dark at its distal end. P2 (B, iii): Cebus cay (morphotype 2) is from the northwestern populations. It has a yellow-white head with black sideburns. It has a light-colored body with contrasting black

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crown and sideburns. There is also a prominent dark dorsal stripe. The limbs are mainly dark to blackish and the upper arms are not lighter than the body. P3 (C, iii): Cebus nigritus (morphotype 3) has a very dark brown or gray to black pelage. If it has a dorsal stripe it is very dull. Its face is white and its sideburns are poorly marked. The limbs are darker than the body and are usually blackish. Pointed crown tufts are present in adults but wear away with age. The karyological characterization showed different structural rearrangements in C. cay (Mudry et al. 1987, Mudry 1990, and Nieves et al., 2008, Table 1). These were confirmed by G-banding technique according to Matayoshi et al., (1986) for C. paraguayanus (=C. cay). These chromosomal rearrangements correspond to five inversions (chromosomes 2, 4, 7, 13 and 17) and only one translocation (chromosomes 3 and 4). These observations agree with foundational work in chromosomal evolution of neotropical primates. This refers to chromosomal inversions as the most frequent rearrangement followed by translocations (Egozcue and Perkins, 1971; Couturier and Dutrillaux, 1981). In this chapter we analyzed 32 new C. cay individuals in which none of the reorganizations previously described were observed (Figure 2 Ai, Bi). It has to be noted that all the C. cay individuals that showed the rearrangements were from Paraguay or a captive center from Northeastern Argentina (Table 1). All of the C. nigritus individuals showed the accepted G-banding pattern (Mudry et al., 1991) (Figure 2, Ci). Regarding heterochromatin, the analysis of the variability of C-band patterns and frequencies of polymorphisms helped us to identify a species-specific pattern and relatedness to the geographic origin of the samples at the intraspecific level (Table 2; Figures 3 and 4). All the individuals showed the centromeric heterochromatic band (C+) for the whole complement. Considering the seven described chromosome pairs with extracentromeric heterochromatic blocks (#4, 6, 11, 12, 13, 17 and 19), only the interstitial block of pair #4 was observed in 100% of the analyzed metaphases. All the C. cay individuals showed the characteristic heterochromatic telomeric block in pair #11 whereas it was absent in CNI individuals in agreement with previous descriptions (Matayoshi et al., 1986; Mudry et al., 1991). At the same time, different types of polymorphisms including absence/presence of the C+ band, block size and inversions were observed among the 275 analyzed individuals. Chromosome pairs #6, 12, 13, 17 and 19 were polymorphic in different degrees at the interspecific level (Figure 3). Paraguay had the greatest number of individuals with each polymorphism (Figure 4). Pair #17 was the most polymorphic for both species (Figure 3). The next most frequent variable pair is different in C. cay and Cebus nigritus. The absence of pair #6 (homozygosis and heterozygosis) corresponds to the 27% of the variability in C. cay (Figure 3a). Among Cebus nigritus individuals the presence of paracentric inversions or absence/presence of the band in pair #13 explains 23% of the variability (Figure 3b). Although the number of analyzed individuals is significantly different for each geographic origin the variability observed can be described as a particular pattern in each case (Figure 4a). We identified two different patterns for the C. cay individuals: one corresponding to the Paraguayan ones (in the X axis, reference 1) and the other correspond to Salta and unknown origin specimens (in the X axis, reference 2 and 6). In the first pattern, chromosomes #12 and #17 were the most variable whereas in the second case, this variability is predominantly for chromosomal pair #19. For Cebus nigritus specimens, two different patterns were associated with the geographic origin (Figure 4b): the Argentinean specimens from Misiones (reference 1 in X axis) and the Brazilian animals (reference 2, X axis). The

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specimens of unknown origin (reference 3 X, axis) showed a pattern similar to the Argentineans although with some particularities (difference in frequency for pairs # 12 and #13).

Figure 3. Percentage of C-band’s polymorphisms observed in the seven chromosome pairs with extra centromeric heterochromatin. (A) C. cay, (B) C. nigritus.

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1: Paraguay; 2: Salta, Argentina; 3: Peru; 4: Bolivia; 5: Brazil; 6: Unknown origin; 7: Captive born individuals

1: Misiones, Argentina; 2: Brazil; 3: Unknown origin Figure 4. Distribution of the number of individuals with each polymorphic pair for each of the analyzed geographic origins. (A) Cebus cay, (B) C. nigritus.

The patterns of heterochromatin variation for both species is associated to the geographic origin of the samples and can be related to the three morphotypes described in this chapter. Morphotype 1 (P1) can be associated with the Paraguayan C. cay and the heterochromatin variation pattern 1 in Figure 4a. Morphotype 2 (P2) can be related to C. cay individuals from Salta, Bolivia, Peru and unknown ones and the heterochromatin pattern referred to as 2 and 6

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(Figure 4a). Morphotype 3 (P3) corresponds to C. nigritus specimens and the heterochromatin pattern referred to as 1 in Figure 4b. Taking into account one of the other genomic variables analyzed in the present chapter, a pool of specimens from different origins were considered for the genome size estimations. This subsample included 18 specimens from PEEP, Posadas, Misiones (Argentina); EFA, Salta (Argentina) and REHM, Tucuman (Argentina) (Table 1). The first analysis of genome size of C. cay was obtained from PEEP samples (3.40pg) (Fantini et al., 2011). In the same study, genome size of Cebus nigritus, also from PEEP specimens, was 3.04pg. These values were significantly different between the two species. The extended sampled including specimens from other institution of Northwestern Argentina decreased the average value of C. cay considerably while significantly increasing its variability. There is no clear distinction between the two C. cay groups in terms of C-value. However, the specimens from Paraguay showed the highest homogeneous values relative to those from Northern Argentina (Fantini L., unpublished data). This significant genome size variability observed in the southernmost populations of C. cay has also been observed by analyzing other variables like molecular genetic studies and geometric morphometrics data obtained from museum skull collections discussed below. Our analysis with molecular genetic tools of the 515bp D-loop sequence within a subset of individuals allowed us to clearly identify five haplogroups. The most common haplogroup mainly included C. cay and Cebus nigritus housed in PEEP and RGO from Misiones, Argentina, three C. cay from ZBA and one specimen from EFA. The second most common haplogroup mostly included C. cay derived from EFA. A third haplogroup included only three C. cay specimens from ZBA with unknown origins. Haplogroup four contained individuals from EFA and one C. cay from ZBA. We suggest that the first haplogroup represents the northeastern region of Argentina. On the other hand the second and fourth haplogroups could represent the northwestern region of Argentina. This situation will be confirmed with the analysis of a great number of individuals along the entire natural distribution (Hassel D., unpublished data). These results are mostly in agreement with the interpretation of the cytogenetic findings regarding C-band patterns and polymorphisms associated with the different geographic origin of the C. cay samples (Figure 4a). Specifically, the individuals from Paraguay (Table 1) showed one C-pattern different from EFA. It also showed specimens of unknown origin sharing the distribution of some of the haplogroups (first and second-fourth). The C. nigritus subset of samples, showed a unique haplogroup in agreement with cytogenetical data previously described (Figure 4b). In addition, the animals from ZBA with unknown origin showed a C-pattern similar to those of EFA, again sharing the above mentioned D-loop haplogroups. This last observation complements the morphometric geometric of the same two species, described below and discussed later. In this context, we explored and quantified skull size patterns and shape variation among the southernmost distributed populations of Cebus using three-dimensional geometric morphometrics techniques. It is necessary to mention that the cranial samples used do not correspond to the same individuals analyzed in the genetic study. We tested for differences among four sets of skull samples from southern Cebus populations (Misiones and Salta-Jujuy in Argentina, Paraguay and Bolivia). We discussed these patterns in an evolutionary and taxonomic context in order to provide new data on the phenotypic variation of Cebus (Arístide et al., 2013). Our results showed that skull size and shape variation among southern C. cay and C. nigritus are indicative of a marked morphological differentiation between them. Also, the degree of morphological differentiation among the C. cay populations with respect

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to C. nigritus was qualitatively related to the geographic distance between them. Individuals from Paraguay were morphologically closer to C. nigritus than the other two populations. The Salta-Jujuy sample showed the biggest morphological distance to both populations. However, geographically close populations were morphologically more similar than distant ones. Even taking this into account, the Salta and Jujuy populations are closely related in shape and geographically to the Bolivia population, yet the former populations had much smaller skull sizes than the latter population (Arístide et al., 2013).

AN INTEGRATIVE ANALYSIS OF CEBUS CAY AND C. NIGRITUS DIVERSITY The extent of morphological divergence can be related to the geographic separation between populations. Furthermore, since cranial morphology is expected to have a strong genetic component, then, divergence observed in a particular region may be indicative of population evolutionary differentiation (Marroig and Cheverud, 2001; Arístide et al., 2013). In this context, it is expected that the extensive geographic distribution of some Cebus species will be accompanied by high levels of variability between populations. This scenario is congruent with the findings we obtained by analyzing the other 275 individuals of C. cay and C. nigritus (reviewed in Table 1) with a cytogenetic methodological approach. The described skull morphological variation patterns support the differentiation between C. nigritus and C. cay and also the differentiation among Northwest Argentinean and Southern Bolivian and Paraguayan forms. The chromosomal analysis, specifically the variability in heterochromatin patterns, also showed a clear differentiation between species (Figure 3). Likewise, there was a differential heterochromatin polymorphisms pattern between Paraguayan and northwestern Argentinean/Bolivian/Peruvian samples of C. cay (Figure 4). In the analysis performed for this chapter, the number of samples from Bolivia and Peru was insufficient to detect any polymorphism associated to its origin. This was also the case for skull variability. Given the geographic proximity between Paraguay and the C. nigritus distribution area and the fact that the skull morphology of C. Cay in Paraguayan populations is more similar to C. nigritus than to any of the other C. cay analyzed populations, may be suggestive of an ongoing or recent genetic flow between the two populations. This proposal gains support from cytogenetics if we take into account the observations we have made considering the first descriptions of the karyotype of C. apella vellerosus = C. nigritus (Mudry, 1990; Mudry et al., 1991) and our study of new specimens with known origins. Why? The lack of information about adjacent species populations disallows species distribution delimitation, thus leading to taxonomic uncertainties and erroneous characterization at the genetic level. C. nigritus populations from Brazil and Argentina have largely been ignored from the cytogenetic level of characterization. Indeed, there is no published works on C. nigritus cytogenetic variability other than the referred karyotype characterization performed by Mudry (1990), Steinberg et al., (2014) and the analysis of 20 new individuals in this chapter. Only recently, Penedo et al., (2014) presented an interesting work of cytogenetic characterization of a considerably large captive colony of Cebus sp. (Sapajus sp. according to Lynch-Alfaro et al., 2012). These authors analyzed the C-banding patterns and the phenotypes of 26 individuals aiming to

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establish a standardized protocol of species diagnosis for management purposes. They identified two (2) phenotypically C. nigritus individuals that presented a total deletion of the heterochromatic block of pair #11 in heterozygosis. The authors proposed that this pattern may be indicative of hybridization or, on the other hand that this deletion derives from sporadic chromosomal mutation in heterozygosis. Based on the scientific literature and particular cases in other Cebus species (Amaral et al., 2008 for Cebus robustus) where the deletion was observed in homozygosis Penedo et al., (2014) concluded that this feature may be an inherited mutation, since the individuals studied were siblings according to the technicians who performed the screening. In addition, they proposed that the deletion described by Mudry (1990) and Mudry et al., (1991) as specific to C. nigritus could be typical of a restricted population in Argentina rather than the entire distribution area of this species, which ranges from Northeastern Argentina to Southeastern Brazil (Vilanova et al., 2005). Based on these variability data and our current state of knowledge about the characterization of C. nigritus populations, we agree with Penedo et al., (2014). Cytogenetic studies of different Brazilian populations of C. nigritus need to be carried out in order to verify if the complete deletion in pair 11 is characteristic of this species or represents a regional geographical polymorphism. Finally, as was previously described by Coyne and Orr (2004), the populations at the extremes of the distribution of a genus or a species frequently exhibit a different pattern at multiple levels than the one observed in the center of the distribution. In this sense, comparing our findings for the southernmost populations of Cebus to published data about the populations of the northernmost species of the genus (Cebus albifrons) a parallel scenario can be identified. The cytogenetic characterization of C. albifrons detected the presence of two chromosomal numbers, 2N = 52 and 2N = 54, with a noticeable variability of C-banding patterns and G-band rearrangements among populations (Koiffmann and Saldanha, 1974; García et al., 1976; Bueno, pers. comm.). Phenotypically, it is one of the most diverse species of the genus with up to 13 subspecies proposed by Hershkovitz (1949). Additionally, RuizGarcía et al. (2010a) analyzed almost 700bp of the mtDNA COII gene in a sample of individuals that included eight of the 13 described subspecies. They found several groups associated with particular geographic distribution areas but none directly related to a specific phenotype. Therefore, there is no correlation among a specific phenotype, karyotype and genotype. It appears that some individuals can share the same karyotype and/or phenotype but different COII haplogroup and vice versa. These observations are in agreement with what we discussed before with Cebus cay and C. nigritus samples and are evidence of a shared pattern associated with the limits of the genus’s distribution. We believe that the ultimate goal should be to consider different complexity levels identifying the properties or characteristics that emerge from each level. Their interaction with many informational strata will allow a more realistic interpretation of the evolutionary process. Thereafter, the establishment of complementary studies about these species in regards to their biology, behavior, morphology, biogeography and genetics is urgent. The aim is to increase our knowledge on Cebus diversity. This will help us to propose strategies for species management and conservation.

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ACKNOWLEDGMENTS This chapter was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Project PIP 0744 and Universidad de Buenos Aires Ciencia y Tecnología grants (UBACyT) Project X154 to MDM. We thank Parque Ecológico El Puma (PEEP), the owners of the Private Reserve Güira Oga (RGO) in Misiones Province, the Estación de Fauna Autóctona (EFA) in Salta and Zoológico de Buenos Aires (ZBA) for providing the corresponding permits and transit guides to access and transport the specimen samples and specially to the veterinarians who helped us manage the animals. The research reported in this chapter met all the guidelines for the study of wild animals set forth by the Ethical Committee of the Argentine Society for Mammalian Studies (SAREM).

REFERENCES Amaral, P.J.S., Finotelo, L.F.M., De Oliveira, E.H.C., Pissinatti, A., Nagamachi, C.Y. and Pieczarka, J.C. (2008). Phylogenetic Studies of the Genus Cebus (Cebidae-Primates) Using Chromosome Painting and G-Banding. BMC Evolutionary Biology 8: 169. Arístide, L., Soto, I.M., Mudry, M.D. and Nieves, M. (2013). Intra and Interspecific Variation in Cranial Morphology on the Southernmost Distributed Cebus (Platyrrhini, Primates) Species. Journal of Mammalian Evolution. doi:10.1007/s10914-013-9249-y. Ascunce, M.S., Hasson, E. and Mudry, M.D. (2003). COII: A Useful Tool for Inferring Phylogenetic Relationships among New World Monkeys (Primates, Platyrrhini). Zoologica Scripta 32(5): 397–406. Ascunce, M.S. (2002). Variabilidad Nucleotípica En El ADN Mitocondrial de Alouatta caraya Del NE de Argentina. Tesis de Doctorado, Buenos Aires - Argentina: Universidad de Buenos Aires. Assumpçao, C. (1983). An Ecological Study of the Primates of Southeastern Brazil with a Reappraisal of Cebus apella Races. Ph.D., University of Edinburgh. http://ethos.bl.uk/ OrderDetails.do?uin=uk.bl.ethos.345737. Avila, I. (2004). Morphological Variation between Two Subspecies of Cebus libidinosus (Primates: Cebidae). Boletin Del Museo Nacional de Historia Natural, Paraguay 15: 1–8. Bi, X., Huang, L. Jing, M., Zhang, L., Feng, P. and Wang, A. (2012). The Complete Mitochondrial Genome Sequence of the Black-Capped Capuchin (Cebus apella). Genetics and Molecular Biology 35(2): 545–52. Cabrera, A. (1957). Catálogo de Los Mamíferos de América Del Sur. Revista Del Museo Argentino de Ciencias Naturales, Ciencias Zoológicas IV(1): 308. Cáceres, N., Meloro, C., Carotenuto, F., Passaro, F., Sponchiado, J., Melo, G.L. and Raia, P. (2014). Ecogeographical Variation in Skull Shape of Capuchin Monkeys. Journal of Biogeography 41(3): 501–12. Casado, F., Bonvicino, C.R., Nagle, C., Comas, B., Manzur, T.D., Lahoz, M.M. and Seuánez, H.N. (2010). Mitochondrial Divergence between 2 Populations of the Hooded Capuchin, Cebus (Sapajus) Cay (Platyrrhini, Primates). The Journal of Heredity 101(3): 261–69. Corach, D., Penacino, G. and Sala, A. (1995). Cadaveric DNA Extraction Protocol Based on Cetyl Trimethyl Amonium Bromide (CTAB). In: Acta Medicinæ Legalis Vol. XLIV 1994,

Diversity of Cebus Species from the Southern Distribution of the Genus

131

edited by Professor Dr Patrice Mangin and Dr Bertrand Ludes, 35–36. Springer Berlin Heidelberg. Couturier, J., and Dutrillaux, B. (1981). Conservation of Replication Chronology of Homologous Chromosome Bands between Four Species of the Genus Cebus and Man. Cytogenetics and Cell Genetics 29(4): 233–40. Coyne, J.A. and Orr, H.A. (2004). Speciation. SINAUER ASSOCIATES, INC Publishers. 545 pp. da Silva, K.V., Moura, E.F., Montenegro, M.M.V., Laroque, P., Ferreira, J.G., and Martins, A.B. (2010). Análise Comparativa Da Variabilidade Cariotípica de Cebus flavius (Schreber, 1774) E Cebus libidinosus Spix, 1823. Anais Do II Seminário de Pesquisa E Iniciação Científica Do Instituto Chico Mendes de Conservação Da Biodiversidade. Dutrillaux, B., Couturier, J., Viegas-Péquignot, E., Chauvier, G. and Trebbau, P. (1978). Presence of abundant heterochromatin in the karyotype of Cebus: C. cappucinus and C. nigrivittatus. Annales de genetique 21(3): 142–48. Egozcue, J. and Perkins, E.M. (1971). Chromosomal Evolution in the Platyrrhini. S. Karger. Fantini, L., Mudry, M.D. and Nieves, M. (2011). Genome Size of Two Cebus Species (Primates: Platyrrhini) with a Fertile Hybrid and Their Quantitative Genomic Differences. Cytogenetic and Genome Research 135: 33–41. Fragaszy, D.M., Visalberghi, E. and Fedigan, L.M. (2004). The Complete Capuchin. Cambridge University Press. García, M., Freitas, L., Miró, R. and Egozcue, J. (1976). Banding Patterns of the Chromosomes of Cebus albifrons. Folia Primatologica 25(4): 313–19. Groves, C.P. (2001). Primate Taxonomy. Washington D.C., USA: Smithsonian Institution Press. Hardie, D.C., Gregory, T.R. and Hebert, P.D.N. (2002). From Pixels to Picograms: A Beginners’ Guide to Genome Quantification by Feulgen Image Analysis Densitometry. The Journal of Histochemistry and Cytochemistry 50(6): 735–49. Hassel, D.L., Mudry, M.D., Argüelles, C.F. and Nieves, M. (2013). Haplotipos mitocondriales en Cebus nigritus y Cebus libidinosus basados en el análisis del d‐loop. XLII Congreso Argentino de Genética y III reunión de la Regional NOA de la Sociedad Argentina de Genética. Salta, Argentina. Hershkovitz, P. (1949). Mammals of Northern Colombia. Preliminary Report No. 4 : Monkeys (Primates), with Taxonomic Revisions of Some Forms. Proceedings of the United States National Museum 98: 323–427. Hewitt, G. (2000). The Genetic Legacy of the Quaternary Ice Ages. Nature 405(6789): 907–13. Hill, WCO. (1960). Primates: Comparative Anatomy and Taxonomy. Vol. VI Cebidae, Part A. Edinburgh: Edinburgh University Press. Koiffmann, C.P. and Saldanha, P.H. (1974). Cytogenetics of Brazilian Monkeys. Journal of Human Evolution 3(4): 275–82. Lynch-Alfaro, J.W., Boubli, J.P., Olson, L.E., Di Fiore, A., Wilson, B., Gutiérrez-Espeleta, G.A., Chiou, K.L. et al., (2012). Explosive Pleistocene Range Expansion Leads to Widespread Amazonian Sympatry between Robust and Gracile Capuchin Monkeys. Journal of Biogeography 39(2): 272–88.

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Lynch-Alfaro, J.W., Sousa, J.D.E., Silva Jr, E. and Rylands, A.B. (2012). How Different Are Robust and Gracile Capuchin Monkeys? An Argument for the Use of Sapajus and Cebus. American Journal of Primatology 74(4): 273–86. Madden, M., Hinley, A.J., Fragaszy, D.M., Ottoni, E.B., Visalberghi, E. and Izar, P. (2007). Modelling the Monkey Habitat in Brazil. http://stoa.usp.br.sci-hub.org/ebottoni/ files/435/6692/2007_GT_Madden%26al.pdf. Mantecón, M.A.F., Mudry, M.D. and Brown, A.D. (1984). Cebus apella de Argentina, Distribución Geográfica, Fenotipo Y Cariotipo. Revista Del Museo Argentino de Ciencias Naturales, Zoología, XIII(41): 399–408. Marroig, G. and Cheverud, J.M. (2001). A Comparison of Phenotypic Variation and Covariation Patterns and the Role of Phylogeny, Ecology, and Ontogeny During Cranial Evolution of New World Monkeys. Evolution 55(12): 2576–2600. Martinez, R., Aguilera, M. and Ferreira, C. (1999). The Karyotype and C-Banding of Cebus Nigrivittatus from the Coastal Cordillera, Venezuela. Folia Primatologica, International Journal of Primatology 70(1): 37–40. Martinez, R.A., Giudice, A.M., Szapkievich, V., Ascunce, M.S., Nieves, M., Zunino, G.E. and Mudry, M.D. (2002). Parameters Modeling Speciogenic Processes in Cebus apella (Primates: Platyrrhini) from Argentina. Maztozoología Neotropical 9(2): 171–86. Martinez, R.A., Torres, E., Nieves, M., Szapkievich, V., Rodríguez, S., Schinini, A., Ascurra, M. and Mudry, M.D. (2004). Genetic Variability in Two Captive Colonies of Cebus apella paraguayanus (primates: Platyrrhini) from Eastern Paraguay. Caryologia 57(4), 332–36. Matayoshi, T., Howlin, E., Nasazzi, N., Nagle, C., Gadow, E. and Seuánez, E.N. (1986). Chromosome Studies of Cebus apella: The Standard Karyotype of Cebus apella paraguayanus, Fischer, 1829. American Journal of Primatology 10(2): 185–93. Matayoshi, T., Seuánez, H.N. and Nasazzi, N. (1987). Heterochromatic Variation in Cebus apella (Cebidae, Platyrrhini) of Different Geographic Regions. Cytogenetic Cell Genetics 44: 158–62. Mudry, M.D. (1990). Cytogenetic Variability within and across Populations of Cebus apella in Argentina. Folia Primatologica 54(3-4): 206–16. Mudry, M., Slavutsky, I., Zunino, G.E., Delprat, A. and Brown, A.D. (1991). A New Karyotype of Cebus apella from Argentina. Revista Brasilera de Genética 14(3): 729–38. Nieves, M. (2007). Heterocromatina Y Evolución Cromosómica En Primates Neotropicales. Tesis de Doctorado, Universidad de Buenos Aires. Nieves, M., De Oliveira, E.H.C., Amaral, P.J.S., Nagamachi, C.Y., Pieczarka, J.C., Mühlmann, M.C. and Mudry, M.D. (2011). Analysis of the Heterochromatin of Cebus (Primates, Platyrrhini) by Micro-FISH and Banding Pattern Comparisons. Journal of Genetics 90(1): 111–17. Nieves, M., Mendez, G., Ortiz, A., Mühlmann, M.C. and Mudry, M.D. (2008). Karyological Diagnosis of Cebus (Primates, Platyrrhini) in Captivity: Detection of Hybrids and Management Program Applications. Animal Reproduction Science 108(1-2): 66–78. Penedo, D.M., Azevedo de Armada, J.L., Santos da Silva, J.F., Marchesi Neves, D., Pissinatti, A. and Monnerat Nogueira, D. (2014). C-Banding Patterns and Phenotypic Characteristics in Individuals of Sapajus (Primates: Platyrrhini) and Its Application to Management in Captivity. Caryologia 67(4): 314–20.

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Ponsà, M., García, M., Borell, A., Garcia, F., Egozcue, J., Gorostiaga, M.A., Delprat, A. and Mudry, M.D. (1995). Heterochromatin and Cytogenetic Polymorphisms in Cebus apella (Cebidae, Platyrrhini). American Journal of Primatology 37(4): 325–31. Ruiz-García, M., Castillo, M.I., Lichilín-Ortiz, N. and Pinedo-Castro, M. (2012). Molecular Relationships and Classification of Several Tufted Capuchin Lineages (Cebus apella, Cebus xanthosternos and Cebus nigritus, Cebidae), by Means of Mitochondrial Cytochrome Oxidase II Gene Sequences. Folia Primatologica 83(2): 100–125. Ruiz-García, M., Castillo, M.I., Váskez, C., Rodriguez, K., Pinedo, M., Shostell, J.M. and Leguizamon, N. (2010). Molecular Phylogenetics and Phylogeography of the WhiteFronted Capuchin (Cebus albifrons; Cebidae, Primates) by Means of mtCOII Gene Sequences. Molecular Phylogenetics and Evolution 57: 1949–1961. Rylands, A.B, Mittermeier, R.A. and Silva Jr, J.S. (2012). Neotropical Primates: Taxonomy and Recently Described Species and Subspecies. International Zoo Yearbook 46(1): 11– 24. Rylands, A.B. (2000). An Assessment of the Diversity of New World Primates. Neotropical Primates 8(2): 61–93. Rylands, A.B., Kierulff, M.C.M. and Mittermeier, R.A. (2005). Notes on the Taxonomy and Distributions of the Tufted Capuchin Monkeys (Cebus, Cebidae) of South America. Lundiana 6(suppl): 97–110. Seuánez, H.N., Armada, J.L., Freitas, L., Rocha E Silva, R., Pissinatti, A. and Coimbra‐Filho, A. (1986). Intraspecific Chromosome Variation in Cebus apella (cebidae, Platyrrhini): The Chromosomes of the Yellow Breasted Capuchin Cebus apella xanthosternos Wied, 1820. American Journal of Primatology 10(3): 237–47. Seuánez, H.N., Bonvicino, C.R. and Moreira, M.A.M. (2005). The Primates of the Neotropics: Genomes and Chromosomes. Cytogenetic and Genome Research 108(1-3): 38–46. Silva Jr., J.S. (2001). Especiação nos macacos-prego e caiararas, gênero Cebus Erxleben, 1777 (Primates, Cebidae). Tesis Doctoral, Rio de Janeiro, Brasil: Universidade Federal do Rio de Janeiro. Steinberg, E.R., Nieves, M., Fantini, L. and Mudry, M.D. (2014). Primates Karyological Diagnosis and Management Programs Applications. Journal of Medical Primatology 43(6): 455–67. Vilanova, R., Silva Jr., J.S., Viveiros Grelle, E.C., Marroig, G. and Cerqueira, R. (2005). Limites Climaticos E Vegetacionais Das Distribuições de Cebus nigritus E Cebus robustus (Cebinae, Platyrrhini). Neotropical Primates 13(1): 14–19. Wilson, D.E. and Reeder, D.M. (2005). Mammal Species of the World. JHU Press. Wright, K.A., Wright, B.W., Ford, S.M., Fragaszy, D., Izar, P., Norconk, M., Masterson, T., Hobbs, D.G., Alfaro, M.E. and Lynch-Alfaro, J.W. (2015). The Effects of Ecology and Evolutionary History on Robust Capuchin Morphological Diversity. Molecular Phylogenetics and Evolution 82PtB, 455–66.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 5

GENETIC STRUCTURE, SPATIAL PATTERNS AND HISTORICAL DEMOGRAPHIC EVOLUTION OF WHITE-THROATED CAPUCHIN (CEBUS CAPUCINUS, CEBIDAE, PRIMATES) POPULATIONS OF COLOMBIA AND CENTRAL AMERICA BY MEANS OF DNA MICROSATELLITES Manuel Ruiz-García and María Ignacia Castillo Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética, Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC., Colombia

ABSTRACT We analyzed 54 white-throated capuchin (Cebus capucinus, Cebidae, Primates) from four different geographical regions (three in Colombia and one in Central America) for nine nuclear DNA microsatellites. This study is complementary to that of Ruiz-García et al., (2012), which showed the first molecular systematic study with mitochondrial data for this species. Our study revealed seven main findings. 1- Gene diversity for C. capucinus was medium to high but lower for the Central American population than for the Colombian ones for both kind of markers. 2- Although a large fraction of the microsatellites showed a neutral behavior, AP74 and D5S111 yielded some evidence of positive selection and D8S165 showed evidence of negative selection. 3- Genetic heterogeneity procedures support very limited genetic differentiation among the Colombian populations and a relatively more differentiated Central American population. However, genetic data suggest that the C. capucinus populations are not genetically disconnected. 4- Genetic assignment analyses detected elevated percentages of misclassified individuals and some relevant cases of first generation migrants among Colombian populations. Additionally, for the majority of cases and based on STRUCTURE analysis, the entire study area had a unique gene pool. 5- No bottleneck 

Correspondence: [email protected], [email protected].

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Manuel Ruiz-García and María Ignacia Castillo events were detected neither for the overall population studied nor for any one of the Colombian populations. In contrast, there was also evidence of population expansions for microsatellites and mitochondrial DNA, which agrees quite well with the Pleistocene Refugia hypothesis. 6- There was no evidence of a spatial trend in genetic structure within Colombia. For both Colombia and Central America taken together, some procedures detected a degree of significant negative autocorrelation at the most distant geographical areas (some Colombian and Guatemalan points). However, other procedures did not record significant genetic structure. In whatever case, this spatial structure was weaker than that detected for mitochondrial markers. 7- From a systematic point of view and taking into account both microsatellite and new mitochondrial data it may be prudent to consider the existence of a unique monotypic species (C. capucinus). There may be as many as two subspecies, one in Colombia (C. c. capucinus) and one in Central America (C. c. imitator), although we consider a single monotypic species a more likely scenario.

Keywords: Cebus capucinus, nuclear DNA microsatellites, mitochondrial DNA, gene diversity, gene heterogeneity, gene flow, genetic assignment and structure, population expansions, spatial autocorrelation, Pleistocene Refugia, systematics

INTRODUCTION The white-throated capuchin, Cebus capucinus (Cebidae, Primates) has the most northern distribution of all capuchin species. It has a weight ranging from 1.5 to 4 kg with males heavier than females. Its group size and population density vary somewhat geographically. Groups range from 10 to 20 individuals (18-24 individuals/km2) in Barro Colorado, Panama (Oppenheimer, 1969; Robinson and Janson, 1987) and from 15 to 20 individuals (30 individuals/km2) in Santa Rosa, Costa Rica (Freese, 1976). Its geographic distribution range extends from the Guatemala–Belize border (personal observation in 2006) and Honduras through Central America into Northern and Western Colombia, to Northern Ecuador (Carchi, Esmeralda, Imbabura). In Colombia, the distribution of this species goes from the country’s border with Panama, extending southward along the Pacific Coast and along the western slope of the Western Andean Cordillera. Its distribution includes the Northwestern Antioquia Department (Uraba), the Departments of Cordoba, Sucre, Bolivar and Southwestern Atlantico (eastward to the west bank of the lower Magdalena River). It also extends eastward to the San Jorge River and includes Gorgona Island on the Pacific coast (Nariño Department), the upper Cauca Valley, and the piedmont area of the Valle del Cauca Department (from coastal areas to 2,100 m above sea level, masl). Some authors have described three C. capucinus subspecies in Colombia. The alleged subspecies are: C. capucinus (Linneus in 1758; origin north of Colombia, in the vicinity of Cartagena de Indias), C. c. curtus (Bangs in 1905; Gorgona Island) and C. c. nigripectus (Eliot in 1909; Las Pavas, Cauca Valley, between Cali and Buenaventura). Geoffroy-SaintHilare (1812) determined a fourth possible subspecies in Colombia (C. c. hypoleucus) located in the Bolivar Department (Sinú River). In Central America, two subspecies have been traditionally considered: C. c. limitaneus (Belize, Honduras, Nicaragua and Guatemala) and C. c. imitator (in Panama, including Coiba Island, and Costa Rica) (Rylands et al., 1997). Nevertheless, Rylands et al., (2000) only considered a total of four subspecies,

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C. c. capucinus in Colombia and Ecuador, C. c. curtus from Gorgona Island, and C. c. limitaneus and C. c. imitator in Central America. Other authors such as Hernández-Camacho and Cooper (1976) and Mittermeier and Combra-Filho (1981) did not recognize any subspecies within C. capucinus because they found pelage characters too variable to permit sub-specific recognition. Identically, Groves (2001), Defler (2010) and Silva Jr (2001) were of the same opinion and they considered this species monotypic. Hernández-Camacho and Cooper (1976) were the first to show the existence of hybrid specimens between this species and C. albifrons, with intermediate phenotypes in the San Jorge River in Colombia. More recently, we have observed hybrids between both Cebus taxa in the same area and in other Colombian areas. This species has been classified by IUCN as in Low Risk with the exception of one of the possible subspecies (the island population of Gorgona; Vulnerable; Rylands et al., 1997). To date only one molecular population genetics and phylogenetic study has been published with C. capucinus (Ruiz-García et al., 2012). That work analyzed 710 base pairs (bp) of the cytochrome c oxidase subunit II mitochondrial gene (mt COII) in 121 C. capucinus specimens sampled in the wild. The animals came from the borders of Guatemala and Belize, Costa Rica, and eight different departments of Colombia (Antioquia, Chocó, Sucre, Bolivar, Córdoba, Magdalena, Cauca, and Valle del Cauca). That study provided five main findings. 1- The Colombian C. capucinus population yielded elevated gene diversity levels (27 haplotypes; haplotypic gene diversity, Hd = 0.941 ± 0.015; nucleotide diversity,  = 0.023 ± 0.001), compared to the Central American population (15 haplotypes; Hd = 0.691 ± 0.049;  = 0.011 ± 0.005). 2- Four different mitochondrial haplogroups were detected in Colombia, two with elevated levels of gene diversity (haplogroups II and III). They were highly heterogeneous. 3- There were three haplogroups consisted of specimens from multiple areas of Colombia (one had a natural hybrid between C. albifrons and C. capucinus coming from the San Jorge River). There were two specimens from the Valle del Cauca Department (sampled near Buenaventura), that did not clearly belong to any of these three genetic ensembles (94%), constituting a fourth haplogroup within Colombia. These three main haplogroups were not restricted to any determined Colombian geographical area and they were sympatric in different Colombian departments where C. capucinus lives. Therefore they could not be clearly assigned to previously defined morphological subspecies (C. c. capucinus, C. c. nigripectus, C. c. hypoleucos). Additionally, the ancestor of one Colombian haplogroup (III) could be the original ancestor from which all the other C. capucinus populations derived. 4- Multiple population expansions events were detected in the different mitochondrial haplogroups. 5- One Colombian haplogroup (II) seems to be the source where the Central American haplogroup originated around 3.66 ± 0.59 millions of year ago (MYA). The total split among the three main Colombian haplogroups ranged from 2.30 to 1.74 MYA. The current haplotype diversification within each one of the Colombian haplogroups ranged from 0.49 to 0.078 MYA. Thus, the temporal haplotype diversification process for C. capucinus in Colombia occurred during the Pleistocene period. In this work, we analyzed a fraction of the specimens originally sequenced for mtDNA in Ruiz-García et al., (2012) but now for a set of nuclear DNA microsatellites, which can offer new results (contribution of males and different evolutionary rates) to understand the evolution of C. capucinus in its geographical distribution range. We used nine hyperpolymorphic microsatellites or STRPs (Short Tandem Repeat Polymorphisms) applied to

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three different geographical Colombian C. capucinus samples and one Central American (Costa Rica and Guatemala) sample (total sample size of 54 across all localities). These kinds of markers are composed of short repetitive elements of one to six nucleotide base pairs in length. They are also randomly distributed, highly polymorphic, and are frequently inside the eukaryotic genomes. An additional and positive property of these markers is the small DNA quantity needed to carry out these molecular analyses (via PCR). The small sample size allows the investigator to use non-invasive procedures to sample wild animals and successfully examine population biology dynamics through the use of molecular genetic techniques (Bruford and Wayne, 1993) as well as to establish gene linkage maps. In this case, we compare the results obtained by means of the microsatellites with those from the mitochondrial DNA. The main aims of this work are as follows. 1- To determine the levels of gene diversity in the C. capucinus populations sampled; 2- To analyze if the microsatellites employed are strictly neutral or if some are affected by natural selection; 3- To estimate the degree of genetic heterogeneity and possible gene flow among the populations by means of microsatellites and to compare these results with those obtained with mtDNA; 4- To estimate the degree of structure and assignation of individuals to their corresponded populations and to analyze if this agrees well with the existence of sympatric mitochondrial haplogroups in the different Colombian Departments; 5- To determine if there were historical demographic changes (bottlenecks and/or population expansions) across the natural history of C. capucinus and if so, to compare them with population expansions detected through the analysis of mtDNA; 6- To estimate the possible existence of spatial patterns in all the geographical area studied as well as only in Colombia and 7- To provide new insights about the systematics of this taxon through pooling together microsatellite and mitochondrial DNA data.

MATERIAL AND METHODS We analyzed 54 C. capucinus for nine DNA microsatellites. The geographical areas where these specimens were sampled were as follows: 19 animals from the Cauca and Valle del Cauca Departments (including three individuals from Gorgona Island), 20 animals from diverse northern Colombian Departments (Turbo, Antioquia; El Banco, Magdalena; Tolu and San Marcos, Sucre; Montería, San Jorge River and Puerto Libertador, Cordoba and Magangue and Zambrano, Bolivar), 11 animals from the Choco Department (Rio Sucio, Acandi, Los Katios National Park and Utria) and four animals from Central America (three from Guatemala, near the Honduras frontier and one from Manuel Antonio National Park in Costa Rica). For brevity, these populations will be named as Cauca, Northern Colombia, Choco, and Central America, respectively. The samples for DNA extraction were from pieces of tissue (muscle) or teeth from hunted animals that were discarded during the cooking process in different Indian and African-ancestry communities living in the Pacific and northern areas of Colombia, or hairs with bulbs plucked from live pets in the same communities. For the Central American individuals we only sampled hairs with bulbs.

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Molecular Procedures DNA from muscle was isolated using standard phenol–chloroform extraction methods (Sambrook et al., 1989), while DNA from teeth and hair were isolated by boiling with 10% Chelex resin following Walsh et al., (1991). We used nine microsatellite markers (AP68, AP74, D5S111, D5S117, D6S260, D8S165, D14S51, D17S804, and PEPC3). The AP68 and AP74 markers were designed for Alouatta palliata and PEPC3 for Cebus apella, while the remaining markers were designed for humans (Ellesworth and Hoelzer 1998). These microsatellites have been successfully used in other Neotropical primates such as Alouatta, Ateles, Lagothrix, Cebus, Saimiri, Aotus and Saguinus (Ruiz-García 2005; Ruiz-García et al., 2006, 2007, 2011). Our final PCR volume and reagent concentrations for the DNA extraction from muscle were 25 μl, with 3 μl of 3 mM MgCl2, 2.5 μl of buffer 10×, 1 μl of 0.04 mM dNTP, 1 μl of each primer (forward and reverse; 4 pmol), 13.5 μl of H2O, 2 μl of DNA, and 1 Taq polymerase unit per reaction (1 μl). For the PCR reactions with hair and teeth, the overall volume was 50 μl, with 20 μl of DNA and twofold amounts of MgCl2, buffer, dNTPs, primers, and Taq polymerase. We performed all PCR reactions in a PerkinElmer Geneamp PCR System 9600 thermocycler for 5 min at 95°C, 30 1min cycles at 95°C, 1 min at the most accurate annealing temperature (50°C for AP68, and 52°C for the remaining markers), 1 minute at 72°C, and 5 min at 72°C. We kept amplification products at 4°C until they were used in a denatured 6% polyacrylamide gels in a Hoefer SQ3 sequencer vertical chamber. Depending upon the size of the markers analyzed, and the presence of 35 W as a constant, we stained the gels with AgNO3 (silver nitrate) after 2–3 h of migration. We used the molecular markers HinfI and ϕ174 (cut with HindIII). We repeated the PCR reactions three times for DNA extracted from hair. Thus, allelic dropout was highly improbable, but we cannot completely exclude the existence of null alleles, which could increase the number of false homozygous genotypes. Nevertheless, it is improbable that all loci were affected in the same way.

Population Genetics Molecular Analyses Genetic Diversity and Natural Selection Several gene diversity statistics were estimated for the microsatellite genotypes. The mean number of alleles per locus and the expected heterozygosity (H) (Nei, 1973) were calculated for the C. capucinus populations and statistically analyzed with a Student t test. The expected heterozygosity values were arcsine transformed prior to statistical analysis (Archie, 1985). To analyze the influence of the natural selection (constrictive or diversifying) on the microsatellites we used the coalescence theory generated by Beaumont and Nichols (1996). We used the LOSITAN Program (Antao et al., 2008) to obtain the observed and expected FST statistical values for each marker we used throughout the samples. Both the infinite allele and the step-wise mutation models were considered. A total of 50,000 iterations were completed to calculate the values that represented the relationship between the FST statistic and the expected heterozygosity of the markers. We used a subsample size of 50 from which the medians and the 5% and the 95% quartiles were calculated and CPU cores of 4. The observed

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FST and the heterozygosity values were superimposed under this distribution consisted of the median and the quartiles. Values that are outside of this theoretical distribution indicate that the microsatellites in question are being affected by natural selection.

Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow The Hardy-Weinberg equilibrium (H-W E) for the C. capucinus populations were estimated using several different strategies. The Weir and Cockerham (1984)’s F and the Robertson and Hill (1984)’s f statistics were used to calculate the degree of excess or deficit, of homo- and heterozygous (complete enumeration) within each one of the populations considered. To measure the exact probabilities of the G test employed, the Markov chain method, with a 10,000 dememorization number, 20 batches and 5,000 iterations per batch, was used, following the Genepop v. 4.2.1 Program (Raymond and Rousset, 1995). The H-W E was simultaneously analyzed by locus and by population using the Fisher´s method (Raymond and Rousset, 1995). The genetic heterogeneity among the C. capucinus populations was studied globally for each marker and all taken together as well as for population pairs. One strategy used the gene frequencies of the nine microsatellites studied, by using exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches, and 5,000 iterations per batch. We also used the Wright F-statistics (Wright, 1951) with the Michalakis and Excoffier (1996)’s procedure. The standard deviations of the F-statistics were calculated using a jackknifing over loci and the 99% confidence intervals were measured by means of bootstrapping over loci. Two procedures were used to measure the significance of FST. The first one used 10,000 randomizations of overall alleles sampled and assumed random mating within populations by means of the G test (Goudet et al., 1996). The second procedure used 10,000 randomizations of genotypes among populations and did not assume random mating within populations by means of the log-likelihood G test (Goudet et al., 1996). The significance of FIS and FIT was also found by using 10,000 randomizations of alleles within samples and in the overall sample. Additionally, the gene diversity analysis of Nei (1973) as well as the repeat number of allele (RST) statistic (Slatkin, 1995; Rousset, 1997) were estimated to measure the gene heterogeneity between the C. capucinus populations analyzed. These analyses are useful to determine which microsatellites more clearly discriminate among the populations of C. capucinus studied and to determine the degree of gene variability within each population relative to the whole species. Possible theoretical gene flow estimates among the C. capucinus populations studied were measured using the private allele model (Slatkin, 1985; Barton and Slatkin, 1986). Additionally, we applied Migrate 3.6 Software to determine possible asymmetrical gene flow among the C. capucinus populations (Beerli, 2006, 2009), (in this case, the scaled migration rate, M = m/ was obtained, where m is the migration rate per generation and  the mutation rate per generation). For the Maximum Likelihood procedure, we employed ten short Markov chains, with 500 recorded steps, 100 increments and a total of 50,000 sampled genealogies with a burn-in of 10,000, and one long Markov chain, with 5,000 recorded steps, 100 increments and a total of 500,000 sampled genealogies with a burn-in of 10,000. The Bayesian procedure was run with one long Markov chain, with 5,000 recorded steps, 100 increments, one concurrent chain and a total of 500,000 sampled genealogies with a burn-in of 10,000.

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Assignation and Genetic Structure Tests We developed diverse assignment analyses by using the GENECLASS 2 Program (Piry et al., 2004). We performed different strategies by employing one Bayesian procedure (Rannala and Mountain, 1997), one frequency procedure (Paetkau et al., 1995) and one genetic distance (standard genetic distance, Nei, 1972). The assignation analyses were carried out without simulations and served to estimate the probabilities of individuals belonging or being excluded from the original populations where they were “a priori” assigned (P < 0.05). Some assignation analyses were also completed with 10,000 resampling simulations by means of the Monte Carlo technique and with the procedure of Paetkau et al., (2004). Additionally, we estimated the possible existence of first generation migrants in the different C. capucinus populations by using the Bayesian, frequency and genetic distance procedures we commented on above without simulations. To determine this, we considered the relationship: L = Lhome/Lmax. This is the ratio of the likelihood computed from the population where the individual was sampled (Lhome) over the highest likelihood value among all population samples including the population where the individual was sampled (Lmax) (Paetkau et al., 2004). Another assignment analysis was applied using Structure 2.3 (Falush et al., 2007), which employs Markov Chain Monte Carlo procedures and the Gibbs sampler, uses multilocus genotypes to infer population structure, and simultaneously assigns individuals to specific populations. The model considers K populations, where K may be unknown, and the individuals are assigned tentatively to one population or jointly to ≥2 populations (if their genotypes are considered admixed). Two analysis groups were carried out. First, we considered the admixture model, wherein the individuals may have mixed ancestry and a noadmixture model with no prior population information to assist with clustering (USEPOPINF = 0). In addition,  was inferred (Dirichlet parameter for degree of admixture; with an initial value of  = 1) using uniform priors for  (its value was the same for all populations). The maximum value of this parameter was 10. Allele frequencies were correlated among populations, assuming different values of FST for each population. We revealed the presence of the most probable number of gene pools by using the increasing likelihood method. The second analysis was undertaken with a model that incorporates informative, geographic origin, individual priors. These assist with the clustering of weakly structured data in order to determine migrants or detect slightly different populations (USEPOPINF = 1) with both admixture and no admixture models. Furthermore, in this case, in order to apply the same conditions to that of the previous case, we introduced LocPrior = 1, Gensback = 2, and Migprior = 0.05. The program was run with 1,000,000 iterations after a burn period of 100,000 iterations for each analysis. Each analysis was performed twice with convergent results. Finally, we used the SAMOVA 1.0 Program (Dupanloup et al., 2002) to investigate population subdivision using analysis of molecular variance in a geographical context. The program SAMOVA 1.0 implements an approach to define groups of populations that are geographically homogeneous and maximally differentiated from each other. Furthermore, this software leads to the identification of genetic barriers between these population groups. The method is based on a simulated annealing procedure that aims at maximizing the proportion of total genetic variance due to differences between groups of populations (SAMOVA, Spatial Analysis of Molecular Variance). This approach starts from individual sampling

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locales as populations and surveys all possible combinations forming two or more broader groups, attempting to identify the most likely position of inferred historical barriers. We employed k = 2, 3, 4 and the statistical significance was estimated using 1,000 permutations.

Possible Historical Demographic Changes One analysis focused on the detection of recent bottleneck events using the most recently derived theory generated by Cornuet and Luikart (1996), and Luikart et al., (1998). The population, which experienced a recent bottleneck, simultaneously decreases the allele number and the expected levels of heterozygosity. Nevertheless, the allele number (ko) is reduced faster than the expected heterozygosity. Therefore, the value of the expected heterozygosity calculated throughout the allele number (Heq) is lower than the obtained expected heterozygosity (He). For neutral markers, within a population in gene mutation drift equilibrium, there is an equal probability that a given locus has a slight excess or deficit of heterozygosity in regard to the heterozygosity calculated from the number of alleles. In contrast, in a bottlenecked population, a large fraction of the loci analyzed will exhibit a significant excess of the expected heterozygosity. To measure this probability, four diverse procedures were used as follows: sign test, standardized difference test, Wilcoxon´s signed rank test and graphical descriptor of the shape of the allele frequency distribution. A population, which did not suffer a recent bottleneck event, will yield an L-shape distribution (such as expected in a stable population in mutation-gene drift equilibrium), whereas a recently bottlenecked population will show a mode-shift distribution. The Wilcoxon´s signed rank test probably has its greatest power when the number of loci analyzed is low, such as in the current case. The BOTTLENECK Program was used for this task. To determine possible population expansions in the C. capucinus populations we used four tests: Locus kurtosis (k) test (Reich and Goldstein, 1998) interlocus (g) test (Reich et al, 1999), the test of Kimmel et al., (1998), and the test of Zivothovsky et al., (2000). The first test (k) is based on the following principles. A population with constant size has gene genealogies, which tend to have a single ancient bifurcation. Therefore, the allele length distributions have multiple discrete peaks. On the contrary, in a growing population, most of the gene genealogy bifurcations tend to date back to the time expansion and as a result the allele length distribution is clearly more smoothly peaked. To measure these differences between the multi-peaked allele distribution of a constant size population and the smooth single-peaked allele distribution of a population in expansion, the k statistic was calculated as follows: k = 2.5 Sig4 + 0.28 Var – (0.95/n) – Gam4. Sig4 is the unbiased variance squared of allele length, Var is the sample variance of the same concept and Gam4 is the unbiased fourth central moment of the allele, respectively. The equations to estimate Sig4 and Gam4 are as follows: Sig 4 

(n 2  3n  3) 1 ( ( Xi  X ) 2 ) 2   ( Xi  X ) 4 n(n  1)(n  2)(n  3) (n  2)(n  3)

Gam4 

(n 2  2n  3) (6n  9)  ( Xi  X ) 4  (n  2)(n  3) ( ( Xi  X ) 2 ) 2 (n  1)(n  2)(n  3)

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where n is the number of chromosomes analyzed, Xi is the number of repeats of each allele found and X is the average repeat number of all the alleles found in a given population for a determined microsatellite. In order to assess significance levels, a binomial distribution is used with the number of trials equal to the number of loci based on the expectation of an almost (P = 0.515) equal probability of negative and positive k-values for the set of loci analyzed. When there is a smaller loci number associated with positive k values than would be expected for a constant-sized population, there is evidence of a population expansion. The significance level of the binomial distribution was measured using the Statistics Sample Program written by Michael H. Kelly. The g test focuses on the following facts. This test shows that for stable populations the allele size variance is highly variable among loci, whereas in expansion populations this variance is usually lower. Therefore, allele sizes with sufficiently low variances are taken as evidence for population expansion. The test used for statistical differences is that proposed by Reich and Goldstein (1998):

g

Var[Vj ] 4 V 2 1 V 3 6

where Var (Vj) is the observed variance of the allele length variances across the markers employed, and V is the average variance across loci. A low value of g is taken as a sign of population expansion. Significance levels for the interlocus test are found on Table 1 of Reich et al., (1999), which shows the fifth-percentile cutoffs for the interlocus test. Luikart et al., (1998) noted that this last test is probably the most powerful for this task. Table 1. Average number of alleles per locus (ANA) and average expected heterozygosity (He) for four Cebus capucinus populations (three Colombian populations: Northern Colombia, Choco and Cauca, and one Central American population) at nine DNA microsatellites. Standard Error (SE) Populations Northern Colombia Cauca Choco Central America

ANA ± SE 6.000 ± 2.739 5.333 ± 2.958 4.333 ± 2.291 1.333 ± 0.714

He ± SE 0.729 ± 0.195 0.646 ± 0.202 0.647 ± 0.207 0.378 ± 0.261

The test of Kimmel et al., (1998) is based on the principle that two different estimates of  (= 4Ne) could be obtained. One is v = V, with V being the variance of the tandem repeat size, whose expression is V = (2  i = 1….n(Xi – X)2)/(n – 1), where n is the number of chromosomes analyzed, Xi is the number of repeats of each allele found and X is the average repeat number of all the alleles found in a microsatellite. The second one is Po = (1/(Po2 – 1))/2 (estimator of the homozygosity), where Po = (n  k = 1…..n (p k2 – 1))/(n -1), with pk being the allele frequency of the kth allele]. An imbalanced  index could be defined as: (t) = E(v)/E(Po) = V(t)/[((1/ Po2) – 1)/2 or by the expression: ln (t) = ln v – ln Po = ln (V) – ln (((1/ Po2) – 1)/2). If a population is in equilibrium, has a constant demographic size, and isn’t

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suffering an expansion, = 1 (ln  = 0). On the contrary, if a population has suffered an expansion coming from a mutation-drift equilibrium situation (constant population size),  < 1 (ln  < 0). If a population has experienced an expansion coming from a previous bottleneck,  > 1 (ln  > 0). This last value will be present for a long time (several thousand generations) before showing the signature of a population expansion, < 1 (ln  < 0). There is an exception to this general rule, when a bottleneck is so intense the population becomes monomorphic before the demographic expansion, in which case,  < 1 (ln  < 0) all the time. All of these  values are consistent in stepwise, logistic or exponential population growth and are not especially affected in diverse mutation models (Kimmel et al., 1998). To determine the statistical significance of the  (ln ), a jackknife procedure (Efron and Tibshirani, 1993) was applied to obtain the variance of  and, with this variance, a Student’s t test and 95 and 99% confidence intervals were estimated. Also, the test of Zivothovsky et al., (2000) was used to calculate an expansion index: Sk = 1 – ((K – (RkV/2)/5V2). K and V are the unnormalized kurtosis (fourth central moment) and the allele size variance is estimated from a sample and corrected for sampling bias, respectively, whereas Rk = km/2m (they are the kurtosis and the variance in the repeat number mutational changes). The expressions used to estimate V and K are: V = i = 1…n pi (Xi – X)2 and K =  i = 1…n pi (Xi – X)4, where X = i = 1…k pi k, and k represents the alleles in a locus given and pi, the allele frequencies. All the other terms were defined in the previous analysis. We used an Rk value of 6.3 because it was obtained for dinucleotide microsatellites by Dib et al., (1996), and because dinucleotide microsatellites were used in the current study. Feldman et al., (1999), used the same data and a geometrical distribution of mutational events, and obtained an estimated 2m of 2.5. This is basically the same as what was obtained by using a truncated Poisson distribution (Zhivothovsky et al., 2000) or by ourselves in the present work (2m = 2.39). The value of Sk is expected to be 0 in a general symmetric stepwise mutation model for a population in equilibrium and of constant size (this was derived by Zhivotovsky and Feldman, 1995). The Sk is positive if an expansion affected the population and is contrarily negative if a bottleneck affected the population. To obtain demographic conclusions of this analysis, the within-population variance and the expansion index are averaged for all the microsatellites studied within each population and their dynamics are compared. Zhivotovsky et al., (2000) showed that a significant correlation existed between V and Sk (r = 0.58) for a human data set. However, this correlation was moderate and, in fact, both statistics could react differently to changes in population size and have dissimilar patterns across populations. A jackknife procedure was performed to obtain the variance of Sk and, with this variance, a Student’s t test and a 95% confidence interval were estimated.

Spatial Genetic Analyses We used two types of software to analyze possible spatial genetic patterns for the Colombian and the Central American C. capucinus individuals. The first was the SGS Version 1.0d (Degen et al., 2001). Analyses with all individuals were carried out using distograms (Degen and Scholz, 1998; Vendramin et al., 1999) with the Gregorious (1978)’s genetic distance and correlograms with the Moran’s I index (Moran, 1950). Three and five distance classes (DC) were used for all individuals taken as a whole (3DC: 0-541 km, 5411,083 km, 1,083-1,624 km. 5 DCs: 0-324 km, 324-648 km, 648-971 km, 971-1,295 km,

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1,295-1,619 km). Other analyses were carried out only with the Colombian individuals for both distogram and correlogram with six DCs (6 DC: 0-123 km, 123-245 km, 245-368 km, 368-490 km, 490-613 km, 613-736 km). The significance of distograms and autocorrelation coefficients were calculated by means of 1,000 Monte-Carlo simulations (Manly, 1997) with an estimated confidence interval of 95% (Streiff et al., 1998). The second program was SPAIDA (Pálsson, 2004). It calculates two estimates of spatial autocorrelation, the Moran’s I index and the Geary’s c coefficient (Geary, 1954), assuming two different mutation models (infinite allele model, IAM and stepwise mutational model, SMM). Moran’s I index weights the covariance among alleles from individuals separated for a certain distance class, with the total variance. Values can oscillate from -1 to 1. Geary’s c coefficient weights the variance within a distance class with the total variance, resulting in values ranging from 0 and up. When there is a positive correlation, there is little variation within distance class and the value is close to 0. In the IAM the variances and the covariances are based only on the frequencies of different alleles, whereas in SMM the variances and covariances are based on differences in the number of repeats. This analysis was only applied to the Colombian individuals with three DCs (3 DC: 0-171 km, 171-342 km, 342-684 km). The significance of the autocorrelation coefficients for each distance class was evaluated by a test with 1,000 permutations. For a selected number of cases, geographical locations of individuals are assigned randomly and the statistics are calculated anew, providing a reference distribution for the observed test statistics.

RESULTS Gene Diversity and Natural Selection Microsatellites AP74 and D8S165 showed the highest number of alleles (14 and 13 alleles respectively). The lowest number of alleles were found in markers AP68 (2 alleles) and D5S117 (3 alleles). The Northern Colombian population showed the highest number of alleles per locus (6.0 ± 2.74) as well as the highest levels of gene diversity (0.729 ± 0.195). In contrast, the Central America population had the lowest number of alleles per locus and the lowest gene diversity level (1.33 ± 1.14 and 0.378 ± 0.261, respectively) (Table 1). The application of the Beaumont and Nichols’s (1996) method using LOSITAN Software (with the infinite allele mutation and the stepwise mutation models) indicated that six out of nine microsatellites analyzed showed neutral behavior (AP68, D5S117, D6S260, D14S51, D17S804 and PEPC3). AP74 and D5S111 showed positive (adaptive) selection and D8S165 indicated negative (balancing) selection (Figure 1).

Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow Two out of nine microsatellites within the Cauca population microsatellites did not demonstrate H-W E (D5S111 and D8S165; Bonferroni’s correction). Similarly, the Northern Colombian population showed three microsatellites without H-W E (AP74, D5S111 and D5S117; Bonferroni’s correction). The Choco and the Central America populations showed

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all the markers in H-W E with Bonferroni’s correction (Table 2). An exact G test with the genic frequencies revealed that only AP74 showed (with the Bonferroni’s correction) significant heterogeneity among the four populations. However, considering all nine microsatellites, the genic heterogeneity was highly significant among the four populations (Table 3). By population pair, all of them yielded significant genetic heterogeneity, save for the Cauca-Northern Colombia pair, which was not statistically significant (Table 4).

(A)

(B)

Figure 1. Application of the LOSITAN software to detect possible natural selection at the nine DNA microsatellites analyzed in the overall Cebus capucinus population studied: for an infinite allele mutation model (A) and for the stepwise mutation model (B). The D8S165 microsatellite was submitted to balancing selection and the AP74 and D5S111 microsatellites were submitted to positive (adaptation) selection.

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Table 2. Probability tests to estimate possible Hardy–Weinberg equilibrium individually and globally for four Cebus capucinus populations (three Colombian populations: Northern Colombia, Choco and Cauca, and one Central American population) at nine DNA microsatellites. * Significant probability with Bonferroni’s correction (P < 0.0055); df = degree of freedom; MLT = Multi locus test Cauca locus P-val S.E. AP68 1.0000 0.0000 AP74 0.0163 0.0036 D5S111 0.0007* 0.0004 D5S117 0.5673 0.0114 D6S260 0.2254 0.0762 D8S165 0.0050* 0.0053 D14S51 0.2650 0.0128 D17S804 1.0000 0.0000 PEPC3 0.3987 0.0222 MLT 0.0000* 0.0000 Northern Colombia locus P-val S.E. AP68 1.0000 0.0000 AP74 0.0002* 0.0002 D5S111 0.0000* 0.0000 D5S117 0.0048* 0.0002 D6S260 0.0110 0.0019 D8S165 0.0104 0.0042 D14S51 0.0460 0.0073 D17S804 0.0181 0.0012 PEPC3 0.2826 0.0155 MLT 0.0000* 0.0000 Choco locus P-val S.E. AP68 0.6643 0.0012 AP74 0.1776 0.0278 D5S111 0.0064 0.0035 D5S117 0.6684 0.0538 D6S260 0.0085 0.0017 D8S165 0.2166 0.0130 D14S51 0.1032 0.0152 D17S804 0.0070 0.0013 PEPC3 1.0000 0.0017 MLT 0.0000* 0.0000 Central America locus P-val S.E. AP68 1.0000 0.0000 AP74 0.2000 0.0556 D5S111 0.3378 0.1147 D5S117 0.2389 0.1756 D6S260 0.7612 0.1762 D8S165 1.0000 0.0000 D14S51 0.4789 0.2437 D17S804 1.0000 0.0000 PEPC3 1.0000 0.0000 MLT 0.2819 0.0032

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Nei’s (1973) gene diversity and Wright’s (1951) F statistics showed the existence of certain homozygote excess at the subpopulation (FIS = 0.273 ± 0.077, P < 0.001, jackknifing over loci; range of FIS, 99% confidence interval with bootstrapping over loci: 0.439-0.053) and total population levels (FIT = 0.328 ± 0.078, P < 0.001, jackknifing over loci; range of FIT, 99% confidence interval with bootstrapping over loci: 0.497-0.114). However, the genetic heterogeneity among the four geographical subpopulations was relatively small (FST = 0.076 ± 0.029, jackknifing over loci; range of FST, 99% confidence interval with bootstrapping over loci: 0.149-0.018). If we assume no random mating within each one of the populations analyzed, the overall probability for all the loci was not significant (P < 0.122-0.092). But if we consider random mating within the subpopulations, then the overall probability for FST was significant (P < 0.001). Table 5 shows the complete results of both Nei (1973) and Wright (1951)’s analyses. The FST and RST statistics per population pairs (Table 6) showed that the differentiation between the Cauca-Northern Colombian and the Cauca-Choco population pairs is practically null (FST = 0.0000 and RST = 0.0107, FST = 0.0124 and RST = 0.0002, respectively). In contrast, the three Colombian populations showed considerable genetic heterogeneity compared to the Central American population (FST = 0.196-0.217 and RST = 0.798-0.843). The genetic heterogeneity between the three Colombian populations and the Central American population was statistically significant for both FST and RST. For the FST statistic, the relationship between the Northern Colombia-Choco population pair was also significant. The theoretical gene flow estimates from the FST and RST statistics (Table 7) were all them above 1, with the exception of the relationships with the Central American population. Thus, the Central American population was differentiated from the three geographical Colombian population considered. The overall gene flow estimate with the method of the private allele was lower than with other methods (Nm = 0.883). Table 3. Exact G tests to determine genic heterogeneity at nine DNA microsatellites among four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) at nine DNA microsatellites. * P < 0.05; ** P < 0.01; *** Significant probability with Bonferroni’s correction; df = degree of freedom P-value across all loci (Fisher’s method) for the three robust capuchin taxa Locus P-Value AP68 0.13601 AP74 0.00001*** D5S111 0.00965** D5S117 0.01622* D6S260 0.02865* D8S165 0.31370 D14S51 0.08288 D17S804 0.00948** PEPC3 0.09074 All markers simultaneously taken: 2 = Infinity (df= 18), highly significant.

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Table 4. Exact G tests to determine genetic heterogeneity among the population pairs of four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) at nine DNA microsatellites taken together (A). Exact G tests to determine genetic heterogeneity among the population pairs of four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) individually analyzed at nine DNA microsatellites (B). * P < 0.05; ** P < 0.01; *** significant with Bonferroni correction; df = degree of freedom; S. E. = Standard Error (A) P-value for each population pair across all loci (Fisher's method) Population pair 2 df P-Value Cauca vs. Northern Colombia 30.14260 18 0.036083* Cauca vs. Choco Infinity 18 0.000001*** Cauca vs. Central America Infinity 18 0.000001*** Northern Colombia vs. Choco 43.80489 18 0.000615*** Northern Colombia vs. C. America 49.02102 18 0.000002*** Choco vs. Central America 31.69605 18 0.001541*** (B) AP68 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.12070 0.15835 0.96733 1.00000 1.00000 1.00000

S.E. 0.00253 0.00236 0.00013 0.00000 0.00000 0.00000

AP74 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.34241 0.00000*** 0.00000*** 0.00258*** 0.00018*** 0.00040***

S.E. 0.00627 0.00000 0.00000 0.00086 0.00010 0.00017

P-Value 0.39575 0.11929 0.00049*** 0.90032 0.00097*** 0.02148*

S.E. 0.00673 0.00429 0.00024 0.00434 0.00045 0.00111

P-Value 0.02469* 1.00000

S.E. 0.00115 0.00000

D5S111 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America D5S117 Population pair Cauca vs. Northern Colombia Cauca vs. Choco

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1.00000 0.02382* 0.10069 1.00000

0.00000 0.00124 0.00185 0.00000

D6S260 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.69331 0.35186 0.18826 0.00661** 0.08241 0.24297

S.E. 0.00347 0.00666 0.00422 0.00085 0.00222 0.00436

D8S165 Population pair P-Value Cauca vs. Northern Colombia 0.06832 Cauca vs. Choco 0.69586 Cauca vs. Central America 0.42642 Northern Colombia vs. Choco 0.48566 Northern Colombia vs. C. America 0.35136 Choco vs. Central America 0.87450

S.E. 0.00347 0.00769 0.00933 0.00631 0.00736 0.00246

D14S51 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.10589 0.15576 1.00000 0.40839 1.00000 1.00000

S.E. 0.00373 0.00363 0.00000 0.00609 0.00000 0.00000

D17S804 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.28902 0.06188 0.11041 0.17234 0.04451* 0.07176

S.E. 0.00621 0.00221 0.00299 0.00281 0.00236 0.00237

PEPC3 Population pair Cauca vs. Northern Colombia Cauca vs. Choco Cauca vs. Central America Northern Colombia vs. Choco Northern Colombia vs. C. America Choco vs. Central America

P-Value 0.48659 0.29227 1.00000 0.02460* 1.00000 1.00000

S.E. 0.00621 0.00412 0.00000 0.00141 0.00000 0.00000

Table 5. Genetic diversity analysis for four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) by means of the Nei’s and the Wright’s F statistics applied to nine DNA microsatellites (Ho = observed gene diversity in the total population; HS = average gene diversity within the subpopulations; HT = expected gene diversity in the total population; DST = absolute gene differentiation among subpopulations; GST = relative genetic differentiation among subpopulations with regard to the total gene diversity; GST’= the same as the previous statistics but corrected by sample size; FIS - FST - FIT = Wright’s F-statistics; RST = Slatkin [1995]’s genetic heterogeneity statistic measured by allele variances). * P < 0.05; ** P < 0.01 Locus AP68 AP74 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804 PEPC3 Overall Jackknife

Ho 0.615 0.393 0.348 0.120 0.324 0.745 0.658 0.383 0.850 0.493

Hs 0.476 0.658 0.727 0.220 0.653 0.905 0.904 0.476 0.862 0.654

Bootstrap 99% Confidence Interval

Ht 0.504 0.874 0.875 0.248 0.725 0.864 0.915 0.497 0.909 0.712

Dst 0.028 0.216 0.148 0.027 0.071 0.000 0.011 0.021 0.047 0.058

Gst 0.055 0.247 0.169 0.110 0.098 0.000 0.012 0.042 0.051 0.082

Gst' 0.080 0.304 0.213 0.141 0.127 0.000 0.017 0.055 0.075 0.118

FIS -0.353 0.302** 0.508** 0.491** 0.487** 0.256** 0.261** 0.319** 0.055 0.273** 0.273 ± 0.077 0.053 0.439

FST 0.083 0.228* 0.086 0.138 0.068 0.000 0.030 0.042 0.053 0.076 0.076 ± 0.029 0.018 0.149

FIT -0.241 0.510** 0.550** 0.561** 0.522** 0.231** 0.283** 0.348** 0.105 0.328** 0.328 ± 0.078 0.114 0.497

RST 0.083 0.825 0.000 0.000 0.100 0.000 0.000 0.021 0.092 0.101

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Table 6. FST (below main diagonal) and RST (above main diagonal) statistic pairs for four Cebus capucinus populations (three Colombian populations: Northern Colombia, Choco and Cauca, and one Central American population) studied at nine DNA microsatellites; ** P < 0.01 Populations Cauca Northern Colombia Choco Central America

Cauca 0.0000 0.0124 0.1967**

Northern Colombia 0.0107 0.0949** 0.2173**

Choco 0.0002 0.0707 0.2009**

Central America 0.7976** 0.7994** 0.8429**

Table 7. Gene flow estimate pairs for four Cebus capucinus populations (three Colombian populations: Northern Colombia, Choco and Cauca, and one Central American population) studied at nine DNA microsatellites from the FST (below main diagonal) and RST (above main diagonal) statistics Populations Cauca Northern Colombia Choco Central America

Cauca Infinite 39.7314 2.0416

Northern Colombia 45.9525 4.7651 1.8005

Choco 2,453.6678 6.5667 1.9884

Central America 0.1268 0.1254 0.0932

Table 8. Bayesian migration rate estimates (m) among four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) pairs using Migrate 3.6 Software with nine DNA microsatellites. 97.5% Confidence Interval in parentheses Populations Cauca

Cauca -

Northern Colombia Choco

31.68 (3.33-38) 37.61 (6.67-42.47) 7.41 (0-22)

Central America

Northern Colombia 42.75 (10.67-45.33) 68.17 (24-77.33) 14.25 (0-26)

Choco 23.72 (0-32.67) 85.65 (37.33-96.67) -

Central America 10.90 (0-24) 17.28 (0-27.33) 20.41 (0-30)

13.81 (0-25.33)

We also determined other historical migration rate estimates by using Bayesian procedures (Migrate 3.6; Table 8) for the four populations considered. These Bayesian estimates showed that the Northern Colombian population yielded higher migration rates towards other populations, than the reverse, although the differences are of low magnitude. As in the previous analysis, the Central American population showed the lowest historical migration rates, although it’s noteworthy to mention that the migration rates from the Central American population toward the Colombian ones are higher than from the Colombian populations towards the Central American one. However, all these historical migration rates are of a relatively elevated magnitude. The maximum likelihood estimates for the three Colombian populations are relatively similar and it’s not possible to determine which Colombian population was the source of migrants to the other populations.

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Genetic Assignment and Genetic Structure Different genetic assignment analyses were carried out using GENECLASS 2.0 Software. The assignment analysis with Nei’s (1972) genetic distance, for the overall C. capucinus sample, yielded 22.22% of individuals incorrectly classified. Within Cauca, four individuals (21.05%) showed multi-genotypes more related with other populations (three more related with Northern Colombia and one to Choco). The Northern Colombian population had 30% of individuals misclassified (three more related to Cauca and three more related to Choco). Choco showed two misclassified individuals (18.2%; two specimens more related to Northern Colombia). With the criteria of Paetkau et al., (1995), 20.37% of the individuals was misclassified. Six individuals (31.58%) were misclassified within Cauca (three more related to Northern Colombia and three more related to Choco), two were misclassified within Northern Colombia (10%; one more related to Cauca and another to Choco), and three were misclassified in Choco (27.27%; all more related to Northern Colombia). With the criteria of Rannala and Mountain (1997), 22.22% of the individuals were misclassified, with four in Cauca (21.05%; three more related with Northern Colombia and one to Choco), five in Northern Colombia (25%; four more related with Cauca and one with Choco) and three in Choco (27.27%; two more related with Cauca and one more related to Northern Colombia). None of the criteria, indicated a misclassification of Central American individuals. These same three criteria were used to detect the first generation of migrants within three Colombian populations. Nei’s (1972) genetic distance did not detect any significant migrant individual of the first generation (0%). Nevertheless, the criteria of Paetkau et al., (1995) detected seven migrant individuals with a probability below P < 0.01 (12.96%; one individual sampled in Cauca coming from Choco, two individuals sampled in Northern Colombia coming from Cauca, two individuals sampled in Northern Colombia coming from Choco, and two individuals sampled in Choco coming from Northern Colombia). Also, the criteria of Rannala and Mountain (1997) detected eight migrant individuals from the first generation (14.81%; one individual sampled in Cauca coming from Northern Colombia, another sampled in Cauca coming from Choco, one sampled in Northern Colombia coming from Cauca, another individual sampled in Northern Colombia coming from Choco, two individuals sampled in Choco coming from Cauca and two individuals sampled in Choco coming from Northern Colombia). Henceforth, there is plenty of evidence supporting the migration of C. capucinus among different regions of Colombia. The Central American individuals seem to be more differentiated. When we considered three different conditions with the STRUCTURE Software (individuals without prior origins with admixture, or no admixture model, and individuals with prior origins and with an admixture model), the highest maximum likelihood corresponded to only one population (Table 9). In the case of individuals with prior geographical information and no admixture model, we detected four different populations, as the most probably case (Figure 2). In this case, four geographical populations were detected, with only one individual from the Northern Colombian population showing a considerably genetic contribution from the Choco population. Thus, we can only differentiate diverse populations of C. capucinus when geographical information is provided.

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Figure 2. Assignation analysis with the STRUCTURE Software to the overall Cebus capucinus sample analyzed (54 individuals studied). In the case of individuals with prior geographical information and no admixture model, four different gene pools were detected agreeing quite well with the four Cebus capucinus populations studied. However, the other Structure analyses revealed a unique gene pool. Green = Northern Colombian population; Red = Cauca population; Blue = Choco population; Yellow = Central American population.

Table 9. Number of possible different gene pools for four Cebus capucinus populations (three Colombian populations, Northern Colombia, Choco and Cauca, and one Central American population) analyzed using the Structure Program with nine microsatellite. * = Most probable number of populations. K = number of populations. Without origin priors and assuming an admixture model (A); Without origin priors and no admixture model (B); With origin priors and assuming an admixture model (C); With origin priors and no admixture model (D) (A) Populations K = 1* K=2 K=3 K=4 K=5 K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -800.2 -908.9 -895.9 -945.5 -962.9 -1,024.9 -969.9 -968.3 -976.3 -1,023.7 -1,016.3 -1,094.6

(B) Populations K = 1* K=2 K=3 K=4 K=5 K=6

Ln Likelihood -803.5 -862.1 -855.9 -899.0 -915.5 -862.4

Genetic Structure, Spatial Patterns and Historical Demographic Evolution … Populations K=7 K=8 K=9 K = 10 K = 11 K = 12

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Ln Likelihood -877.3 -893.0 -927.5 -946.2 -968.4 -976.8

(C) Populations K = 1* K=2 K=3 K=4 K=5 K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -804.2 -868.5 -870.3 -907.5 -900.5 -1,301.3 -1,207.4 -1,233.8 -1,217.8 -1,013.6 -1,059.3 -1,050.3

(D) Populations K=1 K=2 K=3 K = 4* K=5 K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -803.5 -813.8 -827.7 -799.2 -829.1 -818.9 -819.5 -838.0 -828.1 -863.8 -847.4 -876.4

The SAMOVA analysis was carried out with the RST (sum of squared size differences) and FST (number of different alleles) statistics. The analyses were carried out with k = 2, 3 and 4 groups of populations. However, neither value of FCT (differentiation among the groups considered) was significant, although the case of k = 4 showed the highest variance among groups (RST: percentage of variance, 62.90%, FCT = 0.620, P = 0.4213; FST: percentage of variance, 20.52%, FCT = 0.205, P = 0.1163). This means that when k = 2, 3 and 4 it is no better than when considering only one population of C. capucinus in the area considered. This agrees quite well with three out of four analyses previously commented for the STRUCTURE Software. However, after one population, the most probably solution was four populations. Remarkably, the differences among the individuals within the populations (the unique

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variance percentage) was significant for all of the analyses. For instance, for k = 4, these values were highly significant (RST: percentage of variance, 42.40%, FCT = 0.576, P = 0.0001; FST: percentage of variance, 90.83%, FST = 0.092, P = 0.0001). Therefore, the major part of the differences was among individuals and not among populations or geographical regions.

Historical Demographic Changes The BOTTLENECK Software for the overall C. capucinus sample did not show any evidence of bottleneck trends nor for the two-phase mutation model nor for the stepwise mutation model. The sign and the standardized differences tests for the stepwise mutation model were significant (P = 0.0326 and T2 = -1.931, P = 0.0267, respectively) but in a mode contrary to a bottleneck, that is, in favor of a population expansion or different gene pools mixed in the same population. For the Cauca and for the Northern Colombian populations, there were no signals of bottleneck trends affecting these populations. In the case of the Choco population, only one test showed a possible incidence of a bottleneck. This was the case of a shifted mode in the allele frequency distribution. The overall C. capucinus sample studied did not show evidence of historical demographic changes for the k and g tests (P = 0.0759 and g test = 2.7973, respectively). The same was found for each one of the three Colombian populations studied (Cauca: P = 0.7157 and g test = 4.4278; Northern Colombia: P = 0.0757 and g test = 1.3450; Choco: P = 0.2233 and g test = 0.7465). Contrarily, the test of Kimmel et al., (1998) showed a population expansion after little propagules founded the respective populations for all the cases studied. The overall C. capucinus sample and the Northern Colombian population showed statistical evidence in favor of this expansion after the foundation of the population by little propagules ( = 1.7227 and  = 1.7997, P < 0.05). The populations of Cauca and Choco even showed stronger evidence in favor of this mode of population expansion ( = 2.7669 and  = 2.8219, P < 0.01). The test of Zhivotovsky et al., (2000) revealed a significant population expansion for the overall C. capucinus sample and for the Cauca population (SK = 0.5807 and SK = 0.4085, P < 0.01, respectively). In contrast, the Choco and the Northern Colombian populations did not show evidence of significant demographic changes throughout their natural histories (SK = 0.0631 and SK = -0.2791, P > 0.05, respectively). Thus, all of the demographic tests did not reveal any evidence of bottlenecks affecting the C. capucinus populations but there is evidence of population expansions affecting some of the C. capucinus populations.

Spatial Genetic Structure The possible spatial genetic patterns in C. capucinus were analyzed by means of the SGS and the SPAIDA Software. With the SGS Software, two procedures were employed (distograms with Gregorious distance and correlograms with Moran’s I index; Figure 3) analyzing all the Colombian and Central American individuals. With three and five DCs, the distograms did not show any significant spatial trend.

Genetic Structure, Spatial Patterns and Historical Demographic Evolution … Distogram using mean Genetic Distance (Gregorius 1978) 0.9 observed 0.8

0.7

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0.4 324

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Distogram using mean Genetic Distance (Gregorius 1978) 0.75 observed 0.70

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D 0.60 95% CI 0.55

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Distogram using mean Genetic Distance (Gregorius 1978) 0.8 observed 0.7

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Spatial distance (F) Figure 3. Application of the SGS Software to detect possible spatial structure for the overall sample of Cebus capucinus studied and only for the Colombian sample for nine DNA microsatellites: with the Gregorious (1978) genetic distance and three distance classes for the overall sample (A); with the Moran’s I index and three distance classes for the overall sample (B); with the Gregorious (1978) genetic distance and five distance classes for the overall sample (C); with the Moran’s I index and five distance classes for the overall sample (D); with the Gregorious (1978) genetic distance and six distance classes for the Colombian sample (E) and with the Moran’s I index and six distance classes for the Colombian sample (F).

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The overall correlograms were also not significant but the last DC (around 1,620 km) was significantly negative for both, three and five DC. This means that some Central American individuals were spatially differentiated for the remaining individuals analyzed. This pattern was detected by the Moran’s I index but not by the Gregorious distance. Only for the Colombian individuals at a DC of 6 did the distogram with the Gregorious distance not detect any spatial structure. The correlogram with the Moran’s I index detected some significant DCs (3 DC: I = -0.052, P = 0.0000, significant negative DC; 4 DC: I = 0.0097, P = 0.008, significant positive DC), but it showed a “crazy quilt” pattern (Sokal and Oden, 1978b). Therefore, within Colombia, no spatial patterns were detected. This was ratified with the results obtained with the SPAIDA Software. For Colombia, the results for a DC of 3 did not show any significant spatial structure with the Moran’s I index and the Geary’s c coefficient for two mutation models (IAM and SMM) (Table 10). Thus, no spatial structure was detected for C. capucinus inside Colombia and only a slight spatial pattern was detected when Central American specimens were analyzed together with the Colombian specimens. Table 10. Spatial genetic structure by means of SPAIDA Software for the Cebus capucinus individuals sampled in Colombia with three distance classes: with the Moran’s I index and the infinite allele mutation model (IAM) (A), with the Moran’s I index and the stepwise mutation model (SMM) (B), with the Geary’s c coefficient and the infinite allele mutation model (IAM) (C) and with the Geary’s c coefficient and the stepwise mutation model (SMM) (D). * P < 0.05; ** P < 0.01 (A) Locus/Distance Class AP68 AP74 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804 PEPC8 Average

1 DC 0.3817 0.0756 0.0665 0.4222 0.1845 0.0341 0.1070 0.0909 0.1287 0.1688

2 DC 0.3735 -0.0073 0.0670 0.4071 0.2098* 0.0253 0.0920 0.1193 0.1236 0.1592

3 DC 0.3497 0.0467 0.0664 0.3898 0.2080 0.0323 0.0990 0.0680 0.1252 0.15 53

2 DC -0.0154 -0.0063 -0.1000** -0.0002 -0.0008 -0.0009 -0.1001** -0.0674 -0.0004 -0.0118

3 DC -0.0726 -0.0011 -0.0001 -0.0008 -0.0003 -0.0011 -0.0004 0.0148 -0.0015 - 0.0075

(B) Locus/Distance Class AP68 AP74 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804 PEPC8 Average

1 DC 0.0239 0.0045 -0.0016 0.0001 0.0006 -0.0011 -0.0012 0.0095 -0.0008 0.0044

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(C) Locus/Distance Class AP68 AP74 D5S111 Locus/Distance Class D5S117 D6S260 D8S165 D14S51 D17S804 PEPC8 Average

1 DC 0.6403 0.0632 0.1292 1 DC 0.6689 0.1789 0.0899 0.0586 0.2378 0.0464 0.2576

2 DC 0.5796 0.0782 0.1203 2 DC 0.5706 0.1824 0.1194 0.0572 0.2545 0.0399 0.2445

3 DC 0.6970 0.0613 0.1704 3 DC 0.8669 0.2548 0.0840 0.0648 0.2588 0.0444 0.30 46

1 DC 1.1390 0.0321 0.0307 0.0018 0.0062 0.0658 0.0148 0.8554 0.0106 0.2743

2 DC 0.9872 0.0394 0.0267 0.0013 0.0082 0.0613 0.0191 1.5425 0.0099 0.3469

3 DC 1.0694 0.0381 0.0344 0.0017 0.0062 0.0653 0.0178 0.4852 0.0135 0.2181

(D) Locus/Distance Class AP68 AP74 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804 PEPC8 Average

DISCUSSION Population Genetics of C. Capucinus Our study showed that the major part of the microsatellites used yielded a neutral evolutionary dynamic (Kimura, 1983). However, AP74 and D5S111 seem to be submitted to Darwinian (adaptive; selective sweeps) selection, while D8S165 seems to be effected by constrictive selection. Ruiz-García et al., (2004) showed that some microsatellites could be submitted to different kinds of selection in Neotropical Primates and this is reinforced in the current study. Li et al., (2000) determined that edaphic factors may affect microsatellite variation in Triticum dicoccoides either directly by selection or indirectly by affecting mutational mechanisms or hitchhiking. King and Soller (1999) claimed that many microsatellites are functionally integrated within the genome and any changes in length can exert a quantitative regulatory effect on gene transcription activity and due to this have an adaptive potential. Some of these events could affect AP74 and D5S111 in C. capucinus. D8S165 could be in a low recombination area, where the continuous input of deleterious mutations can reduce the neutral diversity of linked loci (purifying or background selection; Charlesworth et al., 1993)

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The microsatellite markers showed medium-elevated gene diversity levels such as it was observed for mitochondrial markers for the overall population of C. capucinus. This fact, together with no bottlenecks, agrees that this species has been generally classified by IUCN as in Low Risk. Also, in an identical fashion, the Colombian populations showed considerably higher gene diversity levels than the Central American population for both kinds of markers. This puts forward that the Central American population was derived from ancestors of some current Colombian population and therefore the migration of this species was from Northern South America towards Central America. The level of genetic heterogeneity among the Colombian C. capucinus populations based on microsatellites is very limited and, in many tests, it was not statistically significant. This agrees quite well with the results of the Migrate 3.6 Program which estimated high migration rates among the Colombian populations. It also determined that a large fraction of individuals were misclassified (18-35%) in respect to their geographical origins detected by the GENECLASS 2.0 Software. Also, a unique gene pool was detected for many of the different conditions performed by the STRUCTURE Software and there was no spatial autocorrelation across the Colombian populations. These results are extremely interesting when they are compared with the mitochondrial ones. Ruiz-García et al., (2012) determined three large and significantly different mitochondrial haplogroups in Colombia (named Colombian haplogroups I, II and III) and a differentiated little cluster (region of Buenaventura). However, with the exception of this last little cluster, each one of these significant haplogroups were composed by animals from different regions of Colombia. For instance, if we classified the animals studied by Ruiz-García et al., (2012) into the three geographical areas of Colombia that we used, then Colombian haplogroup I would be integrated by animals from the Cauca and Northern Colombian populations. Also, the Colombian haplogroup II would include samples from the three populations considered here (Cauca, Northern Colombia and Choco). Furthermore, Colombian haplogroup III would be integrated by individuals from the three quoted populations. This means that in the populations from Cauca and Northern Colombia, the three main Colombian haplogroups are living sympatrically in those regions, whilst in the Choco, two Colombian haplogroups are living sympatrically. Ruiz-García et al., (2012) estimated that the temporal split among these three main Colombian haplogroups were 1.74 ± 0.08 MYA (I-II), 2.02 ± 0.07 MYA (I-III) and 2.30 ± 0.15 MYA (II-III), respectively, but later these haplogroups expanded and intermixed in the same geographical regions. Our microsatellite results clearly support the hypothesis of RuizGarcía et al., (2012) and thus, the genetic heterogeneity among the three geographical areas considered in this work is considerably reduced. Additional support of this view comes from the bottleneck analysis for the overall Colombian population as well as from the test of Kimmel et al., (1998) applied to the overall Colombian population and each of the three geographical areas considered. More support comes from the test of Zhivotovsky et al., (2000) applied to the overall Colombian and to the Cauca populations. Similar to the mitochondrial data all of them detected significant population expansions. These expansions should cancel most of the genetic differences among the different geographical regions in Colombia where C. capucinus inhabits. The existence of some homozygote excess within these geographical regions (as detected by the Wright F statistics) could be due to propagules of each one of these haplogroups living in the same geographical area that did not mate with each other (Wahlund effect). The test of Kimmel et al., (1998) showed that the population expansion in C. capucinus was due to propagules. This correlates with the last idea.

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There is support of the Pleistocene refugia and sea level fluctuations affecting the evolution of C. capucinus. For example, the original split among haplogroups in Colombia began around 2.3-1.7 MYA. Population expansions later occurred by means of propagules (determined by both microsatellites and mitochondrial DNA). Also, haplotype fragmentation within haplogroups ranged from 490,000 to 78,000 YA. During glacial activity peaks, the average sea level was about 100 m lower, while during the interglacials, the sea level rose by about 30–50 m above the present level. This flooded the Maracaibo Basin and great parts of the Northern Colombian Plains and the lower Atrato Valley; Haffer, 1967) and thus affected the evolution of C. capucinus. Humid and dry climatic periods caused drastic contraction and expansion of forest and non-forest areas in Northern Colombia and Southern Central America (Van der Hammen, 1961). In the dry peaks, a few rather restricted forest refuges remained along the Caribbean slopes of Central America (mainly Honduras, Nicaragua and Costa Rica), on the Pacific slopes of Southwestern Costa Rica and also Panama (Chiriquí Refuge). Forest refuges also remained in the central area of Choco (Choco Refuge), at the northern area of the Western and Central Andean cordilleras (Nechí Refuge) and in the eastern area of the Serrania de Perijá (Catatumbo Refuge). Some other additional small refuges existed on the northern and northeastern slopes of the mountains of Eastern Panama, along the area of the mountains on both sides of the Magdalena Valley and on the northern slope of the Sierra Nevada de Santa Marta. In fact, the present connection between the Central American and northwest Colombian forests was interrupted in the Uraba Region during the dry climatic periods (Haffer, 1967). These isolated refuges were broadly connected in Northern Colombia during wet climatic periods. In the wet periods, the Uraba Region was probably covered with heavier forests than today. The Gulf of Uraba disappeared, thus providing a broad connection between Northern South America and Southern Central America. There was a more striking connection of the Western Colombian Andes and the Serranıa del Darien in Eastern Panama. It was during these times when the different C. capucinus populations expanded and possible interbreeding occurred among them, which disrupted the spatial genetic structure. For this reason, the spatial autocorrelation analyses did not detect any positive signal of this within Colombia. However, complementary to this, some of our spatial autocorrelation analyses detected some significantly positive evidence of spatial genetic differentiation between the Colombian populations and the Central American sample although much more moderately than it was established with mitochondrial DNA. This could be explained by two reasons. Males could be major vectors of gene flow compared to females. Alternatively, our sample size for Central America with microsatellites is smaller and less statistically significant than the Central America sample for mitochondrial DNA used by Ruiz-García et al., (2012) to detect such intense spatial structure. Jack et al., (2012) demonstrated that group instability due to group takeovers and small group size (as it should be in the moment of the population expansion) was also related to males leaving their natal group. Another interesting fact is that some of our genetic heterogeneity analyses revealed moderately significant differences of the Choco populations with reference to the other two Colombian populations. This could correlate with the fact that this population is geographically placed in the Choco Refugia.

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Molecular Systematics within C. Capucinus This is the first nuclear DNA study of C. capucinus to be compared with findings of Ruiz-García et al., (2012). These were the first authors to analyze the phylogeography and the molecular systematics of this species by using mitochondrial DNA. To do this comparison, we present a maximum likelihood tree with the mitochondrial COII gene sequences of 128 individuals of C. capucinus, the 121 animals reported in Ruiz-García et al., (2012) plus seven new individuals sampled in December 2013 across Panama by M. Ruiz-García (Figure 4).

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Figure 4. Maximum likelihood tree with 128 Cebus capucinus individuals at the mitochondrial COII genes. The number in the nodes are bootstrap percentages. Pan = Panama, Col = Colombia.

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The first interesting thing in this tree, which differentiated it from those from Ruiz-García et al., (2012), is that five out of seven individuals sampled in Panama were enclosed in the large cluster integrated by Central American individuals (with all the other individuals sampled in Costa Rica and Guatemala). The two remaining individuals (one from the Northern Panamanian area of Boqueron and other from Pacific central area of Panama) were clustered within the Colombian II haplogroup. If we consider the results shown by RuizGarcía et al., (2012), there was no clear relationship of the three large Colombian and the Central American haplogroups. The neighbor-joining tree with the Tamura 3P genetic distance indicated that the ancestor of the Colombian I haplogroup was more related to the ancestor of the Central America haplogroup. The Bayesian tree showed that the ancestor of the little group from Buenaventura was the most related to the Central American one. However, many other results supported that the Colombian II haplogroup should be in the origin of the Central American haplogroup. The less-differentiated Colombian II haplogroup with regard to the Central American showed FST = 0.737. The other two Colombian haplogroups yielded higher genetic differentiation in reference to the Central American haplogroup (FST = 0.803, I; FST = 0.788, III, respectively). The topology of the median joining haplotype network also showed that the Colombian II haplogroup was the most related to the Central American haplogroup. It also presented the lowest of the divergence times in regard to the Central American haplogroup. Another possibility is that the ancestors of the Central America haplogroup appeared before the split of the different Colombian haplogroups. This could agree quite well with the temporal divergence estimates from the median joining network, where these times between the Central American haplogroup and those from Colombia ranged from 4.87 to 3.66 MYA. The temporal divergences among the Colombian haplogroups were more recent and oscillated from 2.30 to 1.74 MYA. Also, the phylogenetic tree that we show (without an outgroup in order to accommodate recently debated issues about molecular dating of recent phylogenetic splits; Ho et al., 2008), had a first divergence for the ancestors of the Central American haplogroup rather than for the Colombian haplogroups. Against this view, is the fact that both microsatellite and mitochondrial data revealed considerably minor gene diversity relative to the Colombian haplogroups and populations. However, this new tree showed two Central American exemplars from Central and Northern Panama belonging to the Colombian II haplogroup, which means that there was, at least, one migration from Northern South America into Central America. These two Panamanian individuals did not show any morphological difference with regard to the other Panamanian exemplars sampled. This should mean that there is no reciprocal monophylia between the Central America C. capucinus and the Colombian C. capucinus haplogroups. This result with mitochondrial data correlates well with the fact that the genetic heterogeneity between the Central America population and the Colombian populations was more moderate using microsatellites (for instance, migration rates were relatively considerable and the spatial autocorrelation was moderated). Clearly, both microsatellites and mitochondrial DNA did not detect differences between the two alleged subspecies in Central America, C. c. limitaneus and C. c. imitator following Rylands et al., (2000). Within Colombia, until four different subspecies have been cited (C. capucinus capucinus-vicinity of Cartagena de Indias; C. c. curtus-Gorgona Island; C. c. nigripectus-between Cali and Buenaventura and C. c. hypoleucus-Bolivar). Both microsatellites and mitochondrial DNA reveal that there is no considerable genetic heterogeneity among different regions of Colombia and that the different mitochondrial haplogroups are mixed in the same geographical areas. Although we have not

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sequenced mitochondrial DNA within individuals from the Gorgona Island, we have analyzed three individuals from this Colombian island for microsatellites. They were completely nondifferentiable from the animals of the Cauca and Valle del Cauca region. Identically, the animals from the Bolivar Department did not form any discrete cluster. Thus, the three alleged subspecies (C. capucinus capucinus, C. c. curtus and C. c. hypoleucus) should be condensed in one unique taxon, C. capucinus capucinus. The work of Ruiz-García et al., (2012) showed that animals from the Buenaventura region formed a small differentiated cluster. Unfortunately, we did not obtain microsatellite data from these two animals, thus, we cannot provide data neither in favor nor against C. c. nigripectus. Animals from that area should be more comprehensively studied before determining the existence of taxa different from C. c. capucinus. With only the data of Ruiz-García et al., (2012), two possible subspecies can be postulated, C. c. capucinus (Colombia) and C. c. imitator (Central America). Nevertheless, although different tests determined the existence of significant genetic heterogeneity between the Central American sample and the Colombian ones, the STRUCTURE Software did not detect strong differentiation among populations and the spatial genetic structure was also moderated with microsatellites. Additionally, the inclusion of new Panamanian individuals showed that there is no total reciprocal monophylia between Central and South American populations of C. capucinus. This shows the importance of sampling a large number of individuals. If future studies corroborates the new results shown here, then one unique taxa should be claimed: C. capucinus as a monotypic species, such as claimed by other authors (Hernández-Camacho and Cooper, 1976; Mittermeier and CombraFilho, 1981; Groves, 2001). Our data clearly disagree with the work of Bobli et al., (2012). They concluded that the Central America C. capucinus should be differentiated as a new species, C. imitator. However, the new mitochondrial data, we present here, showed that haplotypes of both Colombian II haplogroup and Central American haplogroup were mixed in animals in the same troops with no morphological differences in Central and Northern Panama. Furthermore, the nuclear DNA microsatellite results, also showed limited differences between the Colombian and Costa Rica-Guatemalan C. capucinus individuals. Thus, it does not seem realistic to describe the Central America capuchins as a new species.

ACKNOWLEDGMENTS Thanks to Dr. Diana Alvarez and Pablo Escobar-Armel for their respective help in obtaining Cebus capucinus samples during the last 16 years. In Colombia, thanks to Instituto von Humboldt (Villa de Leyva; Janeth Muñoz) as well SDA (Bogotá; Norberto Leguizamón) to provide legal permissions to obtain samples as well as to the diverse Indian and Afrocolombian communities and people of different villages (Embera, Cauca, Waunana, Nuqui, Bahia Solano, Quibdó and Tule) who helped in this task.

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REFERENCES Antao, T., Lopes, A., Lopes, R.J., Beja-Pereira, A., Luikart, G. (2008). LOSITAN: A workbench to detect molecular adaptation based on a Fst-outlier method. BMC Bioinformatics 9:323 doi:10.1186/1471-2105-9-323. Archie, J.W. (1985). Statistical analysis of heterozygosity data: independent sample comparisons. Evolution 39: 623–637. Barton, N.H., Slatkin, M. (1986). A quasi-equilibrium theory of the distribution of rare alleles in a subdivided population. Heredity 56: 409-416. Beerli, P. (2006). Comparison of Bayesian and maximum likelihood inference of population genetic parameters. Bioinformatics 22:341-345. Beerli, P. (2009). How to use migrate or why are markov chain Monte Carlo programs difficult to use? In G. Bertorelle, M. W. Bruford, H. C. Haue, A. Rizzoli, and C. Vernesi, editors, Population Genetics for Animal Conservation, volume 17 of Conservation Biology. (Pp. 42-79). Cambridge University Press, Cambridge UK. Beumont, M., Nichols R. (1996). Evaluating loci for use in the genetics analysis of population structure. Proceedings of the Royal Society of London, Series B 263: 1619-1626. Boubli, J.P., Rylands, A.B., Farias, I., Alfaro, M.E., Lynch Alfaro, J.W. (2012). Cebus phylogenetic relationships: a preliminary reassessment of the diversity of the untufted capuchin monkeys. American Journal of Primatology 74: 381-393. Bruford, M. W., Wayne, R. K. (1993). Microsatellites and their application to population genetics studies. Current Opinion in Genetics and Development 3: 939-943. Charlesworth, B., Morgan, M. T., Charlesworth, D. (1993). The effects of deleterious mutations on neutral molecular variation. Genetics 134: 1289-1303. Cornuet, J. M., Luikart, G. (1996). Description of power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144: 2001-2014. Defler, T.R. (2010). Historia natural de los primates colombianos. Editorial Universidad Nacional de Colombia. Pp. 1-609. Degen, B., Scholz, F. (1998). Spatial genetic differentiation among populations of European beech (Fagus sylvatica L.) in Western Germany as identified by geostatistical analysis. Forest Genetics 5:191-199. Degen, B., Petit, R., Kremer, A. (2001). SGS - Spatial Genetic Software: A computer program for analysis of spatial genetic and phenotypic structures of individuals and populations. Journal of Heredity 92: 447-448. Dib, C., Faure, S., Fizames, C. (1996). A comprehensive genetic map of the human genome base on 5.264 microsatellites. Nature 380: 152-154. Dupanloup, I., Schneider, S., Excoffier, L. (2002). A simulated annealing approach to define the genetic structure of populations. Molecular Ecology 11: 2571-2581. Efron, B., Tibshirani, R. J. (1993). An introduction to the bootstrap. New York, NY: Chapman and Hall. Ellesworth, J. A., Hoelzer, G. A. (1998). Characterization of microsatellite loci in a new world primate, the mantled howler monkey (Alouatta palliata). Molecular Ecology 7: 657–658.

Genetic Structure, Spatial Patterns and Historical Demographic Evolution …

169

Falush, D., Stephens, M., Pritchard, J.K. (2007). Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 7:574–578. Feldman, M. W., Kumm, J., Pritchard, J. K. (1999). Mutation and migration in models of microsatellite evolution. In Goldstein, D. G., and Schlötterer, C. (Eds.). Microsatellites: evolution and applications. Oxford, United Kingdom: Oxford University Press. Freese, C. (1976). Predation on swollen-thorn acacia ants by white-faced monkeys Cebus capucinus. Biotropica 8: 278–281. Geary, R. C. (1954). The contiguity ratio and statistical mapping. The Incorporated Statistician 5: 115–145. Geoffroy-Saint-Hilare, E. (1812). Notes sur trios dessins de Commercon, représentant des Quadrumanes d’un genre inconnu. Paris: Annales du Museum d’Histoire Naturelle. Pp. 171–175. Goudet J., Raymond M., Demeeus T., Rousset F. (1996). Testing differentiation in diploid populations. Genetics 144: 1933–1940. Gregorius, H.R. (1978). The concept of genetic diversity and its formal relationship to heterozygosity and genetic distance. Mathematical Bioscience 41: 253-271. Groves, C.P. (2001). Primate Taxonomy. Smithsonian Institution Press, Washington, DC. Haffer, J. (1967). Speciation in Colombian forest birds west of the Andes. American Museum Novitates 2294:1–57. Hernández-Camacho, J., Cooper, RW. (1976). The nonhuman primates of Colombia. In: Thorington Jr, R.W., Heltne, P.G. (Eds). Neotropical Primates: Field Studies and Conservation. (pp. 35-69). Washington, DC: National Academy of Sciences. Ho, S.Y.W., Saarma, U., Barnett, R., Haile, J., and Shapiro, B. (2008). The effect of inappropriate calibration: three case studies in molecular ecology. PLoS ONE 32: e1615. Jack, K.M., Sheller, C., Fedigan, L.M. (2012). Social factors influencing natal dispersal in male white-faced capuchins (Cebus capucinus). American Journal of Primatology 74: 359-365. Kimmel, M., Chakraborty, R., King, J. P., Bamshad, M., Watkins, W. S., Jorde, L. B. (1998). Signatures of population expansion in microsatellite repeat data. Genetics 148: 19211930. Kimura, M. (1983). The neutral theory of molecular evolution. Cambridge University Press, Great Britain. King, D.G., Soller, M. (1999). Variation and Fidelity: The Evolution of Simple Sequence Repeats as Functional Elements in Adjustable Genes. In: Wasser, S.P., (Ed.). Evolutionary Theory and Processes: Modern Perspectives, Papers in Honor of Eviatar Nevo. Kluwer Academic Publishers Netherlands, Dordrecht. Pp. 65-82. Li, Y.C., Fahima, T., Peng, J.H., Röder, M.S., et al. (2000). Edaphic microsatellite DNA divergence in wild emmer wheat, Triticum dicoccoides, at a microsite: Tabigha, Israel. Theoretical and Applied Genetics 101: 1029-1038. Luikart, G., Allendorf, F., Sherwin, B., Cornuet, J. M. (1998). Distortion of allele frequency distribution provides a test for recent population bottleneck. The Journal of Heredity 86: 319-322. Manly, B.F.J. (1997). Randomization, Bootstrap and Monte Carlo Methods in Biology. Chapman and Hall, London.

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Mittermeier, R.A., Coimbra-Filho, A.F. (1981). Systematics: species and subspecies. In: Coimbra-Filho, A.F., Mittermeier, R.A., (Ed.). Ecology and Behavior on Neotropical Primates, vol. 1. (pp. 29-109). Rio de Janeiro: Academia Brasileira de Ciencias. Moran, P.A.P. (1950). Notes on continuous stochastic phenomena. Biometrika 37: 17-23. Michalakis, Y., Excoffier L. (1996). A generic estimation of population subdivision using distances between alleles with special reference to microsatellite loci. Genetics 142: 1061–1064. Nei, M. (1972). Genetic distance between populations. American Naturalist 106: 283-292. Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the USA 70: 3321–3323. Oppenheimer, J.R. (1969). Cebus capucinus: play and allogrooming in a monkey group. American Zoologist 9:1070. Paetkau D., Calvert, W., Stirling, I., Strobeck, C. (1995) Microsatellite analysis of population structure in Canadian polar bears. Molecular Ecology 4: 347-354. Pálsson, S. (2004). Isolation by distance, based on microsatellite data, tested with spatial autocorrelation (spaida) and assignment test (spassign). Molecular Ecology Notes 4: 143145. Patkeau D., Slade, R., Barden, M., Estoup, A. (2004). Genetic assignment methods for the direct, real-time estimation of migration rate: a simulation based exploration of accuracy and power. Molecular Ecology 13: 55-65. Piry S., Alapetite, A., Cornuet, J. M., Paetkau, D., Baudouin, B and Estoup, A. (2004). GENECLASS2: a software for genetic assignment and first-generation migrant detection. Journal of Heredity 95: 536-539. Rannala B., Mountain, J. L. (1997) Detecting immigration by using multilocus genotypes. Proceedings of the National Academy of Sciences USA 94: 9197-9201. Raymond, M., Rousset, F. (1995). An exact test for population differentiation. Evolution 49:1280-1283. Reich, D. E., Goldstein, D. B. (1998). Genetic evidence for a Paleolitic human population expansion in Africa. Proceedings of the National Academy of Sciences 95: 8119-8123. Reich, D. E., Feldman, M. W., Goldstein, D. B. (1999). Statistical properties of two tests that use multilocus data sets to detect population expansions. Molecular Biology and Evolution 16: 453-466. Robertson, A., Hill, W.G. (1984). Deviations from Hardy-Weinberg proportions, sampling variances and use in estimation of inbreeding coefficients. Genetics 107: 703-718. Robinson, J.G., Janson, C.H. (1987). Capuchins, squirrel monkeys, and atelines: socioecological convergence with Old World primates. In: Smuts, B.B., Cheney, D.L., Seyfarth, R.M., Wrangham, R.W., Struhsaker, T.T., (Eds.). Primate Societies. (pp. 6982). Chicago: University of Chicago Press. Rousset, F. (1997). Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance. Genetics 145: 1219–1228. Ruiz-García, M. (2005). The use of several microsatellite loci applied to 13 Neotropical primates revealed a strong recent bottleneck event in the woolly monkey (Lagothrix lagotricha) in Colombia. Primate Report 71: 27–55. Ruiz-García, M., Castillo, M.I., Alvarez, D. (2004): Evolutionary trends of Neotropical Primates according to the AP68 and AP40 microsatellites. In: Mendes, S.L., Chiarello,

Genetic Structure, Spatial Patterns and Historical Demographic Evolution …

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A.G., (Eds.): A Primatologia no Brasil - Volume 8. (pp. 65-100). Museu de Biologia Prof. Mello Leitao, Santa Teresa, Espirito Santo, Brasil. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P., Shostell, J. M. (2006). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report 73: 3–47. Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., GutierrezEspeleta, G., Shostell, J. M. (2007). Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: Genetic structure and recent bottlenecks. Folia Primatologica 78: 73–87. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L.F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). International Journal of Primatology 32:1218–1241. Ruiz-Garcia, M., Castillo, M.I., Ledezma, A., Leguizamon, N., Sánchez, R., et al. (2012). Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of Primatology 74: 366-380. Rylands, A.B., Mittermeier, R.A., Rodriguez-Luna, E. (1997). Conservation of Neotropical primates: threatened species and an analysis of primate diversity by country and region. Folia Primatologica 68:134–160. Rylands, A.B., Schneider, H., Mittermeier, R.A., Groves, C.P., Rodriguez-Luna, E. (2000). An assessment of the diversity of New World Primates. Neotropical Primates 8: 61-93. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. Silva, Jr J de S. (2001). Especiacao nos macacos-pregos e caiararas, género Cebus Erxleben, 1777 (Primates, Cebidae). Doctoral Thesis. Pp.377. Universidade Federal do Rio de Janeiro, Rio de Janeiro. Slatkin, M (1985). Rare alleles as indicators of gene flow. Evolution 39: 53-65. Slatkin, M. (1995). A measure of population subdivision based on microsatellite allele frequencies. Genetics 139: 457–462. Streiff, R., Labbe, T., Bacilieri, R., Steinkellner, H., Glossl, J., Kremer A. (1998). Withinpopulation genetic structure in Quercus robur L. and Quercus petraea (Matt.) Liebl. assessed with isozymes and microsatellites. Molecular Ecology 7:317-328. Van der Hammen, T. (1961). The Quaternary climatic changes of northern South America. Annual New York Academy of Sciences 95:676–683. Vendramin, G.G., Degen, B., Petit, R.J., Anzidei, M., Madaghiele, A., Ziegenhagen, B. (1999). High level of variation at Abies alba chloroplast micrsatellite loci in Europe. Molecular Ecology 8: 1117-1126. Walsh, P.S., Metzger, D.A., Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506513. Weir, B.S., Cockerham, C.C. (1984). Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370.

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Wright, S. (1951). The genetical structure of populations. Annals of Eugenics 15: 323–354. Zhivotovsky, L. A., Feldman, M. W. (1995). Microsatellite variability and genetic distances. Proceedings of the National Academy of Sciences 92: 11549-11552. Zhivotovsky, L. A., Bennett, L., Bowcock, A. M., Feldman, M. W. (2000). Human population expansion and microsatellite variation. Molecular Biology and Evolution 17: 757-767.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 6

INVALIDATION OF THREE ROBUST CAPUCHIN SPECIES (CEBUS LIBIDINOSUS PALLIDUS, C. MACROCEPHALUS AND C. FATUELLUS; CEBIDAE, PRIMATES) IN THE WESTERN AMAZON AND ORINOCO BY ANALYZING DNA MICROSATELLITES Manuel Ruiz-García1,, María Ignacia Castillo1, Kelly Luengas-Villamil1 and Norberto Leguizamón2 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva. Unidad de Genética. Departamento de Biología. Facultad de Ciencias. Pontificia Universidad Javeriana. Bogotá DC., Colombia 2 Secretaria Distrital Ambiental (SDA), Bogotá DC., Colombia

ABSTRACT We analyzed 163 robust capuchins for eight nuclear DNA microsatellites representing three different “a priori” morphological taxa (47 pallidus individuals from Bolivia, 24 macrocephalus individuals from the Peruvian and Southern Colombian Amazon and 92 fatuellus individuals from the Colombian Eastern Llanos and Northern Colombian Amazon). There were seven main findings to come out of this study. 1Levels of gene diversity were high for all three populations but the most southern population, pallidus, had the highest whereas the most northern population, fatuellus, showed the lowest; 2- The major part of the microsatellites we used were neutrally dynamic, although some did show evidence of positive natural selection; 3- Deviations from the Hardy-Weinberg equilibrium were detected especially for fatuellus and pallidus (Wahlund effect). We also detected six different populations of widely intermixed individuals. The populations were not related with the morphological taxa traditionally considered by primatologists. 4- Some evidence of significant genetic differentiation was found for different markers among these three taxa. However, the relative genetic heterogeneity found was of a small magnitude but the estimations of gene flow were very 

Correspondence: [email protected], [email protected].

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Manuel Ruiz-García, María Ignacia Castillo, Kelly Luengas-Villamil et al. high and even superior to those obtained for populations of Cebus capucinus, a unique and recognized species by all primatologists. 5- The assignment analysis showed that a considerable amount of the analyzed individuals were misclassified within their respective taxa (around 33%). 6- No bottleneck events were detected for any of the robust capuchin populations but population expansions were detected which support an explosive expansion of these monkeys during the Pleistocene. 7- None of the different spatial genetic structure analyses detected any significant genetic patches or evidence of gene flow interruption that would be expected if diverse species were living in the same geographical area. In contrast, very smooth monotonic clines were detected which are typical of gradual differentiation without reproductive isolation. Our results do not support the existence of three well-differentiated robust capuchin species within the study area. We propose that these three alleged morphological taxa be classified in two different subspecies within Cebus apella, C. a. macrocephalus and C. a. fatuellus.

Keywords: Cebus apella, pallidus, macrocephalus, fatuellus, gene diversity, genetic heterogeneity and gene flow, genetic assignment, population expansions, Pleistocene, spatial genetic structure

INTRODUCTION The tufted (robust) capuchins (Cebus apella, Linnaeus, 1758) were considered as a single species (Cabrera, 1957 and Hill, 1960) that has the largest geographical distribution of all the New World primate species. Its range extends from the Eastern Andes Cordillera in Colombia (but with a population in the upper Magdalena River Basin within the Colombian Departments of Huila and Tolima) to 27° south within the southern Brazilian states. It also inhabits the Misiones, Salta and Jujuy provinces of Argentina. The taxonomy of the robust capuchin is extremely confusing and is as diverse as the authors that have worked on the subject (Table 1). Elliot (1913) and Hershkovitz (1949, 1955) each recognized 12 species of robust capuchins, with Cabrera (1957) being the first author to include all the robust capuchin taxa within a single unique species, C. apella, with 11 subspecies. Hill (1960) also suggested a single unique species, C. apella, with 16 subspecies. Torres de Assumpcao (1983, 1988) identified phenotypic differences between robust capuchins from each of five distinct geographic regions. She also identified an extensive sixth area (that included Central and Northeastern Brazil) where there was more phenotypic variation in the animals inhabiting this area compared to the other five areas. This variation is due to different selective habitat pressures, extensive hybridization with different surrounding forms or less genetic speciation differentiation within this sixth area. Torres de Assumpcao (1983, 1988) did not assign any specific or sub-specific nomenclature to the robust capuchins of these geographic areas. Groves (2001) determined four robust capuchin species (C. apella, C. libidinosus, C. nigritus and C. xanthosternos) and 14 subspecies, whereas Silva Jr (2001) distinguished between robust and gracile capuchins, assigning the former to the genus Sapajus (Kerr, 1792) and including seven species (S. apella, S. macrocephalus, S. libidinosus, S. cay, S. nigritus, S. robustus and S. xanthosternos (see also Fragaszy et al., 2004). He assigned gracile capuchins to the genus Cebus. Sapajus has also been recently supported by Lynch-Alfaro et al., (2012) based on alleged morphological, genetic, behavioral, ecological

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and biogeographic evidence. However, also in the current book, Ruiz-García et al., (2016a) show that these data (especially the molecular and karyotype ones) don’t justify the nomenclature split between Cebus and Sapajus. Table 1. Different morphological classifications for the robust capuchins obtained by diverse authors. The taxonomic review of Groves (2001, 2005) of the tufted capuchins is identical to that of Rylands et al., (2000). C = Cebus; S = Sapajus Elliot (1913) C. apella C. fatuellus C. f. fatuellus C. f. peruanus C. macrocephalus C. azarae C. azarae C. azarae pallidus C. libidinosus C. frontatus C. variegatus C. versuta C. cirrifer C. crassiceps C. caliginosus C. vellerosus Hill (1960) C. apella C. a. apella C. a. margaritae C. a. fatuellus C. a. tocantinus C. a. macrocephalus C. a. magnus C. a. juruanus C. a. maranonis C. a. peruanus C. a. pallidus C. a. cay C. a. libidinosus C. a. robustus C. a. frontatus C. a. nigritus C. a. xanthosternos

Cabrera and Yepes (1940) C. xanthosternos C. paraguayanus C. libidinosus C. macrocephalus C. frontatus C. nigritus C. vellerosus C. fatuellus C. apella

Cabrera (1957) C. apella C. a. apella C. a. libidinosus C. a. macrocephalus C. a. margaritae C. a. nigritus C. a. pallidus C. a. paraguayanus C. a. robustus C. a. vellerosus C. a. versutus C. a. xanthosternos

Groves (2001) and Rylands et al., (2000) C. apella C. a. apella C. a. fatuellus C. a. macrocephalus C. a. peruanus C. a. tocantinus C. a. margaritae C. libidinosus C. l. libidinosus C. l. pallidus C. l. paraguayanus C. l. juruanus C. nigritus C. n. nigritus C. n. robustus C. n. cucullatus C. xanthosternos

Silva Jr (2001) S. apella S. macrocephalus S. libidinosus S. cay S. nigritus S. robustus S. xanthosternos

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In the current work, we used molecular genetics procedures and data to validate or negate three proposed taxa of robust capuchins. The first taxa we analyzed consisted of individuals sampled in Bolivia and that had been classified by using very different nomenclature. Elliot (1913) considered these Bolivian animals as belonging to C. azarae pallidus. Differently, Cabrera (1957) and Hill (1960) classified them as C. apella pallidus, but Groves (2001, 2005) and Rylands et al., (2000) considered them as C. libidinosus pallidus. Silva Jr (2001) also included them within S. libidinosus but without any sub-specific status. The Peruvian and Southern Colombian Amazon individuals have been analyzed and recognized by all the authors interested in the question. However, some of them considered it as a full species, C. or S. macrocephalus (Elliot, 1913 and Silva Jr, 2001), but others considered it as a subspecies (C. apella macrocephalus; Cabrera, 1957; Hill, 1960; Groves, 2001, 2005; Rylands et al., 2000). Finally, a third individual group sampled in the Colombian Eastern Llanos and Northern Colombian Amazon was also considered. Elliot (1913) considered the animals from that area as C. fatuellus and Cabrera (1957) considered them within C. apella apella. Hill (1960) and Groves (2001, 2005) determined these animals as C. apella fatuellus while Silva Jr (2001) assumed that they were within S. macrocephalus. Due to the uncertainty of these nomenclatures, we employed the names of pallidus, macrocephalus and fatuellus but without the status of full species or subspecies until the present findings are discussed. We have studied eight hyper-polymorphic STRPs (Short Tandem Repeat Polymorphisms) or microsatellites to analyze the genetic differences among these three robust capuchin taxa. These kinds of markers are composed of short repetitive elements, one to six nucleotide base pairs in length. They are also randomly distributed, highly polymorphic, and are frequently inside the eukaryotic genomes. An additional and positive property of these markers is the small DNA quantity needed to carry out these molecular analyses (via PCR). The small sample size allows the investigator to use non-invasive procedures to sample wild animals and successfully examine population biology dynamics through the use of molecular genetic techniques (Bruford and Wayne, 1993) as well as to establish gene linkage maps. Therefore, the main aim of the current work is to bring forward new evidence with nuclear DNA markers and determine the “real” systematic status of these three robust capuchin taxa by analyzing the gene diversity levels of each taxa and the degree of genetic heterogeneity and estimations of gene flow among them. Additionally we aim to determine if these three taxa suffered the same evolutionary demographic changes and if they developed a spatial genetic structure compatible with the existence of three full species.

MATERIALS AND METHODS The DNA were obtained from samples of hair, teeth, muscle and blood, which were collected from animals found alive or dead in diverse Indian communities in the Bolivian, Peruvian (including frontier with Brazil) and Colombian Amazon as well as in the Eastern Colombian Llanos (Orinoco). We requested permission to collect biological materials from either carcasses or live animals that were already present in the community. We sampled small pieces of tissue (muscle or blood) or teeth from hunted animals that were discarded during the cooking process, or hairs with bulbs plucked from live pets. Communities were visited only once, all sample donations were voluntary, and no financial or other inducement

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was offered for supplying specimens for analysis. All the pets and the hunted animals were obtained by the Indian communities at a maximum of around 10-15 km from their specific community. A total of 163 robust capuchins were sampled in the wild in three South American countries: Bolivia, Peru and Colombia. In Bolivia, 47 pallidus individuals were sampled, including the following localities: Carrasco (Cochabamba), El Carmen (Santa Cruz), San Ignacio de Moixos, San José de Ibiato, Trinidad, Santa Ana de Yacumo, Exaltación, Portento, San Lorenzo, Guayaramerín, Yata River and Ribera Alta (Beni), Nuanilla, Santa Ana de Madidi (La Paz) and Santa Rosa (Pando). In Peru and Southern Colombian Amazon, 24 macrocephalus were sampled, including the following localities: the area between Leticia and San Juan de Atacuarí in the Amazonas Department in Colombia, localities near Puerto Asís (Putumayo Department, Colombia), Ucayali River and affluent as well as Napo and Curaray rivers in the Northern Peruvian Amazon and Javarí River (frontier between Peru and Brazil) in Peru. Finally, 92 fatuellus were sampled in Colombian lands: La Macarena and different points in the Meta Department, Araracuara (Caqueta Department), Vaupes Department, Ocuné and El Tuparro (Vichada Department), San José and other points in the Guaviare Department and Puerto Valencia and other points in the Guainia Department.

Molecular Procedures DNA from muscle and blood was isolated using standard phenol–chloroform extraction methods (Sambrook et al., 1989), while DNA from teeth and hair were isolated by boiling with 10% Chelex resin following Walsh et al., (1991). We used eight microsatellite markers (AP68, D5S111, D5S117, D6S260, D8S165, D14S51, D17S804, and PEPC8). The AP68 marker was designed for Alouatta palliata and PEPC8 for C. apella, while the remaining markers were designed for humans (Ellesworth and Hoelzer, 1998). These microsatellites have been successfully used in other Neotropical primates such as Alouatta, Ateles, Lagothrix, Cebus, Saimiri, Aotus and Saguinus (RuizGarcía, 2005; Ruiz-García et al., 2006, 2007, 2011). Our final PCR volume and reagent concentrations for the DNA extraction from muscle and blood were 25 μl, with 3 μl of 3 mM MgCl2, 2.5 μl of buffer 10×, 1 μl of 1 mM dNTP, 1 μl of each primer (forward and reverse; 4 pmol), 13.5 μl of H2O, 2 μl of DNA, and 1 Taq polymerase unit per reaction (1 μl). For the PCR reactions with hair and teeth, the overall volume was 50 μl, with 20 μl of DNA and twofold amounts of MgCl2, buffer, dNTPs, primers, and Taq polymerase. We performed all PCR reactions in a PerkinElmer Geneamp PCR System 9600 thermocycler for 5 min at 95°C, 30 1-min cycles at 95°C, 1 min at the most accurate annealing temperature (50°C for AP68, and 52°C for the remaining markers), 1 minute at 72°C, and 5 min at 72°C. We kept amplification products at 4°C until they were used in a denatured 6% polyacrylamide gels in a Hoefer SQ3 sequencer vertical chamber. Depending upon the size of the markers analyzed, and the presence of 35 W as a constant, we stained the gels with AgNO3 (silver nitrate) after 2–3 h of migration. We used the molecular markers HinfI and ϕ174 (cut with HindIII). We repeated the PCR reactions three times for DNA extracted from hair. Thus, allelic dropout was highly improbable, but we cannot completely exclude the existence of null alleles, which could increase the number of false homozygous genotypes. Nevertheless, it is improbable that all loci were affected in the same way.

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Population Genetics Molecular Analyses Genetic Diversity and Natural Selection Several gene diversity statistics were estimated through the microsatellite genotypes obtained. The mean number of alleles per locus and the expected heterozygosity (H) (Nei, 1973) were calculated for the three robust capuchin taxa studied and statistically analyzed with a student t test. The expected heterozygosity values were arc sin transformed prior to statistical analysis (Archie, 1985). To analyze the influence of natural selection (constrictive or diversifying) on the microsatellites used, the coalescence theory generated by Beaumont and Nichols (1996) was employed for the overall robust capuchin sample. We used the LOSITAN program (Antao et al., 2008) and obtained the observed and expected FST statistical values for each marker used throughout the samples. Both the infinite allele and the step-wise mutation models were considered. A total of 50,000 iterations were completed to calculate the values that represented the relationship between the FST statistic and the expected heterozygosity of the markers. There were subsample sizes of 50 from which the medians and the 5% and the 95% quartiles were calculated and CPU cores of 4. The observed FST and the heterozygosity values were superimposed under this distribution and consisted of the median and the quartiles. Values outside of this theoretical distribution indicate that the microsatellites in question are being affected by natural selection. Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow The Hardy-Weinberg equilibrium (H-W E) for each one of the three robust capuchin populations studied were estimated using several different strategies. The F statistics of Weir and Cockerham (1984) and Robertson and Hill (1984) were used to calculate the degree of excess or deficit, of homo- and heterozygous (complete enumeration) within each one of the populations. To measure the exact probabilities of the G test, we used the Markov chain method, with a 10,000 dememorization number, 20 batches and 5,000 iterations per batch, following the Genepop v. 4.2.1 Program (Raymond and Rousset, 1995). The H-W E was simultaneously analyzed by locus and by taxon using the Fisher’s method (Raymond and Rousset, 1995). The genetic heterogeneity among the three robust capuchin taxa was analyzed globally for each marker and analyzed together as well as by population pairs. One strategy used the gene frequencies of the eighth microsatellites studied and included exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches, and 5,000 iterations per batch. We also used the Wright F-statistics (Wright, 1951) with the Michalakis and Excoffier (1996) procedure. The standard deviations of the F-statistics were calculated using a jackknifing over loci procedure. Also, the 99% confidence intervals were measured by means of bootstrapping over loci. Two procedures were used to measure the significance of FST. The first one used 10,000 randomizations of overall alleles sampled and assumed random mating within populations by means of the G test (Goudet et al., 1996). The second procedure used 10,000 randomizations of genotypes among populations and did not assume random mating within populations by means of the log-likelihood G test (Goudet et al., 1996). The significance of FIS and FIT was also found by using 10,000 randomizations of alleles within samples and in the overall sample. Additionally, the gene diversity analysis of Nei (1973) as well as the

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repeat number of the allele (RST) statistic (Slatkin, 1995; Rousset, 1997) were estimated to measure the gene heterogeneity between the three robust capuchin populations. These analyses are useful to determine which microsatellites more clearly discriminate among the populations of the robust capuchins and to determine the degree of gene variability within each population relative to the whole sample. Possible theoretical gene flow estimates among the three populations studied were measured using the private allele model (Slatkin, 1985; Barton and Slatkin, 1986). Additionally, we applied Migrate 3.6 Software to determine possible asymmetrical gene flow among the robust capuchin populations (Beerli, 2006, 2009), (in this case, the scaled migration rate, M = m/µ was obtained, where m is the migration rate per generation and µ the mutation rate per generation). For the Maximum Likelihood procedure, we employed ten short Markov chains, with 500 recorded steps, 100 increments and a total of 50,000 sampled genealogies with a burn-in of 10,000, and one long Markov chain, with 5,000 recorded steps, 100 increments and a total of 500,000 sampled genealogies with a burn-in of 10,000. The Bayesian procedure was run with one long Markov chain, with 5,000 recorded steps, 100 increments, one concurrent chain and a total of 500,000 sampled genealogies with a burn-in of 10,000.

Assignation and Genetic Structure Tests We developed diverse assignment analyses by using the GENECLASS 2 program (Piry et al., 2004). We performed different strategies by employing one Bayesian procedure (Rannala and Mountain, 1997), one frequency procedure (Paetkau et al., 1995) and one genetic distance (standard genetic distance, Nei, 1972). The assignation analyses were carried out without simulations and served to estimate the probabilities of individuals belonging or being excluded from the original populations where they were “a priori” assigned (P < 0.05). Some assignation analyses were also completed with 10,000 resampling simulations by means of the Monte Carlo technique and with the procedure of Paetkau et al., (2004). Additionally, we estimated the possible existence of first generation migrants in the different populations by using the Bayesian, frequency and genetic distance procedures we commented on above without simulations. To determine this, we considered the relationship: L = Lhome/Lmax. This is the ratio of the likelihood computed from the population where the individual was sampled (Lhome) over the highest likelihood value among all population samples including the population where the individual was sampled (Lmax) (Paetkau et al., 2004). Another assignment analysis was applied using STRUCTURE 2.3 (Falush et al., 2007), which employs Markov Chain Monte Carlo procedures and the Gibbs sampler, uses multilocus genotypes to infer population structure, and simultaneously assigns individuals to specific populations. The model considers K populations, where K may be unknown, and the individuals are assigned tentatively to one population or jointly to ≥2 populations (if their genotypes are considered admixed). Two analysis groups were carried out. First, we considered the admixture model, wherein the individuals may have mixed ancestry and the no-admixture model with no prior population information to assist with clustering (USEPOPINF = 0). In addition, α was inferred (Dirichlet parameter for degree of admixture; with an initial value of α = 1) using uniform priors for α (its value was the same for all populations). The maximum value of this parameter was 10. Allele frequencies were correlated among populations, assuming different values of FST for each population. We

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revealed the presence of the most probable number of gene pools by using the increasing likelihood method. The second analysis was undertaken with a model that incorporates informative geographic origin individual priors to assist with the clustering of weakly structured data in order to determine migrants or detect slightly different populations (USEPOPINF = 1) with both admixture and no admixture models. Furthermore, in this case, in order to apply the same conditions to that of the previous case, we introduced LocPrior = 1, Gensback = 2, and Migprior = 0.05. The program was run with 1,000,000 iterations after a burn period of 100,000 iterations for each analysis. Each analysis was performed twice with convergent results.

Possible Historical Demographic Changes One analysis was focused on the detection of recent bottleneck events using the most recently derived theory generated by Cornuet and Luikart (1996), and Luikart et al., (1998). The population, which experienced a recent bottleneck, simultaneously decreases the allele number and the expected levels of heterozygosity. Nevertheless, the allele number (ko) is reduced faster than the expected heterozygosity. Therefore, the value of the expected heterozygosity calculated throughout the allele number (Heq) is lower than the obtained expected heterozygosity (He). For neutral markers, in a population in gene mutation drift equilibrium, there is an equal probability that a given locus has a slight excess or deficit of heterozygosity in regard to the heterozygosity calculated from the number of alleles. In contrast, in a bottlenecked population, a large fraction of the loci analyzed will exhibit a significant excess of the expected heterozygosity. To measure this probability, four diverse procedures were used as follows: sign test, standardized difference test, Wilcoxon´s signed rank test and graphical descriptor of the shape of the allele frequency distribution. A population, which did not suffer a recent bottleneck event, will yield an L-shape distribution (such as expected in a stable population in mutation-gene drift equilibrium), whereas a recently bottlenecked population will show a mode-shift distribution. The Wilcoxon´s signed rank test probably has its greatest power when the number of loci analyzed is low, such as in the current case. The BOTTLENECK Program was used for this task. To determine possible population expansions in the three robust capuchin populations, four tests were carried out. Two of them were the within locus kurtosis (k) test and the interlocus (g) test proposed by Reich and Goldstein (1998) and Reich et al., (1999). The first test is based on the following principles. A population with constant size has gene genealogies, which tend to have a single ancient bifurcation. Therefore, the allele length distributions have multiple discrete peaks. On the contrary, in a growing population, most of the gene genealogy bifurcations tend to date back to the time expansion and as a result the allele length distribution is clearly more smoothly peaked. To measure these differences between the multi-peaked allele distribution of a constant size population and the smooth single-peaked allele distribution of a population in expansion, the k statistic was calculated as follows: k = 2.5 Sig4 + 0.28 Var – (0.95/n) – Gam4. Sig4 is the unbiased variance squared of allele length, Var is the sample variance of the same concept and Gam4 is the unbiased fourth central moment of the allele, respectively. In order to assess significance levels, a binomial distribution is used with the number of trials equal to the number of loci based on the expectation of an almost (P = 0.515) equal probability of negative and positive k-values for the set of loci analyzed. When there is a smaller loci number associated with positive k values

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than would be expected for a constant-sized population, there is evidence of a population expansion. The g test focuses on the following facts. This test shows that for stable populations the allele size variance is highly variable among loci, whereas in expansion populations this variance is usually lower. Therefore, allele sizes with sufficiently low variances are taken as evidence for population expansion. The test used for statistical differences is that proposed by Reich and Goldstein (1998):

g

Var[Vj ] 4 V 2 1 V 3 6

where Var (Vj) is the observed variance of the allele length variances across the markers employed, and V is the average variance across loci. A low value of g is taken as a sign of population expansion. Significance levels for the interlocus test are found on Table 1 of Reich et al., (1999), which shows the fifth-percentile cutoffs for the interlocus test. Luikart et al., (1998) noted that this last test is probably the most powerful for this task. The test of Kimmel et al., (1998) is based on the principle that two different estimates of θ (=4Ne) could be obtained. One is θv = V, with V being the variance of the tandem repeat size, whose expression is V = (2 Σ i = 1….n (Xi – X)2)/(n – 1), where n is the number of chromosomes analyzed, Xi is the number of repeats of each allele found and X is the average repeat number of all the alleles found in a microsatellite. The second one is θPo = (1/(Po2 – 1))/2 (estimator of the homozygosity), where Po = (n Σ k = 1…..n (p k2 – 1))/(n -1), with pk being the allele frequency of the kth allele. An imbalanced β index could be defined as: β(t) = E(θv)/E(θPo) = V(t)/[((1/ Po2) – 1)/2 or by the expression: ln β(t) = ln θv – ln θPo = ln (V) – ln (((1/ Po2) – 1)/2). If a population is in equilibrium, has a constant demographic size, and isn’t suffering an expansion, β= 1 (ln β = 0). On the contrary, if a population has suffered an expansion coming from a mutation-drift equilibrium situation (constant population size), β < 1 (ln β < 0). If a population has experienced an expansion coming from a previous bottleneck, β > 1 (ln β > 0). This last value will be present for a long time (several thousand generations) before showing the signature of a population expansion (β< 1 (ln β < 0). There is an exception to this general rule, when a bottleneck is so intense the population becomes monomorphic before the demographic expansion, in which case, β < 1 (ln β < 0) all the time. All of these β values are consistent in stepwise, logistic or exponential population growth and are not especially affected in diverse mutation models (Kimmel et al., 1998). To determine the statistical significance of the β (ln β), a jackknife procedure (Efron and Tibshirani, 1993) was applied to obtain the variance of β and, with this variance, a Student’s t test and 95 and 99% confidence intervals were estimated. Also, a test of Zivothovsky et al., (2000) was used to calculate an expansion index: Sk = 1 – ((K – (RkV/2)/5V2). K and V are the unnormalized kurtosis (fourth central moment) and the allele size variance is estimated from a sample and corrected for sampling bias, respectively, whereas Rk = km/σ2m (they are the kurtosis and the variance in the repeat number mutational changes). The expressions used to estimate V and K are: V = Σi = 1…n pi (Xi – X)2 and K = Σ i = 4 1…n pi (Xi – X) , where X = Σi = 1…k pi k. The k variable represents the alleles in a locus given and pi represents the allele frequencies. All the other terms were defined in the previous

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analysis. The value of Rk employed was 6.3 because this value was obtained for dinucleotide microsatellites by Dib et al., (1996), and because dinucleotide microsatellites were used in the current study. Feldman et al., (1999), used the same data and a geometrical distribution of mutational events. They obtained an estimated σ2m of 2.5, which is basically the same as what was obtained by using a truncated Poisson distribution (Zhivothovsky et al., 2000) and by ourselves in the present work (σ2m = 2.39). The value of Sk is expected to be 0 in a general symmetric stepwise mutation model for a population in equilibrium and of constant size (this was derived by Zhivotovsky and Feldman, 1995). The Sk is positive if an expansion affected the population and is contrarily negative if a bottleneck affected the population. To obtain demographic conclusions of this analysis, the within-population variance and the expansion index are averaged for all the microsatellites studied within each population and their dynamics are compared. Zhivotovsky et al., (2000) showed that a significant correlation existed between V and Sk (r = 0.58) for a human data set, but this correlation was moderate and, in fact, both statistics could react differently to the changes in population size and have different patterns in different populations. To measure the statistical significance of the Sk values, a jackknife procedure was performed to obtain the variance of Sk and, with this variance, a Student’s t test and a 95% confidence interval were estimated.

Spatial Genetic Analyses Two software were employed to analyze possible spatial genetic patterns for the Bolivian, Peruvian and Colombian robust capuchin individuals studied. The first was the SGS version 1.0d (Degen et al., 2001). The analyses with all the individuals analyzed were carried out by using distograms (Degen and Scholz, 1998; Vendramin et al., 1999) with the genetic distance of Gregorious (1978) and distograms with the common alleles (Surles et al., 1990; Hamrick et al.,1993). Three and 10 distance classes (DC) were employed for all individuals taken as a whole (3 DC: 0-718 km, 718-1,437 km, 1,437-2,155 km; 10 DC: 0-216 km, 216431 km, 431-647 km, 647-862 km, 862-1,078 km, 1,078-1,293 km, 1,293-1,509 km, 1,5091,724 km, 1,724-1,940 km, 1,940-2,155 km). The significances of distograms were calculated by means of 1,000 Monte-Carlo simulations (Manly, 1997) with an estimated confidence interval of 95% (Streiff et al., 1998). The second program used was SPAGeDi 1.4 (Hardy and Vekemans, 2002). The correlograms were obtained for four different statistics. They were the Moran’s I index (Moran, 1950), the relationship coefficient of Queller and Goodnight (1989), the pairwise Rousset (2000)’s distance between individuals and the pairwise correlation coefficients of allele size (Streiff et al., 1998), with jackknifed estimators over loci and 5,000 random permutations of gene copies for probabilities. These analyses were applied to three data sets. The first was for all the samples analyzed (area of distribution pallidus, macrocephalus and fatuellus), where six DC were defined with an identical number of pairs per CD (1,291 pairs) (0-194 km; 194-412 km; 412-637 km; 637-1,327 km; 1,327-1,622 km and 1,622-2,589 km). The second analysis was inside Bolivia (for pallidus) and four DC were defined with an identical number of pairs per CD (259 pairs) (0-230 km; 230-379 km; 379-473 km and 4731,140 km). The third analysis was inside Colombia (for fatuellus) and four DC were defined with an identical number of pairs per CD (488 pairs) (0-29 km; 29-332 km; 332-469 km and 469-970 km). For macrocephalus, no analyses were carried out due to the reduced sample size of this taxon.

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Figure 1. Application of LOSITAN Software to detect possible natural selection at the eight DNA microsatellites analyzed in the overall robust capuchin population studied: for an infinite allele mutation model (A) and for the stepwise mutation model (B). The D6S260 microsatellite was outside of both figures.

RESULTS Gene Diversity and Natural Selection The average number of alleles for fatuellus, macrocephalus and pallidus were 9.75 ± 5.52, 5.38 ± 2.83 and 7.50 ± 2.98, respectively. Clearly, this statistic is correlated with the sample sizes of each population analyzed. The gene diversity was somewhat different in the three robust capuchin taxa analyzed (fatuellus: H = 0.607 ± 0.305; macrocephalus: H = 0.639 ± 0.225; pallidus: H = 0.749 ± 0.222). These gene diversity differences could be very related

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with the fact that pallidus generated macrocephalus and this in turn fatuellus following the hypotheses of Lynch-Alfaro et al., (2012) and Ruiz-García et al., (2012, 2016a). Table 2. Probability tests to estimate possible Hardy–Weinberg equilibrium individually and globally for the three robust capuchin taxa studied (fatuellus, macrocephalus and pallidus) at eight microsatellites. * P < 0.05; ** P < 0.01; *** significant with Bonferroni correction (P < 0.0021); df = degree of freedom; S. E. = Standard Error

The application of the Beaumont and Nichols (1996)’ method by using the LOSITAN Software with the infinite allele mutation and the stepwise mutation models showed, in the

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first case, one microsatellite having positive adaptation natural selection (D14S51), whilst, for the second model, two microsatellite had positive adaptation natural selection (D14S51 and PEPC8). All the other microsatellites showed neutral behavior (Figure 1).

Hardy-Weinberg Equilibrium, Genetic Heterogeneity and Gene Flow Some of the loci analyzed showed some deviations from the H-W E (Bonferroni’s correction: α = 0.0021) (Table 2). In the case of fatuellus, seven out of eight microsatellites showed homozygote excess. The other two robust capuchin showed a different perspective. In the case of macrocephalus, no marker showed significant H-W E deviations, while for pallidus, three microsatellites showed significant homozygote excess (D5S111, D5S117 and D6S260). Taking all the markers and populations together, there was no H-W E for the robust capuchin sample analyzed (2 = infinite; df = 36; P = 0.00000001). Table 3. Exact G tests to determine genic heterogeneity at eight DNA microsatellites among three taxa of robust capuchins (fatuellus, macrocephalus and pallidus). * P < 0.05; ** P < 0.01; *** significant with Bonferroni correction (P < 0.0021); df = degree of freedom

All markers simultaneously taken: 2 = Infinity (df= 16), highly significant.

The genic differentiation by means of exact G tests yielded five of the eight microsatellites showing significant heterogeneity among the three taxa considered (D5S111, D5S117, D6S260, D17S804 and PEPC8) with the Bonferroni correction. Taking all the markers together, the genetic heterogeneity was also highly significant (2 = infinite; df = 16; P = 0.00000001) (Table 3). By population pairs, taking all the markers simultaneously, the three population pairs showed a significant genetic heterogeneity, although the most outstanding differentiation was between fatuellus and pallidus. By markers, AP68 showed significant differences for fatuellus-pallidus and D5S111 yielded significant differences for fatuellus-macrocephalus and for macrocephalus-pallidus. D5S117 showed significant differences for fatuellus-pallidus and D6S260 showed significant differences for fatuelluspallidus. Additionally, D8S165 yielded significant differences for fatuellus-macrocephalus and D14S51 did not show significant differences for any population pair. D17S804 showed significant differences for fatuellus-macrocephalus and for fatuellus-pallidus and PEPC8 yielded significant differences for fatuellus-macrocephalus and for macrocephalus-pallidus (Table 4). However, although there was clear evidence of significant heterogeneity among the

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three studied robust capuchin taxa, this genetic heterogeneity was relatively small as it was shown by the gene diversity (Nei 1973) and the F statistic (Wright 1951) analyses (Table 5). The first analysis showed values of GST = 0.024 and GST’= 0.035, whilst FST = 0.039 ± 0.010 (99% confidence interval: 0.015-0.061). This means that, on average, only around 3-4% of the gene differentiation is among the three robust capuchin taxa considered and around 9697% of the gene differentiation is among individuals within each one of these taxa. The RST statistic ranged from 0.053 to 0.116 depending of the procedures employed. If we do not consider random mating within samples using a log-likelihood G test, no microsatellite showed clear evidence of genetic heterogeneity (only evidence of significant heterogeneity taken all loci simultaneously, P = 0.006-0.000). In contrast, if we consider random mating within samples many microsatellites showed significant genetic heterogeneity (D5S111, D5S117, D6S260, D17S804, PEPC8 and all the loci taken together). The other F-statistics showed clear evidence of a certain significant homozygote excess both at the global level (FIT = 0.323 ± 0.042; 99% confidence interval: 0.208-0.418; all the microsatellites showed significant homozygote excess with the exception of AP68 and D17S804 using Bonferroni’s correction) as well as within each robust capuchin taxon (FIS = 0.295 ± 0.044; 99% confidence interval: 0.170-0.393; all the microsatellites showed significant homozygote excess with exception of AP68 and D17S804 using Bonferroni’s correction). Thus, neither the overall sample of the three robust capuchin taxa nor the individual taxa were in H-W E. The overall gene flow estimate with the method of the private allele was Nm = 1.31, which could be interpreted as these populations not being totally reproductively disconnected as would be expected if they were really different species. The Bayesian migration rate estimates were consistently higher than the maximum likelihood migration rate estimates (Table 6). This also was observed in another study with different populations of C. capucinus (Ruiz-García et al., 2016b). However, although the magnitude of these estimates are different, the trends are the same. In all the cases, these migration rates were very elevated. For instance, the average values of the Bayesian and maximum likelihood migration rates were 54.68 and 15.27, respectively, whilst the same estimates for different populations of an alleged same species as C. capucinus were considerably lower, 31.14 and 7.06, respectively.

Genetic Assignment and Genetic Structure Different genetic assignment analyses were carried out by means of the GENECLASS 2.0 Software. The assignment analysis with Nei’s (1972) genetic distance was not an adequate procedure for this task because all the individuals analyzed were incorrectly classified. With the criteria of Paetkau et al., (1995), 32.5% of the individuals was misclassified. Out of 92 fatuellus individuals, 13 appeared classified as pallidus and eight as macrocephalus (22.83% of misclassified individuals). Out of 24 macrocephalus individuals, four were classified as fatuellus and three as pallidus (29.17% of misclassified individuals). Out of 47 pallidus individuals, 16 were classified as fatuellus and eight as macrocephalus (51% of misclassified individuals). The Rannala and Mountain (1997)’s criteria offered 32.5% of misclassified individuals similar to the previous procedure. In the case of fatuellus, 12 individuals appeared classified as pallidus and 10 as macrocephalus (23.91% of misclassified individuals). For macrocephallus, 5 individuals appeared classified as pallidus

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and three as fatuellus (33.33% of misclassified individuals), meanwhile for pallidus, 16 individuals appeared classified as fatuellus and eight as macrocephalus (51% of misclassified individuals). Two of the same criteria were applied to detect the first generation migrant individuals. For the Nei’s (1972) genetic distance procedure, 63 individuals with a probability below 0.05 were detected (38.65% of migrant individuals of the first generation), whilst for the criteria of Rannala and Mountain (1997), 53 individuals were first generation migrants (32.51%). Clearly, the results show that these three robust capuchin taxa are not independent units and cannot be considered different species as is also supported by the Migrate 3.6 analysis. Table 4. Exact G tests to determine genetic heterogeneity among the population pairs of three robust capuchin taxa (fatuellus, macrocephalus and pallidus) analyzed at eight DNA microsatellites taken together (A). Exact G tests to determine genetic heterogeneity among the population pairs of three robust capuchin taxa (fatuellus, macrocephalus and pallidus) individually analyzed at eight DNA microsatellites (B). * P < 0.05; ** P < 0.01; *** significant with Bonferroni correction (P < 0.0021); df = degree of freedom; S. E. = Standard Error

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Table 5. Genetic diversity analysis for three robust capuchin taxa (fatuellus, macrocephalus and pallidus) by means of the Nei’s and the Wright’s F statistics applied to DNA microsatellites (Ho = observed gene diversity in the total population; HS = average gene diversity within the subpopulations; HT = expected gene diversity in the total population; DST = absolute gene differentiation among subpopulations; GST = relative genetic differentiation among subpopulations with regard to the total gene diversity; GST’= the same as the previous statistics but corrected by sample size; FIS - FST - FIT = Wright’s F-statistics; RST = Slatkin [1995]’s genetic heterogeneity statistic measured by allele variances). * P < 0.05; ** P < 0.01 Locus AP68 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804 PEPC8 Overall Jackknife

Ho 0.603 0.551 0.304 0.811 0.557 0.341 0.133 0.579 0.485

Hs 0.645 0.799 0.574 0.948 0.745 0.888 0.162 0.830 0.699

Ht 0.662 0.839 0.584 0.951 0.747 0.903 0.165 0.874 0.716

Dst 0.017 0.040 0.011 0.003 0.002 0.016 0.003 0.044 0.017

Gst 0.025 0.048 0.018 0.004 0.003 0.017 0.018 0.050 0.024

Gst' 0.037 0.070 0.027 0.005 0.005 0.026 0.027 0.073 0.035

Bootstrap 99% Confidence Interval

FIS 0.041 0.288** 0.475** 0.289** 0.259** 0.424** 0.134 0.305** 0.295** 0.295 ± 0.044 0.170 0.393

FST 0.052 0.059 0.076 0.046 0.000 0.005 0.084 0.042 0.039** 0.039 ± 0.010 0.015 0.061

FIT 0.091 0.330** 0.515** 0.321** 0.259** 0.427** 0.207** 0.334** 0.322** 0.323 ± 0.042 0.208 0.418

RST 0.000 0.044 0.008 0.113 0.000 0.249 0.030 0.010 0.053

Table 6. Bayesian migration rate estimates (m) among three robust capuchin taxa (fatuellus, macrocephalus and pallidus) pairs by means of the Migrate 3.6 software (A). Maximum likelihood migration rate estimates (m) among three robust capuchin taxa (fatuellus, macrocephalus and pallidus) pairs by means of the Migrate 3.6 software (B); 1 = fatuellus; 2 = macrocephalus; 3 = pallidus (A) Robust capuchin taxa 1 2 3

1 57.77 73.29

2 33.32 20.22

3 91.54 51.96 -

(B) Robust capuchin taxa 1 2 3

1 10.44 13.49

2 6.49 19.48

3 9.53 32.21 -

The STRUCTURE Software was run with four different conditions. In each case the result was the same, with the highest maximum likelihood for six different populations (Table 7 and Figure 2), which did not correspond with the three morphological robust capuchin taxa. When we consider the three suggested taxa as priors, it is clear that the multi-genotypes of the

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different individuals analyzed were totally intermixed (Figure 3). This is further evidence that these three “a priori” robust capuchin taxa are not different species.

Figure 2. Assignation analyses with the Structure Program of the overall robust capuchin sample analyzed (163 individuals studied). There was no prior population information to assist clustering (USEPOPINF = 0), no admixture and allele frequencies correlated among populations. We assumed different values of FST for each population. The best result was K = 6 (i.e., 6 different populations) (A). This considered informative geography, admixture and allele frequencies correlated among populations, assuming different values of FST for each population and different robust capuchin taxa priors (USEPOPINF = 1). The best result was identified for K = 6 with extensive migration between them (B). Thus, six different gene pools were detected as the most probable condition with extensive gene flow among them. The three alleged different robust capuchin species were not confirmed (fatuellus, macrocephalus, pallidus).

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Figure 3. Assignation analyses with the Structure Program and assuming three gene pools correlated with the three possible robust capuchin species (fatuellus, macrocephalus, pallidus): Without prior population information to assist clustering (USEPOPINF = 0), admixture and allele frequencies correlated among populations, assuming different values of FST for each population (A); in a triangle figure (B). Red = fatuellus; green = macrocephalus; blue = pallidus. Clearly multi-genotypes of the three alleged robust capuchin species were intermixed, which is against the idea that they are three different and full species.

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Table 7. Number of possible different gene pools for three robust capuchin taxa (fatuellus, macrocephalus and pallidus) analyzed using the Structure Program with eight microsatellite loci. * = Most probable number of populations. K = number of populations. Without “a priori” origins and with admixture (A); With “a priori” origins and with admixture (B) (A) Populations K=1 K=2 K=3 K=4 K=5 K = 6* K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -2,382.6 -2,317.1 -2,307.6 -2,471.3 -2,243.0 -2,230.4 -2,329.5 -2,330.0 -2,535.9 -2,538.3 -2,601.4 -2,596.2

(B) Populations K=1 K=2 K=3 K=4 K=5 K = 6* K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -2,351.6 -2,318.6 -2,308.6 -2,496.8 -2,233.6 -2,208.1 -2,285.3 -2,362.7 -2,441.5 -2,532.5 -2,649.0 -2,639.9

Historical Demographic Changes The BOTTLENECK Software for each one of the three robust capuchin taxa analyzed showed the following scenario. For fatuellus, when the two-phase mutation model was employed, no test detected any bottleneck sign. When the stepwise mutation model was employed, three microsatellites (D5S111, D14S51 and PEPC8) showed significant values. Furthermore, the sign test, the standardized differences test, and the Wilcoxon test showed significant departures (Table 8). However, these significant deviations were negative and thus don’t support bottleneck events but rather population subdivision or population expansions. The mode shift test did not reveal any sign of a bottlenecked population for either mutation model. For macrocephalus, for the two-phase mutation model, D5S111 showed a significant value although no test revealed any significant deviation. By using the stepwise mutation model, D5S111 and D8S165 and the standardized differences test also showed negative

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significant values. Thus, with this mutation model, there was some evidence of population subdivision or population expansion as it was shown in the previous robust capuchin population. A similar situation was revealed for pallidus. The two-phase mutation model did not detect any significant deviation (with the exception of D17S804 that had a negative value), whilst the stepwise mutation model, as in the previous two cases, D8S165 and D17S804 as well as the standardized differences test offered significant negative values. The three robust capuchin populations we analyzed showed the same trend by means of this analysis. Also, there could be a certain degree of genetic fragmentation within the three populations, which did not correlate with the morphological designation of the three alleged taxa. This agrees with the STRUCTURE analysis which detected six different populations but with individuals highly mixed among them. The k test was applied to the overall sample (included fatuellus and pallidus but not macrocephalus because of its small sample size). It revealed that the overall sample and fatuellus individuals experienced significant population expansions (for both p = 0.029), whilst pallidus did not reach a significant probability for a population expansion (p = 0.126). The g test did not show any evidence of population expansions (overall sample: g = 0.775, p > 0.05; fatuellus: g = 0.755, p > 0.05; pallidus: g = 1.276, p > 0.05). The test of Kimmel et al., (1998) showed a population expansion after little propagules founded the respective populations of the three robust capuchins we studied (Table 9). In the case of fatuellus (β = 1.44, p < 0.05), this significant population expansion was of a minor degree compared to macrocephalus (β = 4.872, p < 0.01) and to pallidus (β = 3.551, p < 0.01). The test of Zhivotovsky et al., (2000) detected a significant population expansion for fatuellus (Sk = 0.337, p < 0.05), but it did not detect any significant population change for macrocephalus (Sk = 0.068, p > 0.05) and pallidus (Sk = -0.317, p > 0.05). Therefore, some of the different tests used to estimate population changes in these robust capuchin populations positively detected population expansions. These findings agree quite well with the hypotheses of Lynch-Alfaro et al., (2012) and Ruiz-García et al., (2012, 2016a) of relatively recent population expansions during the Pleistocene.

Spatial Genetic Structure We analyzed for possible spatial genetic patterns in the robust capuchins using SGS and SPAGeDi 1.4 Software. With the SGS Software, both distograms (with Gregorious genetic distance and with common alleles) with three DCs did not show any significant spatial trend for all the individuals (total sample) of the three alleged robust capuchin taxa (Figure 4). With 10 DCs, both distograms showed the same spatial trends. For brevity, we only comment on the distogram with the Gregorious genetic distance. At 646-862 km (4 DC), there was a significant positive genetic similarity (p = 0.000) among the individuals separated by these distances, which could indicate important long-distance migration during the colonization of the robust capuchins into the Western area of South America. Later, the six (1,077-1,293 km), seven (1,293-1,509 km) and nine (1,724-1,940 km) DCs showed significant negative genetic relationships among the individuals separated by these distances. However, the 10 DCs (last; 1,940-2,155 km) showed positive similarity among the individuals separated far away. Thus, although the 10 DC distogram yielded significant spatial structure, this was not what was

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expected for three different species, where significant genetic patches should be obtained separating the alleged three species. Table 8. Application of the Bottleneck Software to detect possible recent bottleneck events in the fatuellus capuchin population studied (A); in the macrocephalus capuchin population studied (B) and in the pallidus capuchin population studied (C). Ko = Number of observed alleles; He = expected heterozygosity; TPM = Two Phase Mutation Model; SMM = Step Mutation Model; SD = standard deviation; Prob = Probability. * Significant cases (P < 0.05). In this analysis, all the significant cases were contrary to a recent bottleneck event (A) Markers

Observed ko

AP68 D5S111 D5S117 D6S260 D8S165 DS14S51 D17S804 PEPC8 Sign Test

7 12 4 15 9 17 1 13 TPM: Prob = 0.1008 SMM: Prob = 0.0228* Standardized TPM: T2 = - 0.806, differences Prob = 0.2102 test SMM: T2 = - 4.684, Prob = 0.0000*

Observed Under the He TPM: He ±SD

Under the Under the TPM: Prob SMM: He ± SD

0.742 0.805 0.424 0.906 0.745 0.857 0.000 0.818 Wilcoxon test

0.4189 0.2581 0.2801 0.1333 0.3306 0.0714 0.1719

Modeshift

0.708 ± 0.086 0.825 ± 0.048 0.493 ± 0.152 0.877 ± 0.030 0.761 ± 0.071 0.896 ± 0.025 0.850 ± 0.040 Prob (one tail for H excess) = 0.8516 (TPM), 0.9961 (SMM) Normal Lshaped distribution

0.772 ± 0.053 0.868 ± 0.025 0.579 ± 0.107 0.902 ± 0.021 0.819 ± 0.039 0.916 ± 0.019 0.884 ±0.022

Under the SMM: Prob 0.2300 0.0241* 0.0940 0.5251 0.0523 0.0045* . 0.0130*

(B) Markers

Observed ko

AP68 D5S111 D5S117 D6S260 D8S165 DS14S51 D17S804 PEPC8 Sign Test

4 10 3 6 8 2 3 7 TPM: Prob = 0.5877 SMM: Prob = 0.6507

Standardized TPM: T2 = - 0.965, differences Prob = 0.1672 test SMM: T2 = - 2.810, Prob = 0.0025*

Observed Under the Under the Under the Under He TPM: He ± SD TPM: SMM: He ± SD the Prob SMM: Prob 0.660 0.4374 0.571 ± 0.125 0.2721 0.623 ± 0.100 0.711 0.0006* 0.841 ± 0.042 0.0150* 0.867 ± 0.027 0.569 0.5687 0.509 ± 0.132 0.4702 0.541 ± 0.120 0.929 0.7235 0.918 ± 0.016 0.6917 0.919 ± 0.016 0.774 0.0158* 0.851 ± 0.040 0.0508 0.866 ± 0.031 0.536 0.3705 0.397 ± 0.128 0.3267 0.409 ± 0.127 0.234 0.0580 0.457 ± 0.149 0.1333 0.505 ± 0.127 0.909 0.3836 0.882 ± 0.031 0.2966 0.889 ± 0.026 Wilcoxon Prob (one tail test for H excess) = 0.6796 (TPM), 0.6796 (SMM) ModeNormal Lshift shaped distribution

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Markers

Observed ko

Observed Under the TPM: He He ± SD

AP68 D5S111 D5S117 D6S260 D8S165 DS14S51 D17S804 PEPC8 Sign Test

4 12 5 11 9 8 5 6 TPM: Prob = 0.6197 SMM: Prob = 0.1857

0.532 0.869 0.708 0.907 0.703 0.908 0.254 0.732 Wilcoxon test

Standardized TPM: T2 = - 1.011, differences Prob = 0.1559 test SMM: T2 = - 3.592, Prob = 0.0002*

Modeshift

0.525 ± 0.140 0.860 ± 0.036 0.610 ± 0.119 0.886 ± 0.037 0.795 ± 0.057 0.875 ± 0.036 0.640 ± 0.107 0.752 ± 0.071 Prob (one tail for H excess) = 0.5273 (TPM), 0.9629 (SMM) Normal L-shaped distribution

Under the TPM: Prob 0.4339 0.5026 0.2061 0.2776 0.0719 0.1527 0.0041* 0.3138

Under the SMM: He ± SD 0.597 ± 0.105 0.885 ± 0.023 0.678 ± 0.082 0.900 ± 0.021 0.835 ± 0.035 0.886 ± 0.025 0.695 ± 0.078 0.782 ± 0.054

Under the SMM: Prob 0.2133 0.1943 0.4330 0.4672 0.0061* 0.2226 0.0005* 0.1507

Table 9. Statistics calculated for the imbalance index (Kimmel et al., 1998) (A) and for the test of Zhivotovsky et al. (2000) (Sk) (B) to detect demographic changes applied to eight microsatellites in the overall robust capuchin population studied in the fatuellus, macrocephalus and pallidus taxa. * Significant population expansion (A) Statistics V = v Po Po  ln  Probability

fatuellus 10.5916 0.2523 7.3543 1.4402 0.3648 0.05*

macrocephalus 22.9776 0.3096 4.7158 4.8725 1.5836 0.001*

pallidus 12.0583 0.3070 4.8039 3.5509 1.2672 0.001**

(B) Statistics V K SK Probability

fatuellus 7.5772 214.1108 0.3373 0.05

pallidus 9.6275 640.5108 -0.3166 >0.05

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Table 10. Application of SpaGeDi Software to detect possible spatial structure in the overall robust capuchin population studied for eight DNA microsatellite markers with six distance classes (DC) defined: with the Moran‘s I index, with the Queller and Goodnight (1989)’ coefficient, with the Rousset (2000) pairwise distance and with the correlation of allele size (Streiff et al., 1998). * P < 0.05 Statistics

1 DC: 0- 2 DC: 194194 km 412 km 0.0437* 0.017*

Moran ‘s I index Queller and 0.047* Goodnight (1989) coefficient Rousset 0.3098 (2000) pairwise distance Correlation 0.0289 of allele size (Streiff et al., 1998)

3 DC: 412637 km 0.0215*

4 DC: 6371,327 km -0.005

5DC:1,3271,622 km -0.0049*

6DC:1,6222,589 km -0.0508*

0.0083

0.0121

0.0066

-0.0967*

-0.0820*

0.3454

0.3620

0.3426

0.3945

0.4764*

0.0051

-0.0207

-0.0153

-0.0285

0.0078

Distogram using mean Genetic Distance (Gregorius 1978) 0.75 observed

0.70 95% CI

D 0.65

0.60 718

95% CI

Reference/ Mean 1437

Spatial distance Figure 4. (Continued).

2155

196

Manuel Ruiz-García, María Ignacia Castillo, Kelly Luengas-Villamil et al. Number of alleles/haplotypes in common 0.80 observed

0.75

0.70 95% CI

D

0.65

0.60

95% CI

0.55

0.50 718

Reference/ Mean 1437

2155

Spatial distance Distogram using mean Genetic Distance (Gregorius 1978) 0.80 observed

0.75

0.70 95% CI

D

0.65

0.60

95% CI

0.55

0.50 216 431 647 862 1078 1293 1509 1724 1940 2155

Spatial distance

Reference/ Mean

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Number of alleles/haplotypes in common 1.0 observed

0.9

0.8 95% CI

D

0.7

0.6

95% CI

0.5

0.4 216

Reference/ Mean 431

647

862 1078 1293 1509 1724 1940 2155

Spatial distance Figure 4. Application of SGS Software to detect possible spatial structure for the overall sample of robust capuchins studied (163 individuals) for eight DNA microsatellites: with the Gregorious (1978) genetic distance and three distance classes (A); with the common shared alleles and three distance classes (B); with the Gregorious (1978) genetic distance and 10 distance classes (C); with the common shared alleles and 10 distance classes (D). The absence of clear spatial structure is not supportive of three full and differentiated species of robust capuchins.

The SpaGeDi Software, defining six DCs with the Moran’s I index and for the overall robust capuchin sample, showed a significant average correlogram typical of a monotonic cline (1 DC: 0.0437; 2 DC: 0.017; 3 DC: 0.0215; 4 DC: -0.005; 5 DC: -0.0494; 6 DC: 0.0508; Table 10) with five out of six DCs statistically significant. By markers, AP68 (three significant DCs) and D5S117 (four significant DCs) showed a relative clear monotonic cline; D5S111 (one significant DC), D14S51 (two significant DCs), D17S804 (one significant DC) and PEPC8 (two significant DCs) showed some significant but erratic spatial trends, while D6S260 and D8S165 did not show any spatial trend. Globally, there was a significant spatial structure for this data set (27.08% = 13/48, significantly higher than the type I error of 5%; p < 0.05). The results with the coefficient of Queller and Goodnight (1989) also yielded a significant average correlogram (three significant DCs) related to a monotonic cline and a global spatial structure (33.33% = 16/48, significantly higher than a type I error of 5%; p < 0.05). The same was found for the pairwise distance suggested by Rousset (2000) (one significant DC) with an average significant correlogram related with a monotonic cline and an overall significant spatial structure (29.17% = 14/48, significantly higher that the error type I of 5%; p < 0.05). The correlation coefficient of allele size (Streiff et al., 1998) did not show any significant spatial structure (14.58% = 7/48, p > 0.05). The same analyses were carried out for pallidus with four DCs (Table 11). The average correlogram with the Moran’s I index showed a significant pattern of a monotonic cline as it

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was found for the overall robust capuchin sample with two significant DCs (1 DC: 0.0533; 2 DCs: -0.0084; 3 DCs: -0.0105; 4 DCs: -0.0606). By markers, D5S117 (two significant DCs), D17S804 (three significant DCs) and D14S51 (no significant DC) showed a clear trend in agreement with a monotonic cline. The other microsatellites did not reveal any significant spatial trend. Indeed, the overall number of significant DCs was not significant (18.75% = 6/32; p >0.05). Also, the coefficient of Queller and Goodnight (1989) and the pairwise distance of Rousset (2000)’s showed average correlograms with a monotonic clinal pattern for pallidus in Bolivia but with no significant number of significant DCs (18.75% = 6/28; p > 0.05 and 15.62% = 5/28; p > 0.05, respectively). However, the correlation coefficient for allele size (Streiff et al., 1998) did not show any significant spatial trend regarding DC with no significant number of significant DC (6.25% = 2/32; p > 0.05). Table 11. Application of SpaGeDi Software to detect possible spatial structure in the pallidus population for eight DNA microsatellite markers with four defined distance classes (DC): with the Moran‘s I index, with the coefficient of Queller and Goodnight (1989), with the Rousset (2000) pairwise distance and with the correlation of allele size (Streiff et al., 1998). * P < 0.05 Statistics Moran ‘s I index Queller and Goodnight (1989) coefficient Rousset (2000) pairwise distance Correlation of allele size (Streiff et al., 1998)

1 DC: 0230 km 0.0533* 0.0635

2 DC: 230379 km -0.0084 -0.0093

3 DC: 379-473 km -0.0105 -0.0032

4 DC: 4731,140 km -0.0606* 0.076

0.3755

0.4547

0.4752

0.5861

0.0105

-0.0287

0.0183

0.0003

Table 12. Application of SpaGeDi Software to detect possible spatial structure in the fatuellus population studied for eight DNA microsatellite markers with four distance classes defined (DC): with the Moran ‘s I index, with the Queller and Goodnight (1989)’ coefficient, with the Rousset (2000) pairwise distance and the correlation of allele size (Streiff et al., 1998). * P < 0.05 Statistics Moran ‘s I index Queller and Goodnight (1989) coefficient Rousset (2000) pairwise distance Correlation of allele size (Streiff et al., 1998)

1 DC: 029 km 0.0322* 0.0258

2 DC: 29332 km -0.0206 -0.0252

3 DC: 332469 km -0.0266 -0.0758*

4 DC: 469970 km -0.0005 0.0259

0.3129

0.2791

0.3057

0.2704

-0.0132

-0.0048

-0.0030

-0.0245

For fatuellus, the average correlogram with the Moran‘s I index showed that the first DC was significantly positive (individuals significantly related), but the remaining DCs did not

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show any spatial genetic structure (Table 12). By marker, AP68 (one significant DC) and PEPC8 (one significant DC) showed correlograms related to monotonic clines. D5S111 and D14S51 showed some significant DCs but no clear spatial structure. The remaining markers did not show any spatial trend. There was no significant spatial genetic structure (17.85% = 5/28; p > 0.05). The coefficient of Queller and Goodnight (1989), Rousset’s (2000) pairwise distance, and the correlation coefficient of allele size (Streiff et al., 1998) did not show a clear pattern of spatial structure. However, the first two statistics showed a significant number of significant DCs (39.29% = 11/28, significantly higher than a type I error of 5%; p < 0.05 and 21.43% = 6/28, significantly higher than a type I error of 5%; p < 0.05, respectively). For the third statistic the number of significant DCs was not significant (3.57% = 1/28; p > 0.05). Thus, only some individual markers showed significant spatial genetic patterns, which agrees quite well with smooth monotonic clines for the overall sample as well as for pallidus and fatuellus. And, only for the overall and the pallidus samples, smooth monotonic clines were discovered with the average correlograms obtained with the eight microsatellites we used. These spatial patterns are more related with subspecies or populations which moderately differed in recent times and not with full separated species. These could have generated strong significantly different genetic patches or a stair cline due to the inexistence of gene flow, or a very narrow hybrid zone influenced by natural selection among these populations (Hewitt, 1993).

DISCUSSION All the population genetics results herein obtained disagree with the fact that pallidus, macrocephalus and fatuellus are full separated species such as was claimed by Elliot (1913) and Silva Jr (2001) or partially by Groves (2001, 2005) and Rylands et al., (2000, 2005) and followed by Lynch-Alfaro et al., (2012) and Nascimento et al., (2015). The population of pallidus had the highest level of microsatellite gene diversity (H = 0.75), whereas fatuellus (H = 0.61) had the lowest gene diversity. For the mitochondrial COII gene, Ruiz-García et al., (2012) also showed that the pallidus-macrocephalus clade showed considerably higher gene diversity (five haplotypes, H = 0.60 ± 0.13 and nucleotide diversity = 0.17%) than the fatuellus clade (two haplotypes, H = 0.14 ± 0.11 and nucleotide diversity = 0.02%). Thus, both kinds of molecular markers showed the same trend. Dobzhansky (1971) showed that ancestral populations contained more gene diversity than the more recent ones (see, for instance, Tishkoff et al., 1996, 2009, for humans). This result agrees quite well with the hypothesis that Atlantic forest robust capuchins generated the robust capuchins from the Chaco and Cerrado and later this population generated the robust capuchin population in Bolivia. In turn this generated the more northern robust capuchin populations in the current Western Amazon of Peru, Ecuador and Brazil but south of the Amazon River. Directly, or more probably, indirectly, these populations generated the most northern robust capuchin populations at the northern side of the Amazon River. The most recent of these is probably fatuellus. Therefore, DNA microsatellites are correlated with the “Reinvasion of the Amazon,” a hypothesis of Lynch-Alfaro et al., (2012a), and with the more complete hypothesis called “North Amazon origin, Eastern expansion and subsequent reinvasion of the Amazon,” described by Ruiz-García et al., (2016a).

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Within the three assumed morphological robust capuchin taxa, there are other subtle molecular subdivisions revealed by the fact that no H-W E was found (Wahlund effect; Wahlund, 1928). This agrees quite well with the results produced by the STRUCTURE Software, which detected six different populations. However, these subdivisions are not related with morphological coat differences. Additionally, the H-W E deviation was stronger in fatuellus than in pallidus and macrocephalus, probably, because fatuellus is a more recent population than the other two. There was significant heterogeneity affecting some of the DNA microsatellites. The most remarkably significant genetic differentiation was between pallidus and fatuellus, the most extreme, geographically speaking, of the populations studied. Nevertheless, these significant genetic heterogeneities are relatively minor. Indeed, if we consider the non-existence of random mating within populations (fact demonstrated with the absence of H-W E), no marker showed significant differences. Related to this, the historical gene flow estimates showed that three populations were not disconnected. Furthermore, in turn, the Bayesian and maximum likelihood estimates among them were considerably high and significantly higher than the same estimates for different populations of C. capucinus in Colombia, which are considered by all authors as a unique species (Ruiz-García et al., 2016b). This disagrees with the possibility that these three taxa are fully differentiated species. In an identical way, the assignment analyses showed that around 33% of the individuals were misclassified. In each one of the populations considered there were probably multigenotype individuals from the other two populations. This likely happened in all three populations. Indeed, around 33-39% of the individuals could represent first generation migrants, which indirectly supports reproductive cohesiveness among these three populations a fact that again disagrees with three fully differentiated species. No analyses detected any bottleneck event in the three populations and all of them showed significant evidence of population expansions as is predicted by the hypotheses of Lynch-Alfaro et al., (2012) and Ruiz-García et al., (2012, 2016a). The diverse population genetics tests detected population expansions for the overall sample (with exception of the g test). By population, all tests, with the exception of that of Kimmel et al., (1998), showed fatuellus as the population with more evidence of population expansion (even for the nonsignificant g test). This is probably because this population experienced the most recent population expansion. The test of Kimmel et al., (1998) was more significant for pallidus and macrocephalus than for fatuellus. This could be related with the fact that the colonization from the Chaco (a dry biome very different to that of the Amazon rain forests) by other robust capuchin taxa, such as C. a. cay (Casado et al., 2010), should be in a more propagule mode. Contrarily, consider the more recent and massive crossing of the Negro River by the ancestors of fatuellus (more numerous propagules or propagules containing more individuals). Also related with the other analyses, the spatial genetic analyses did not show typical correlograms for full differentiated species in the geographical area analyzed. The smooth monotonic clines obtained for several markers and especially for the overall sample and the pallidus sample are typical of gradual differentiation within a species conformed by subspecies or populations which gradually differ because of isolation-by-distance. Clearly, these results did not show the abrupt genetic and reproductive discontinuities that we would expect if there were well fully differentiated species. There was a slightly higher genetic structure in pallidus than in fatuellus, which supports that the first was an older formed taxon and its colonization was accomplished more through propagules. Differently, the second one

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was more recent, rapid, and massive and came from the other side of the Negro River. These migration and colonization events occurred 0.2-0.5 million years ago (Pleistocene) as we discuss elsewhere (Ruiz-García et al., 2016a).

Figure 5. Maximum likelihood tree with 87 robust capuchins studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are bootstrap percentages.

If we take all of these population genetics results into account together with the molecular phylogenetic trees obtained by Ruiz-Garcia et al., (2012; 2016a) (Figure 5) by means of mitochondrial markers, the three robust capuchin taxa studied here should be classified as follows. Both pallidus and macrocephalus could be unified in one unique taxa, C. apella macrocephalus (macrocephalus: Spix, 1823, Cactua lake, Amazon River, Brazil; pallidus: Gray, 1866, Beni River, Bolivia). The mitochondrial results of Ruiz-García et al., (2012, 2016a) clearly showed that individuals of both macrocephalus and pallidus conformed a unique clade even sharing the same mitochondrial haplotypes. The existence of

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morphological color and coat differences is evidence in support of the strong and rapid actuation of natural selection on some external phenotype traits when the robust capuchins colonized new habitats and biomes. This situation could be compared with the fact that in humans, Asians and indigenous American are of the same molecular branch but they show some external physical characteristics which allows them to be clearly distinguishable from each other (Rosenberg et al., 2002, 2005). If pallidus was classified by Groves (2001, 2005) within C. libidinosus and, molecularly speaking, pallidus and macrocephalus are undifferentiated, then, C. apella and C. libidinosus should be a unique species, C. apella. The third population we studied, together with the mitochondrial results of Ruiz-García et al., (2012, 2016a), ratified C. apella fatuellus as a real entity. This population was taken into account by Elliot (1913), Cabrera and Yepes (1940), Hill (1960) and Groves (2001, 2005), but it was not considered by Cabrera (1957), Silva Jr (2001), Lynch-Alfaro et al., (2012) and Nascimento et al., (2015). Our population genetics study showed that this population, although only slightly, was the most differentiated of the three populations studied. This, together with the works of Ruiz-García et al., (2012, 2016a), allow us to hypothesize that C. a. fatuellus does not come directly from the most Western C. a. macrocephalus populations. We can also hypothesize that it is from some propagule of some robust capuchin form from the Brazilian Cerrado, which crossed the middle or eastern area of the Amazon River and generated C. a. apella. This taxon in turn—having a geographic barrier to the Negro River, generated the most recent C. apella lineage, C. a. fatuellus. It’s interesting to note that the squirrel monkey taxon (Saimiri cassiquiarensis albigena) was also recently generated and detected by molecular procedures (Ruiz-García et al., 2015). It inhabits the Colombian Eastern Llanos with C. a. fatuellus. Thus, both genera of primates colonized the Colombian Eastern Llanos coming from the Amazon in recent times. The comparative use of nuclear and mitochondrial DNA markers as well as the application of different population genetics tests allow us to analyze if different populations have the same or different evolutionary trajectories. Populations of the same species have the same trajectories whereas as those of different species have different trajectories. Furthermore, if the populations are indirectly connected or disconnected from a reproductive point of view, the above mentioned tools are essential to the study of systematics regarding Neotropical primates (and many other organisms). Such applications will help to validate or negate the many species proposed by use of the Phylogenetic species concept (PSC; Cracraft, 1983). This was named by Isaac et al., (2004) as “taxonomic inflation.” Additionally, Zachos et al., (2013) showed that some of the proposed new mammal species in recent years are completely unjustifiable. Thus, more prudence is required by primatologist before defining “new” species of primates.

ACKNOWLEDGMENTS Thanks to Dr. Diana Alvarez, Pablo Escobar-Armel, Nicolás Lichilín, Luisa CastellanosMora, Fernando Nassar, Hugo Gálvez, and Alan Velarde for their respective help in obtaining wild robust capuchin monkey samples during the last 20 years. Thanks to Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Muñoz), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de Extracción y Procesamiento Pesquero),

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Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales from Peru, and to the Colección Boliviana de Fauna (Dr. Julieta Vargas) and to CITES Bolivia for their role in facilitating the obtainment of collection permits in Colombia, Peru and Bolivia. We also thank the many people of diverse Indian tribes in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana and Alamas), Bolivia (Sirionó, Canichana, Cayubaba and Chacobo) and Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna) for their support in obtaining samples of robust capuchins.

REFERENCES Antao, T., Lopes, A., Lopes, R. J., Beja-Pereira, A., Luikart, G. (2008). LOSITAN: A workbench to detect molecular adaptation based on a Fst-outlier method. BMC Bioinformatics 9:323 doi:10.1186/1471-2105-9-323. Archie, J. W. (1985). Statistical analysis of heterozygosity data: independent sample comparisons. Evolution 39: 623–637. Barton, N. H., Slatkin, M. (1986). A quasi-equilibrium theory of the distribution of rare alleles in a subdivided population. Heredity 56: 409-416. Beerli, P. (2006). Comparison of Bayesian and maximum likelihood inference of population genetic parameters. Bioinformatics 22:341-345. Beerli, P. (2009). How to use migrate or why are markov chain Monte Carlo programs difficult to use? In G. Bertorelle, M. W. Bruford, H. C. Haue, A. Rizzoli, and C. Vernesi, editors, Population Genetics for Animal Conservation, volume 17 of Conservation Biology. (pp. 42-79). Cambridge University Press, Cambridge UK. Beumont, M., Nichols R. (1996). Evaluating loci for use in the genetics analysis of population structure. Proceedings of the Royal Society of London, Series B 263: 1619-1626. Bruford, M. W., Wayne, R. K. (1993). Microsatellites and their application to population genetics studies. Current Opinion in Genetics and Development 3: 939-943. Cabrera, A. (1917). Notas sobre el género Cebus. Rev. Real Acad. Cs. Exac Fis Nat Madrid 16: 1-24. Cabrera, A. (1957). Catálogo de los mamíferos de América del Sur. I (Metatheria, Unguiculata, Carnívora). Revista del Museo Argentino de Ciencias Naturales “Bernardo Rivadavia,” Zoología 4:1-307. Cabrera, A., Yepes, J. (1940). Historia Natural Ediar. Mamíferos Sud-Americanos. Compañía Argentina de Editores, Buenos Aires, Argentina. Casado, F., Bonvicino, C. R., Nagle, C., Comas, B., Manzur, T. D., Lahoz, M. M., Seuánez, H. N. (2010). Mitochondrial divergence between 2 populations of the Hooded Capuchin, Cebus (Sapajus) cay (Platyrrhini, Primates). Journal of Heredity 101:261–269. Cornuet, J. M., Luikart, G. (1996). Description of power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144: 2001-2014. Cracraft, J. (1983). Species concepts and speciation analysis. In Johnston RJ (Ed.). Current ornithology. Vol I. p. 159-187. Plenum Press, New York.

204

Manuel Ruiz-García, María Ignacia Castillo, Kelly Luengas-Villamil et al.

Degen, B., Scholz, F. (1998). Spatial genetic differentiation among populations of European beech (Fagus sylvatica L.) in Western Germany as identified by geostatistical analysis. Forest Genetics 5:191-199. Degen, B., Petit, R., Kremer, A. (2001). SGS - Spatial Genetic Software: A computer program for analysis of spatial genetic and phenotypic structures of individuals and populations. Journal of Heredity 92: 447-448. Dib, C., Faure, S., Fizames, C. (1996). A comprehensive genetic map of the human genome base on 5.264 microsatellites. Nature 380: 152-154. Dobzhansky, T. (1971). Evolutionary oscillations in Drosophila pseudoobscura.. In Ford, E. B. (Ed.). Ecological Genetics and Evolution (pp 109–133). Oxford, Blackwell Scientific. Efron, B., Tibshirani, R. J. (1993). An introduction to the bootstrap. New York, NY: Chapman and Hall. Ellesworth, J. A., Hoelzer, G. A. (1998). Characterization of microsatellite loci in a new world primate, the mantled howler monkey (Alouatta palliata). Molecular Ecology 7: 657–658. Elliot, D. G. (1913). A Review of Primates. Monograph Series, American Museum of Natural History, New York. Falush, D., Stephens, M., Pritchard, J. K. (2007). Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 7:574–578. Feldman, M. W., Kumm, J., Pritchard, J. K. (1999). Mutation and migration in models of microsatellite evolution. In Goldstein, D. G., and Schlötterer, C. (Eds.). Microsatellites: evolution and applications. Oxford, United Kingdom: Oxford University Press. Fragaszy, D. M., Visalberghi, E., Fedigan, L., Rylands, A. B. (2004). Taxonomy, distribution and conservation: Where and what are they, and how did get there? In: Fragaszy, D. M., Fedigan, L., Visalberghi, E. (Eds.). The Complete Capuchin: The Biology of the Genus Cebus. (pp. 13-35). Cambridge University Press, Cambridge. Goudet J., Raymond M., Demeeus T., Rousset F. (1996). Testing differentiation in diploid populations. Genetics 144: 1933–1940. Gregorius, H. R. (1978). The concept of genetic diversity and its formal relationship to heterozygosity and genetic distance. Mathematical Bioscience 41: 253-271. Groves, C. P. (2001). Primate Taxonomy. Smithsonian Institution Press, Washington, DC. Groves, C. P. (2005). Order primates. In: Wilson, D. E., Reeder, D. M. (Eds.). Mammal species of the world: a taxonomic and geographic reference. 3rd Ed. Baltimore: Johns Hopkins Univ Press. Pp. 111–184. Hamrick, J. L., Murawski, D. A., Nason, J. D. (1993). The influence of seed dispersal mechanisms on the genetic structure of tropical tree populations. Vegetatio 107: 281-297. Hardy, O. J., Vekemans, X. (2002). SPAGeDi: a versatile computer program to analyse spatial genetic structure at the individual or population levels. Molecular Ecology Notes 2: 618-620. Hershkovitz, P. (1949). Mammals of northern Colombia. Preliminary report n° 4: Monkeys (Primates), with taxonomic revisions of some forms. Proceedings of the United States National Museum 98: 323-427.

Invalidation of Three Robust Capuchin Species …

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Hershkovitz, P. (1955). Notes on the American monkeys of the genus Cebus. Journal of Mammalogy 36: 449-452. Hewitt, G. W. (1993). After the ice: Parallelus meets Erythropus in the Pyrenees. In Harrison RG (Ed.): Hybrid zones and the evolutionary process. (pp. 140-164). Oxford University Press, New York and Oxford, US. Hill, W. C. O. (1960). Primates: Comparative Anatomy and Taxonomy. IV. Cebidae, Part A. Edinburgh University Press, Edinburgh. Isaac, J. B., Mallet, J., Mace, G. M. (2004). Taxonomic inflation: its influence on macroecology and conservation. Trends Ecology and Evolution 19: 464–469. Kimmel, M., Chakraborty, R., King, J. P., Bamshad, M., Watkins, W. S., Jorde, L. B. (1998). Signatures of population expansion in microsatellite repeat data. Genetics 148: 19211930. Luikart, G., Allendorf, F., Sherwin, B., Cornuet, J. M. (1998). Distortion of allele frequency distribution provides a test for recent population bottleneck. The Journal of Heredity 86: 319-322. Lynch Alfaro, J. W., Boubli, J. P., Olson, L. E., Di Fiore, A., Wilson, B., Gutiérrez-Espeleta, G. A., Chiou, K. L., Schulte, M., Neitzel, S., Ross, V., Schwochow, D., Nguyen, M. T. T., Farias, I., Janson, C. H., Alfaro, M. E. (2012). Explosive Pleistocene range expansion leads to widespread Amazonian sympatry between robust and gracile capuchin monkeys. Journal of Biogeography 39: 272–288. Manly, B. F. J. (1997). Randomization, Bootstrap and Monte Carlo Methods in Biology. Chapman and Hall, London. Moran, P.A.P. (1950). Notes on continuous stochastic phenomena. Biometrika 37: 17-23. Michalakis, Y., Excoffier L. (1996). A generic estimation of population subdivision using distances between alleles with special reference to microsatellite loci. Genetics 142: 1061–1064. Nascimento, F. F., Lazar, A., Seuánez, H. N., Bonvicino, C. R. (2015). Reanalysis of the biogeographical hypothesis of range expansion between robust and gracile capuchin monkeys. Journal of Biogeography. http://wileyonlinelibrary.com/journal/jbi doi:10.1111/jbi.12448. Nei, M. (1972). Genetic distance between populations. American Naturalist 106: 283-292. Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the USA 70: 3321–3323. Paetkau D., Calvert, W., Stirling, I., Strobeck, C. (1995) Microsatellite analysis of population structure in Canadian polar bears. Molecular Ecology 4: 347-354. Patkeau D., Slade, R., Barden, M., Estoup, A. (2004). Genetic assignment methods for the direct, real-time estimation of migration rate: a simulation based exploration of accuracy and power. Molecular Ecology 13: 55-65. Piry S., Alapetite, A., Cornuet, J. M., Paetkau, D., Baudouin, B and Estoup, A. (2004). GENECLASS2: a software for genetic assignment and first-generation migrant detection. Journal of Heredity 95: 536-539. Queller, D. C., Goodnight, K. F. (1989). Estimating relatedness using genetic markers. Evolution 43: 258-275.

206

Manuel Ruiz-García, María Ignacia Castillo, Kelly Luengas-Villamil et al.

Rannala B., Mountain, J. L. (1997) Detecting immigration by using multilocus genotypes. Proceedings of the National Academy of Sciences USA 94: 9197-9201. Raymond, M., Rousset, F. (1995). An exact test for population differentiation. Evolution 49:1280-1283. Reich, D. E., Goldstein, D. B. (1998). Genetic evidence for a Paleolitic human population expansion in Africa. Proceedings of the National Academy of Sciences 95: 8119-8123. Reich, D. E., Feldman, M. W., Goldstein, D. B. (1999). Statistical properties of two tests that use multilocus data sets to detect population expansions. Molecular Biology and Evolution 16: 453-466. Robertson, A., Hill, W. G. (1984). Deviations from Hardy-Weinberg proportions, sampling variances and use in estimation of inbreeding coefficients. Genetics 107: 703-718. Rosenberg, N. A., Pritchard, J. K., Weber, J. L., Cann, H. M., Kidd, K. K., Zhivotovsky, L. A., Feldman, M. W. (2002) Genetic structure of human populations. Science 298: 23812385. Rosenberg, N. A., Mahajan, S., Ramachandran, S., Zhao, C., Pritchard, J. K., and Feldman, M. W. (2005) Clines, clusters, and the effect of study design on the inference of human population structure. PLoS Genetics 1: 660-671. Rousset, F. (1997). Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance. Genetics 145: 1219–1228. Rousset, F. (2000). Genetic differentiation between individuals. Journal of Evolutionary Biology. 13: 58-62. Ruiz-García, M. (2005). The use of several microsatellite loci applied to 13 Neotropical primates revealed a strong recent bottleneck event in the woolly monkey (Lagothrix lagotricha) in Colombia. Primate Report 71: 27–55. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P., Shostell, J. M. (2006). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report 73: 3–47. Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., GutierrezEspeleta, G., Shostell, J. M. (2007). Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: Genetic structure and recent bottlenecks. Folia Primatologica 78: 73–87. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L. F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). International Journal of Primatology 32:1218–1241. Ruiz-García, M., Castillo, M. I., Lichilin, N., Pinedo-Castro, M. (2012). Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, C. xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene sequences. Folia Primatologica 83: 100-125. Ruiz-García, M., Luengas, K., Leguizamón, N., Thoisy, B., Gálvez, H. (2015). Molecular Phylogenetics and Phylogeography of all the Saimiri species (Cebidae, Primates) inferred from mt COI and COII gene sequences. Primates 56: 145-161. Ruiz-García, M., Castillo, M. I., Luengas, K. (2016a). It is misleading to use Sapajus (robust capuchins) as a genus?: A review of the evolution of the capuchins and suggestions on their systematics. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular

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Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I. (2016b). Genetic structure, spatial patterns and historical demographic evolution of white-throated capuchin (Cebus capucinus, Cebidae, Primates) populations of Colombia and Central America by means of DNA microsatellites. In RuizGarcía, M., and Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Rylands, A. B., Schneider, H., Mittermeier, R. A., Groves, C. P., Rodriguez-Luna, E. (2000). An assessment of the diversity of New World Primates. Neotropical Primates 8: 61-93. Rylands, A. B., Kierulff, M. C. M., Mittermeier, R. A. (2005). Notes on the taxonomy and distributions of the tufted capuchin monkeys (Cebus, Cebidae) of South America. Lundiana 6: 97-110. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. Silva, Jr J de S. (2001). Especiacao nos macacos-pregos e caiararas, género Cebus Erxleben, 1777 (Primates, Cebidae). Doctoral Thesis. Pp. 377. Universidade Federal do Rio de Janeiro, Rio de Janeiro. Slatkin, M (1985). Rare alleles as indicators of gene flow. Evolution 39: 53-65. Slatkin, M. (1995). A measure of population subdivision based on microsatellite allele frequencies. Genetics 139: 457–462. Streiff, R., Labbe, T., Bacilieri, R., Steinkellner, H., Glossl, J., Kremer A. (1998). Withinpopulation genetic structure in Quercus robur L. and Quercus petraea (Matt.) Liebl. assessed with isozymes and microsatellites. Molecular Ecology 7:317-328. Surles, S. E., Arnold, J., Schnabel, A., Hamrick, J. L., Bongarten, B. C. (1990) Genetic relatedness in open pollinated families of two leguminous tree species, Robinia pseudoacacia L. and Gleditsia triacanthos L.. Theoretical and Applied Genetics 80: 4956. Torres de Assumpcao, C. (1983). An ecological study of the primates of southeastern Brazil, with a reppraisal of Cebus apella races. Doctoral Thesis. University of Edinburgh, Edinburgh. Torres de Assumpcao, C. (1988). Resultados preliminares de reavaliacao das racas do macaco-prego Cebus apella (Primates: Cebidae). Rev. Nordestina Biol. 6: 15-28. Vendramin, G. G., Degen, B., Petit, R. J., Anzidei, M., Madaghiele, A., Ziegenhagen, B. (1999). High level of variation at Abies alba chloroplast micrsatellite loci in Europe. Molecular Ecology 8:1117-1126. Wahlund, S. (1928). Zusammensetzung von Population und Korrelationserscheinung vom Standpunkt der Vererbungslehre aus betrachtet. Hereditas 11:65–106. Walsh, P. S., Metzger, D. A., Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506513. Weir, B. S., Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370. Wright, S. (1951). The genetical structure of populations. Annals of Eugenics 15: 323–354. Zachos, F. E., Apollonio, M., Barmann, E. V., Festa-Bianchet, M., Gohlich, U., Habel, J. C., Haring E., Kruckenhauser, L., Lovari, S., McDevith, A. D., Pertoldi, C., Rossner, G, E.,

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Sánchez-Villagra, M. R., Scandura, M., Suchentrunk, F. (2013). Species inflation and taxonomic artefacts—A critical comment on recent trends in mammalian classification. Mammalian Biology 78: 1-6. Zhivotovsky, L. A., Feldman, M. W. (1995). Microsatellite variability and genetic distances. Proceedings of the National Academy of Sciences 92: 11549-11552. Zhivotovsky, L. A., Bennett, L., Bowcock, A. M., Feldman, M. W. (2000). Human population expansion and microsatellite variation. Molecular Biology and Evolution 17: 757-767.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 7

IT IS MISLEADING TO USE SAPAJUS (ROBUST CAPUCHINS) AS A GENUS? A REVIEW OF THE EVOLUTION OF THE CAPUCHINS AND SUGGESTIONS ON THEIR SYSTEMATICS Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética. Departamento de Biología. Facultad de Ciencias, Pontificia Universidad Javeriana. Bogotá DC., Colombia

ABSTRACT The systematics and the evolutionary history of the capuchin monkeys is highly controversial. Recently, Lynch-Alfaro et al., (2012a, b) proposed to split the traditional Cebus genus into two genera, Cebus (gracile capuchins) and Sapajus (robust capuchins) and they also proposed a hypothesis to explain the evolution of these Neotropical primates (“Reinvasion of the Amazon”). Additionally, Bobli et al., (2012) suggested splitting the gracile capuchins into at least 12 species, although traditionally they had been classified into four species. Nevertheless the work of Lynch-Alfaro et al., (2012a) was criticized because of the small number of genes used and limited sample size (Nascimento et al., 2015). Good resolution of a species tree requires the correct identification of species, data from several loci, a high number of individuals per species, and careful analysis of ancient DNA data from museum specimens. Herein, we analyzed 452 capuchin monkeys (both gracile and robust groups) for four mitochondrial genes and a subset of 27 individuals for 16 mitochondrial genes. There were four main findings: 1Genetic distance values between Cebus and Sapajus were within the range of different species within a genus but significantly less than the values among different genera of Neotropical primates. 2- Genetic diversity was considerably higher in the gracile capuchins than in the robust capuchins. 3- Neither genetic tree showed the monophylia 

Correspondence: [email protected], [email protected].

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil between Cebus and Sapajus. 4- The evolutionary history of the mitochondrial haplotypes within gracile capuchins and robust capuchins began around 4-5 and 3 million years ago (MYA) respectively. Our results along with a review of karyological, morphological, and ethological data of capuchin monkeys do not support a split of the capuchins into two different genera. Also, our hypothesis, “North Amazon gracile origin, Eastern expansion and subsequent reinvasion of the Amazon,” more completely explains the evolution of capuchins than does “Reinvasion of the Amazon” hypothesis of Lynch-Alfaro et al., (2012a). The Biological Species Concept (BSC) should be applied to the capuchins more than the Phylogenetic Species Concept (PSC), because we have enough data on many biological aspects of this group. The gracile capuchins should also be classified in a unique species, C. capucinus (including the traditional C. capucinus, C. albifrons and C. olivaceus) with different lineages intermixed in many geographical areas, whilst the robust capuchins should be classified into at least three species C. xantosthernos, C. nigritus and C. apella. It is difficult to apply traditional rules of systematic nomenclature to capuchins. Gracile capuchins underwent many migration events, had high gene flow and there is evidence of continuous mixture among different lineages. In contrast, robust capuchins had explosive Pleistocene radiation, colonized a wide array of extremely different biomes, and generated extreme diversity in morphology. The situation of C. apella should be similar to that of our own species. Explosive radiation occurred in humans within the last 0.1-0.2 MYA allowing us to conquer extremely different ecological conditions, even using tools. Thus, from a phylogenetic perspective, it’s probably not important to focus on coat morphological differences for capuchins.

Keywords: Cebus, Sapajus, mitochondrial genes, evolution of capuchin monkeys, speciation, Biological Species Concept, Phylogenetic Species Concept

INTRODUCTION The systematics and the reconstruction of the evolutionary history of the capuchin monkeys has been a central topic for many Neotropical primatologists. These monkeys have long captured our attention due to their creative and sophisticated behavior (including use of tools), their highly developed social trends and the wide array of different biomes that they colonized (Fragaszy et al., 2004). Traditionally, two groups of these monkeys have been defined, the gracile (or untufted) and the robust (tufted) capuchins. Hershkovitz (1949) recognized three species of gracile capuchins, which have been widely accepted. They are the white-fronted capuchin, Cebus albifrons (Humboldt, 1812), the white-faced capuchin, C. capucinus (Linnaeus, 1758), and the wedge-capped or weeper capuchin, C. olivaceus (Schomburgk, 1848). More recently, a fourth species, the ka’apor capuchin, C. kaapori, was described by Queiroz (1992). Hershkovitz (1949) also defined 13 subspecies of C. albifrons, five of C. capucinus and five of C. olivaceus. Here is a breakdown of the 13 subspecies of C. albifrons. 1- C. a. albifrons was identified by Humboldt (1812) in the Maipurés and Atures rapids along the border of the Orinoco River in Colombia and Venezuela. Defler and Hernández-Camacho (2002) determined that its Colombian distribution was in the lower Tuparro (left bank), Tuparrito, Tomo, Bita and Meta (right bank) rivers. 2- C. a. hypoleucus is within the Bolivar Department (Sinu River) in

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Northern Colombia. It was later assigned to C. capucinus. 3- C. a. malitiosus is distributed in the deciduous and humid forests of the northern and eastern slopes of the Santa Marta Sierra Nevada to elevations as high as 1300 m above sea level including Tayrona National Park in Colombia. 4- C. a. cesarae is distributed within the Magdalena Department south of Santa Marta Ciénaga Grande and in the lowlands of the Cesar Department from the vicinity of El Banco and Tamalameque northwards to the deciduous and gallery forests of the Rancheria River in the southern Guajira Department. Its geographical distribution encloses the Perijá Serrania, east of Valledupar, the Calancala and Ariguaní rivers and the east bank of the Magdalena River in Colombia. 5- C. a. pleei is distributed in swamps around Norosi, in the extreme north of Central Cordillera until Monpós, and along the west bank of the Magdalena River, in Colombia. 7- C. a. versicolor inhabits the Tolima Department and the middle Magdalena Valley (including the Monpós Island) in the Cesar Department. It is also distributed in the southeastern Sucre Department between the San Jorge and lower Cauca rivers, in the northern part of the Antioquia Department eastward from the Cauca River, including the Nechí River and in the eastern Boyacá Department as well as in the Caldas, Santander and the western Cundinamarca Departments in Colombia. 7- C. a. leucocephalus has a distribution range that extends from the eastern bank of the Magdalena River to the humid lowland Catatumbo region of the Norte de Santander Department in Colombia. 8- C. a. adustus was first found at the Cogollo River, five km northwest of Machiques, in the Zulia Department of Venezuela. It is distributed in the Maracaibo Lake region (Venezuela) and from the eastern base of the Sierra de Perija in Venezuela and Colombia. It’s possible that this taxon is distributed across the piedmont forests of the western Arauca Department, at the northern tip of the Boyacá Department and in the eastern tip of the Norte de Santander Department. 9- C. a. unicolor was first observed near the mouth of the Tefé River in the Brazilian Amazon. It also, has a distribution range that includes the east bank of the Ucayali River and a large fraction of the Peruvian Amazon River, including the Alto Purús River. It also includes the Northern Bolivian Amazon. 10- C. a. yuracus was first described in Montalvo along the north bank of the Bobonaza River, 45 km from its confluence with the Pastaza River in Eastern Ecuador. In Colombia, this taxon is distributed in the southwestern Putumayo Department, south of the Guamués River. In the Peruvian Amazon, this taxon also inhabits the northern areas, including the Marañon and Napo Rivers as well as south of the Marañón River including the Ucayali River, and both banks of the Huallaga River until the Pachitea River, near Pucallpa. 11- C. a. cuscinus inhabits the southeastern Peruvian Amazon from the Alto Purús River up to Northern Bolivia as well as the lower parts of the Junin and Cuzco Peruvian Departments. This taxon seems to be frequently within the Manú National Park (upper Madre de Dios River, Peru; Terborgh, 1983). 12- C. a. aequatorialis is from the Pacific Coast in Western Ecuador and also in the Tumbes region in Northern Peru. 13- C. a. trinitatis, the last one, is from the Trinidad Island. Hernández-Camacho and Cooper (1976) reinterpreted the arrangement of the Colombian forms proposed by Hershkovitz (1949). They included leucocephalus and pleei within C. a. versicolor because in 1958, the first author observed individuals representing the color-phases of the three previously defined subspecies (dark phase – C. a. leucocephalus; light phase – C. a. pleei and intermediate phase – C. a. versicolor) in a single troop (in the region of Barrancabermeja at the eastern bank of the middle Magdalena River). Later, Groves (2001, 2005) further reduced the number of subspecies of C. albifrons to six, recognizing just one northern Colombian form, C. a. versicolor (leucocephalus, malitiosus, adustus, cesarae, and

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pleei as synonyms), and three Amazonian forms, C. a. albifrons, C. a. cuscinus (yuracus as a junior synonym), and C. a. unicolor, along with C. a. trinitatis from Trinidad, and C. a. aequatorialis from the Pacific coast in Ecuador and Peru. Defler and Hernández-Camacho (2002) confirmed, from a phenotypical perspective, that C. a. albifrons and C. a. unicolor were synonymous. They also stated that the Arauca Department populations originally classified as C. a. adustus and later enclosed by Groves (2001) inside C. a. versicolor, could also be enclosed within C. a. albifrons. However, Defler (2003; 2010) recognized C. a. malitosus and C. a. cesarae as two well-differentiated taxa from C. a. versicolor, disagreeing in this aspect from Groves (2001). Table 1. Different morphological classifications for the robust capuchins obtained by diverse authors. The taxonomic review of Groves (2001, 2005) for the tufted capuchins is identical to that of Rylands et al., (2000). C = Cebus; S = Sapajus Elliot (1913) C. apella C. fatuellus C. f. fatuellus C. f. peruanus C. macrocephalus C. azarae C. azarae C. azarae pallidus C. libidinosus C. frontatus C. variegatus C. versuta C. cirrifer C. crassiceps C. caliginosus C. vellerosus Hill (1960) C. apella C. a. apella C. a. margaritae C. a. fatuellus C. a. tocantinus C. a. macrocephalus C. a. magnus C. a. juruanus C. a. maranonis C. a. peruanus C. a. pallidus C. a. cay C. a. libidinosus C. a. robustus C. a. frontatus C. a. nigritus C. a. xanthosternos

Cabrera and Yepes (1940) C. xanthosternos C. paraguayanus C. libidinosus C. macrocephalus C. frontatus C. nigritus C. vellerosus C. fatuellus C. apella

Cabrera (1957) C. apella C. a. apella C. a. libidinosus C. a. macrocephalus C. a. margaritae C. a. nigritus C. a. pallidus C. a. paraguayanus C. a. robustus C. a. vellerosus C. a. versutus C. a. xanthosternos

Groves (2001) C. apella C. a. apella C. a. fatuellus C. a. macrocephalus C. a. peruanus C. a. tocantinus C. a. margaritae C. libidinosus C. l. libidinosus C. l. pallidus C. l. paraguayanus C. l. juruanus C. nigritus C. n. nigritus C. n. robustus C. n. cucullatus C. xanthosternos

Silva Jr (2001) S. apella S. macrocephalus S. libidinosus S. cay S. nigritus S. robustus S. xanthosternos

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Hershkovitz (1949) proposed five subspecies of C. capucinus. 1- C. capucinus, was originally found in Northern Colombia, probably near to Cartagena de Indias. 2- C. c. curtus was found in Gorgona Island within the Pacific area of Colombia. 3- C. c. nigripectus was first found in Las Pavas, Cauca Valley, between Cali and Buenaventura in Colombia. 4- C. c. limitaneus is from Belize, Honduras, Nicaragua and Guatemala and 5- C. c. imitator is from Panama, including the islands of Coiba, and Costa Rica. Nevertheless, Hershkovitz (1949) suggested that there were several subspecies in Colombia, “pending a thorough study of ample material.” More recently, Rylands et al., (2000) considered four subspecies, C. c. capucinus in Colombia and Ecuador, C. c. curtus from the Gorgona Island, and C. c. limitaneus and C. c. imitator in Central America. However, Hernández-Camacho and Cooper, (1976) and Mittermeier and Combra-Filho, (1981), for example, did not recognize these subspecies because they found pelage characters too variable to permit recognition of any subspecies. Groves (2001) was of the same opinion and he considered this species monotypic. Similarly, neither Defler (2003, 2010) (in Colombia) nor Silva Jr (2001) recognized any C. capucinus subspecies. In the case of C. olivaceus (also named C. nigrivittatus), Hershkovitz (1949) suggested the following five subspecies: 1- C. o. nigrivittatus from the upper Branco River, in the Northern Brazilian Amazon; 2- C. o. olivaceus from the southern foot of Monte Roraima, in the Northern Brazilian Amazon; 3- C. o. castaneus described from Cayenne, French Guiana; 4- C. o. apiculatus from Cuara River, Venezuela; and 5- C. o. brunneus from the Northwestern coast of Venezuela. Neither Silva Jr. (2001) nor Groves (2001, 2005) considered any of the subspecies to be valid. The systematics of the robust capuchins have been at least as diverse as that of the gracile capuchins (see Table 1). For example, Groves (2001) used coat color variation and tuft shape to divide the robust capuchins into four species: C. apella, C. libidinosus, C. nigritus, C. xanthosternos: 1- C. apella, is the tufted or brown capuchin, and has six subspecies, C. a. apella, C. a. fatuellus, C. a. macrocephalus, C. a. peruanus, C. a. tocantinus and C. a. margaritae that are distributed in Venezuela, Surinam, Guianas, Brazil, Colombia, Ecuador, Peru and Bolivia. 2- C. libidinosus, is the bearded or black-striped capuchin that has four subspecies, C. l. libidinosus, C. l. pallidus, C. l. paraguayanus and C. l. juruanus. It is distributed in Brazil, Peru, Bolivia, Paraguay and Argentina. 3- C. nigritus is the black or black horned capuchin. It has three subspecies, C. n. nigritus, C. n. robustus and C. n. cucullatus and is distributed in Brazil, Argentina and Paraguay. 4- The fourth species is C. xanthosternos. It is the yellow-breasted or buff-headed capuchin and is endemic in the Atlantic Forest of Southern Bahia, north of the Jequitinhonha River. It is found at least as far north as the Paraguacú River, near Salvador in Brazil. Silva Jr (2001) placed the robust capuchins into its own subgenus, Sapajus (Kerr, 1792), with seven species: 1- C. (Sapajus) macrocephalus and 2- C. (Sapajus) apella in the Amazon; 3- C. (Sapajus) libidinosus and 4- C. (Sapajus) cay in the Caatinga, Cerrado and Chaco habitats, and 5- C. (Sapajus) robustus, 6- C. (Sapajus) nigritus and 7- C. (Sapajus) xanthosternos, in the Atlantic Coastal Forest. The Marcgrave’s capuchin, Cebus flavius, also a robust capuchin, was recently rediscovered in Northeast Brazil (Mendes Pontes et al., 2006; Oliveira and Langguth, 2006). Several studies have recently come forward to help clarify the systematics and the evolution of the capuchin monkeys. Here we summarize some of the most noteworthy molecular genetics studies:

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil 1. Ruiz-García et al., (2010) were the first to analyze the intra-specific phylogeny of C. albifrons at the molecular level. They analyzed 118 specimens at the mitochondrial (mt) COII gene and showed the existence of three well defined groups in Northern Colombia: malitosus, versicolor-pleei-cesarae and leucocephalus. They arose from at least, three distinct migrations from different Amazonian groups. Five different Amazonian and Eastern Llanos C. albifrons’s groups (I, II, III, IV, and V) were also found. Many of these groups did not correspond with the traditional morphological subspecies previously described. In many Amazonian localities, some of these groups live in sympatry probably by secondary expansion after their respective formations. 2. In a second study, focusing on the mt Cyt-b gene, Casado et al., (2010) estimated the molecular divergence of two separate populations of C. cay, and estimated its time of separation from C. apella. Twenty-three C. cay individuals from Brazil and nine from Paraguay showed 24 haplotypes (20 and 4, respectively), accounting for 29 variable sites (19 transitions and 10 transversions). The genetic distance between haplotypes averaged 0.5%, with 1.1% between C. cay populations. Mismatch distribution indicated that this species suffered a recent demographic expansion. Divergence time estimates suggested that the two populations of C. cay split in the Pleistocene. 3. Ruiz-García et al., (2012a) were the first to analyze, molecularly, the intra-specific phylogeny of C. capucinus (121 individuals analyzed). Four different and significant haplotype lineages were found in Colombia living sympatrically in the same Departments of this country. They all presented high levels of gene diversity but the III Colombian mitochondrial haplogroup was determined likely to be the most ancestral lineage. The II Colombian mitochondrial haplogroup was probably the source of origin of the unique Central America mitochondrial haplogroup that was detected. These molecular population genetics data do not agree with the existence of two well-defined subspecies in Central America (limitaneus and imitator). This Central America mitochondrial haplogroup showed significantly less genetic diversity than the Colombian mitochondrial haplogroups. All the C. capucinus analyzed showed evidence of historical population expansions. 4. Lynch-Alfaro et al., (2012a), analyzed 53 specimens of capuchin monkeys (eight C. capucinus, 12 C. olivaceus, 13 C. albifrons, six S. microcephalus, two S. apella, three S. cay, two S. libidinosus, two S. xanthosternos and five S. nigritus) at two mt genes (12S rRNA and Cyt-b), and determined that the capuchin monkeys contained two well supported monophyletic clades, the morphologically distinct gracile and robust groups. They considered these groups to be two well-separated genera of capuchins (Cebus and Sapajus). They estimated a late Miocene divergence between Cebus and Sapajus and a subsequent Plio-Pleistocene diversification within each of the two clades. A Bayesian analysis indicated that the current wide-ranging sympatry of Cebus and Sapajus across much of the Amazon Basin was the result of a single explosive late Pleistocene invasion of Sapajus from the Atlantic Forest into the Amazon, where Sapajus is now sympatric with gracile capuchins across much of their range. They considered three different hypotheses to explain the distribution of capuchin monkeys and the sympatry of both robust and gracile groups in the Amazon. The first hypothesis, “Out of the Amazon,” explains the sympatry of robust

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and gracile capuchins because the Amazon is the origin focus for all capuchin monkeys. They evolved by allopatric or sympatric speciation within the Amazon Basin. The gracile capuchins subsequently radiated north and the robust capuchins radiated south. This hypothesis agrees quite well with three assumptions. First, the Amazon Basin is the ancestral origin of the capuchin monkeys. Second, the diversification into gracile and robust capuchins also occurred within the Amazon. Third, a recent invasion of these two capuchin forms occurred towards northern South America, Central America, the Cerrado and the Atlantic Forest. This hypothesis predicts that the basal division for capuchins was north (gracile forms) and south (robust forms) of the Amazon River. The second hypothesis is the “Atlantic versus Amazon.” This hypothesis is based in the vicariance between the Atlantic coastal forest and the Amazon Basin, the major force shaping capuchin monkey distributions. Different examples with Neotropical primates (Callithrix vs. Mico + Cebuella; Tagliaro et al., 1997) and many other vertebrates (with divergence between Atlantic coastal forest and the Amazon) can be related (Patton et al., 2000). This hypothesis predicted that the robust capuchins would be the ancestral condition, and the gracile capuchins would have evolved relatively recently from a robust ancestor in the Amazon. Three predictions emerge from this hypothesis. First, there is a basal split within capuchins between robust Atlantic and robust Amazon taxa. Second, there is paraphyly of robust capuchins, with the robust Atlantic clade as the sister group to the Amazon gracile capuchins + Amazon robust capuchins. This means that gracile capuchins are a subclade of the Amazon robust capuchins). Third, there is a relatively recent origin of gracile capuchins. There is a third hypothesis: “Reinvasion of the Amazon.” This hypothesis affirms that after a basal gracile Amazon–robust Atlantic Forest divergence, the robust Atlantic Forest capuchin evolved in allopatry from the Amazon gracile capuchin, and later re-established sympatry when the robust form expanded across the Cerrado and into the Amazon. Here are some predictions of this hypothesis. First, there is initial divergence of a robust Atlantic capuchin clade and a gracile Amazon capuchin clade. Second, the robust Amazon capuchins forming a recently evolved subclade are nested with the Atlantic Forest robust clade. Lynch-Alfaro et al., (2012a) performed two tests of the dispersal rate to evaluate these three hypotheses. If the “out of the Amazon” hypothesis is true, the prediction is that both robust and gracile capuchins began diversifying at the same time, and that each dispersed at relatively equal rates. Following this hypothesis, the Atlantic Forest robust capuchins, as the most distant dispersal group from the Amazon, might be expected to have the highest dispersal rate. If the “Atlantic versus Amazon” hypothesis is true, then, they predicted that the robust capuchins have been diversifying longer in both the Amazon and the Atlantic forests, which could explain their large distribution without recourse to rapid dispersal. Furthermore, the gracile capuchins, as a recently evolved subclade of the Amazonian robust capuchins, would have rapidly colonized the areas where they currently live. This predicts a more rapid rate of dispersal for gracile compared with robust capuchins. If the “reinvasion of the Amazon” hypothesis is true, the gracile Amazon and the robust Atlantic Forest capuchins have been diversifying and dispersing the longest amount of time. But the

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5.

6.

7.

8.

relatively recent reinvasion and dispersal of robust capuchins throughout the Cerrado and the Amazon encompasses the largest area. This requires a more rapid dispersal rate for robust Amazon capuchins than that for either the gracile Amazon or the Atlantic Forest robust capuchins. They concluded that the “reinvasion of the Amazon” hypothesis was the best fit. Lynch-Alfaro et al., (2012b) reviewed extensive morphological, genetic, behavioral, ecological, and biogeographic evidence and stated that there was sufficient data to split the capuchin monkeys into two full genera (Cebus and Sapajus). Bobli et al., (2012) analyzed two mt genes (Cyt-b and D-loop) from 50 gracile capuchin samples. Their data indicate that the gracile capuchins underwent a radiation about 2 MYA ago and quickly diversified in both the Andes and the Amazon. These authors used an extreme typological PSC and split the gracile capuchin taxa into many species. They suggested at least two Amazonian species (C. yuracus and C. unicolor), a species from the Guiana Shield (C. albifrons), two Northern Andean species (C. versicolor and C. cesarae), C. brunneus on the Venezuelan coast, C. adustus in the region of Lake Maracaibo, C. capucinus in Northwestern Ecuador, Colombia and Panama, C. imitator in Central America, C. olivaceus and C. castaneus occupying a large part of the Guiana Shield and C. kaapori in the Eastern Amazon, south of the Amazon River. Ruiz-García et al., (2012b) analyzed 49 robust capuchins that had exact geographic origins from diverse areas of Colombia, Peru, Bolivia, French Guiana, Brazil, Argentina and Paraguay. They determined different findings. First, they found two established and related taxa in the northern Amazon River area. They named C. a. apella and C. a. fatuellus. C. a. apella is distributed from French Guiana until, at least, the Negro River in the northern Brazilian Amazon. C. a. fatuellus is distributed throughout the Colombian Eastern Llanos and the northern Colombian Amazon. Second, they determined two other C. apella taxa in the southern Amazon area: C. a. macrocephalus and C. a. cay. C. a. macrocephalus has a western and southern Amazon distribution, while C. a. cay has a more southern distribution outside the Amazon Basin. In the upper and western Amazon Basin, there was a unique lineage (C. a. macrocephalus) with one widely distributed haplotype. Therefore, the four morphological subspecies or species described for this area (C. a. maranonis, C. a. macrocephalus, C. a. peruanus, C. l. pallidus) and maybe a fifth unknown subspecies were molecularly undifferentiated at least for the mitochondrial gene analyzed. They were all identified as C. a. macrocephalus. Third, the specimens classified as C. nigritus and C. xanthosternos were clearly differentiated from the other specimens. These two lineages were assigned to the status of full species. Nascimento et al., (2015) criticized the work of Lynch-Alfaro et al., (2012a). They re-analyzed the data of these authors, including additional mt Cyt-b data from S. xanthosternos and S. flavius. They placed S. xanthosternos in a monophyletic clade representing the most basal lineage of the robust capuchins (this had already been demonstrated by Ruiz-García et al., 2012b). Their analyses indicated polyphyletic arrangements for several capuchin species (a fact observed in the previously cited works). They concluded that the molecular data available at that moment lacked the adequate variation for accurately resolving species relationships. A better resolution

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of the species tree is required along with the correct identification of species, data from several unlinked nuclear loci from a higher number of individuals per species, and careful analysis of ancient DNA data from museum specimens. In the current work we employed the nomenclature proposed by Lynch-Alfaro et al., (2012a, b) with Cebus and Sapajus as separated genera. However, pending our results we will either maintain or change this nomenclature. Many of the most modern systematic changes in capuchin monkeys, as well as in many other organisms, vary depending on the species criteria used. For example, the BSC is arguably the strongest concept of species for sexually reproducing taxa (Mayr, 1942, 1963). This concept holds that species are sets of interbreeding populations that are reproductively isolated (with no fertile hybrids) from other similar sets. This means that this concept has taken into account the existence of pre-zygote isolation reproductive processes, which is a paradigm within the Neodarwinism synthesis (Barton and Bengtsson, 1986; Coyne et al., 1994; Antonovics, 2006). The BSC has two major operational problems. First, it is difficult to evaluate among populations in extreme allopatry. Second, the researchers must demonstrate if there are fertile hybrids among different taxa in the wild. Other alternative species definitions are the genetic species concept (GSC; Baker and Bradley, 2006) and the PSC (Cracraft, 1983). The first one defines a species as a group of populations that are genetically isolated from other groups (two different genetic pools with independent evolutionary fates). Thus, this definition does not necessarily imply pre-zygote isolation reproductive processes. The second one defines a species as the smallest monophyletic and diagnosable cluster of individuals with a parental pattern of ancestry and descent by means of molecular or morphological characteristics. Operationally, this concept is easier to apply than the BSC, because a researcher could define new species without having to demonstrate if there are pre and/or post-zygote isolation reproductive mechanisms, strong karyotype differences, “normal” hybrids in the wild, etc. It’s clear that if the researcher has reproduction data for a determined taxon or for a taxa set, the BSC is a more complete species concept than the PSC. To help to clarify many of the controversy about systematics and the evolutionary history of the capuchin monkeys, we analyzed a set of 452 individuals (using the previous nomenclature: 124 C. capucinus, 240 C. albifrons, one C. olivaceus, two S. xanthosternos, two S. nigritus, four S. cay, 22 S. a. macrocephalus, one S. robustus, five S. a. apella and 51 S. a. fatuellus) sequenced for four mt genes (D-loop, Cyt-b, COI and COII). We included a greater number of specimens, geographic localities and genes compared to any previous capuchin study. Furthermore, a sub-set of these individuals (27 specimens: 14 C. albifrons, two S. a. cay, seven S. a. macrocephalus, one S. a. apella and three S. a. fatuellus) were sequenced for 16 mitochondrial genes. Our study has four main aims: 1- To determine if the genetic distances of neutral or quasi neutral molecular markers among different taxa of gracile and robust capuchins are within the range of values among species of the same genus or within values among different Neotropical primate genera; 2- To analyze if the alleged monophylia between Cebus and Sapajus claimed by Lynch-Alfaro et al., (2012a) is maintained when the sample size, number of geographical sights sampled, and the number of molecular markers are increased; 3- To determine if the “reinvasion of the Amazon” hypothesis and the temporal splits found by Lynch-Alfaro et al., (2012a) are maintained when the sample size, geographical localities and

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number of genes are increased; 4- To analyze if there is a correlation between the traditional morphological species and subspecies of capuchin monkeys and the results using molecular genetics data; and 5- If there is no correlation, to propose a new systematics for the capuchin monkeys.

MATERIALS AND METHODS It’s important to know the exact geographical origins of the individuals analyzed in order to resolve the evolutionary history of the capuchins (or, other taxa), (Nascimento et al., 2015). Indeed, sometimes it is easy to observe mistakes concerning the origin and distributions of some samples. For instance, Figure 1 in Lynch-Alfaro et al., (2012a) showed incorrect distributions of robust capuchins. Similarly, Nascimento et al., (2015) criticized the same study for erroneously classified some specimens (one from Ayacucho, two from the Loreto Department in the Peruvian Amazon, and two from the Meta Department in Colombia) as macrocephalus. In disagreement, Nascimento et al., (2015) concluded that they are apella. However, we have directly sampled many capuchins in these areas of Peru and Colombia. Based on our data, the Peruvian specimens are macrocephalus, agreeing with Lynch-Alfaro et al., (2012a). Again based on our data, neither work correctly classified the two Colombian individuals. They are fatuellus. For this reason, we have directly sampled a total of 452 wild capuchin monkeys and documented their geographical origins. The DNA was obtained from samples of hair, teeth, muscle and blood from animals found alive or dead in diverse Indian communities. We requested permission to collect biological materials from either carcasses or live animals that were already present in the community. We sampled small pieces of tissue (muscle or blood) or teeth from hunted animals that were discarded during the cooking process, or hairs with bulbs plucked from live pets. Communities were visited only once, all sample donations were voluntary, and no financial or other inducement was offered for supplying specimens for analysis. All the pets and the hunted animals analyzed were obtained by the Indian communities at a maximum of 15 km from the community. These 452 individuals were from the following taxa. There were 124 C. capucinus (58 from Colombia representing the four haplogroups detected by Ruiz-García et al., 2012a, and 66 from diverse countries of Central America: Panama, Costa Rica and Guatemala). Another 240 were C. albifrons. This included 78 C. a. versicolor, 14 C. a. pleei, eight C. a. cesarae, five C. a. adustus, 34 C. a. leucocephalus, one C. a. malitosus, 47 C. a. albifrons (including 40 C. a. unicolor if we follow the nomenclature of Hershkovitz, 1949), 45 C. a. yuracus, three C. a. cuscinus, two C. a. aequatorialis, one individual from a undescribed subspecies from the Peruvian San Martin Department and two natural hybrids between C. albifrons and C. capucinus. All of these animals were sampled in Colombia, Ecuador, Peru, Bolivia and Brazil. Also there were: one C. olivaceus castaneus (French Guiana), two S. xanthosternos (Bahia, Brazil), two S. nigritus (Misiones, Argentina), four S. cay (South Brazil and Paraguay), one S. robustus (Espirito Santo, Brazil), 22 S. apella macrocrocephalus (frontier between Argentina and Bolivia, Bolivia, Brazil, Peru and Colombia), five S. apella apella (French Guiana and Brazil) and 51 S. apella fatuellus (Colombia). We used a sample of Aotus

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azarae boliviensis (Santa Cruz Department, Bolivia) as an outgroup. All samples were completely sequenced for four mitochondrial genes. A subset of these capuchin monkeys were sequenced for 16 mitochondrial genes. The subset included 27 capuchin monkeys (six C. a. versicolor, one C. a. pleei, one C. a. cesarae, one C. a. malitosus, three C. a. albifrons/unicolor, one C. a. yuracus, one C. a. aequatorialis, three S. apella fatuellus, one S. apella apella, two S. cay and seven S. apella macrocephalus). It also contained 19 Ateles geoffroyi (Costa Rica), 10 Lagothrix lagotricha cana (Madeira River, Brazil), 10 Alouatta palliata (Costa Rica), 17 Saimiri cassiquiarensis albigena (Colombia), two Aotus nancymaee (Peru), seven Aotus vociferans (Colombia), one Saguinus labiatus (Peru), one Saguinus fuscicollis (Peru), 20 Saguinus leucopus (Colombia) and three Saguinus oedipus (Colombia).

Figure 1. (Continued).

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Figure 1. (Continued).

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Figure 1. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 452 gracile and robust capuchin monkeys for four concatenated mitochondrial genes (control region, COI, COII and Cyt-b). The number in the nodes are bootstrap percentages. The roubst capuchin clade begins with C. xanthosternos and continues thereafter. There are 13 individuals of different taxa of C. albifrons (albifrons/unicolor, yuracus and malitosus) within the robust capuchin’s clade.

Molecular Procedures The DNA from muscle and blood was extracted using the phenol-chloroform procedure (Sambrook et al., 1989), while DNA samples from hair and teeth were extracted with 10% Chelex resin (Walsh et al., 1991). The 452 capuchin individuals sampled were sequenced for four mt genes (COI, COII, Cyt-b and D-loop). For the mt COI amplification (polymerase chain reaction, PCR), we used the forward primer LCO1490 (5’GGTCAACAAATCATAAAGATATTGG-3’), and the reverse primer HCO2198 (5’TAAACTTCAGGGTGACCAAAAAATCA-3’) (657 base pairs, bp) (Folmer et al., 1994)

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under the following PCR profile: 94°C for 5 min, followed by 39 cycles of 94°C for 30 s, 44°C for 45 s, 72°C for 45 s, and a final cycle of 72°C for 5 min. For the amplification of the mt COII gene (located in the lysine and asparagine tRNAs) we used the forward primer L6955 (5’ -AACCATTTCATAACTTTGTCAA-3’) and the reverse primer H7766 (5’ CTCTTAATCTTTAACTTAAAAG-3’) (720 bp) (Ashley and Vaughn, 1995; Collins and Dubach, 2000a; Ruiz-Garcia et al., 2010, 2012a,b). We used the following temperatures: 95°C for 5 min, 35 cycles of 45 s at 95°C, 30 s at 50°C and 30 s at 72°C and a final extension time for 5 min at 72°C. For both genes, the PCRs were performed in a 25 l volume with reaction mixtures including 4 l of 10 x buffer, 6 l of 3 mM MgCl2, 2 l of 5 mM dNTPs, 2l (8 mM) of each primer, 2 units of Taq DNA polymerase, 5 l of ddH2O and 2 l (20–80 ng/l) of DNA. The mt Cyt-b was amplified by PCR using the procedure of Montgelard et al., (1997) (1,140 bp) and the mt D-loop was amplified using the primers L15400 (5’TCCACCATTAGCACCCAAAG-3’) and H15940 (5’-CCTGAAGTCGGAACCAGATG-3’) (610 bp) (Kocher et al., 1989). The conditions for Cyt-b and D-loop amplification were performed in 25 l reactions including 2 l of DNA, 2 l of 10 x buffer, 13 l ddH20, 2 l (25 mM) MgCl2, 1 l (10 mM) each of forward and reverse primers, 2l (10 mM) dNTPs, and 2 units of Taq DNA polymerase. The standard thermal cycling program consisted of 10 min at 95°C, 35 cycles of 35 s at 94°C, 35 s at 55°C and 30 s at 70°C and a final extension time for 10 min at 72°C. The total length of the sequences studied were 3,127 bp. All amplifications, including positive and negative controls, were checked in 2% agarose gels. The gels were visualized in a Hoefer UV Transilluminator. Both mt DNA strands were sequenced directly using BigDye Terminator v3.1 (Applied Biosystems, Inc.). We used a 377A (ABI) automated DNA sequencer. For a subset of 27 individuals, DNA of high quality was extracted and isolated with the QIAamp DNA Mini Kit (Qiagen, Inc.) for blood samples (DNA Purification from blood or body Fluids; Spin Protocol) and for muscle samples (DNA Purification from tissues). To carry out the amplifications of the mt DNA of the capuchin specimens studied, we used the LongRange PCR Kit (Qiagen, Inc.), with final volume reactions of 25 l. The reaction mix was composed of a 80-200 ng DNA template, 2 units of Long-Range PCR Enzyme, 3 l of 10 x LongRange PCR Buffer, 4 μl (15 pmol) of each primer, and 2 μl of 10 mM dNTPs. The cycling conditions were as follows: 95°C for 3 min, followed by 50 cycles denaturing at 95°C for 20 s, primer annealing at 53–58°C (depending on primer set, with a decrease of 0.1°C every cycle) for 30 s, and extension at 72°C for 10 min. This was followed by 30 cycles of denaturing at 95°C for 20 s, annealing at 48–53°C (depending on primer set) for 30 s, and extension at 72°C for 5 min, with a final extension at 72°C for 10 min. Using four sets of primers to generate overlapping amplicons from 3,345 bp to 5,049 bp in length, allowed us to carry out a quality test for genome circularity (Bensasson et al., 2001; Thalman et al., 2004). There was an increased possibility of the reduction of amplifying nuclear mitochondrial pseudogenes (numts) in our analyses. Herein, we show the results of 16 mitochondrial genes (two rRNA, D-loop and 13 protein codifying genes; D-loop, 610 bp 12S rRNA, 930 bp; 16S rRNA, 1,580 bp; ND1, 950 bp; ND2, 1,035 bp; COI, 657 bp; COII, 720 bp; ATP8, 205 bp; ATP6, 695 bp; COIII, 775 bp; ND3, 340 bp; ND4L, 305 bp; ND4, 1,380 bp; ND5, 1,810 bp; ND6, 530 bp and Cyt-b, 1,140 bp). The sequences were concatenated by means of the SequenceMatrix v. 1.7.6 (Vaidya et al., 2011). Thus, our analyses were undertaken with 13,662 bp which represents about 80-85% of the total mitochondrial DNA length.

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Overlapping regions were examined for irregularities, such as frameshift mutations and premature stop codons. The lack of such irregularities agrees quite well with the absence of numt sequences in our mitochondrial sequences. We used a 377A (ABI) automated DNA sequencer. The samples were sequenced in both directions to ensure sequence accuracy.

Data Analyses We used the Kimura 2P genetic distance (Kimura, 1980) to determine the percentage of genetic differences between the alleged Cebus and Sapajus genera, among different species within different genera (Alouatta, Ateles, Aotus and Saguinus) and among different genera (Alouatta, Lagothrix, Ateles, Aotus, Saimiri and Saguinus). The Kimura 2P genetic distance is a standard measurement for barcoding tasks (Hebert et al., 2003, 2004). In the current work, we only show these genetic distances with the mt COII gene because the other genetic distances will be shown elsewhere. For Neotropical primates and Hominoidea, there is an average genetic distance at the COII gene of around 5.82% ± 1.64% among species within a genus, around 2-4% for subspecies within species and around 15.68% ± 1.73% among genera (Ascunce et al., 2003; Collins and Dubach, 2000b). To analyze the genetic relationships among the 452 capuchin individuals for four mt genes as well as for 27 capuchin individuals for 16 mt genes studied, we used two neighborjoining (NJ; Saitou and Nei, 1987) trees with the Kimura 2P genetic distance. Other phylogenetic trees are shown elsewhere, but the results were basically identical. We used the Network 4.6.10 Software (Fluxus Technology Ltd.) to form a median joining network (MJN) to estimate possible divergence times among the haplotypes (joined COI, COII, Cyt-b, and D-loop sequences) in capuchin monkeys (Bandelt et al., 1999). The  statistic (Morral et al., 1994) was estimated and transformed into years. To determine the temporal splits, it is necessary to estimate the mutation rate at these mt genes. For mt COI, an average mutation rate of 1% per million years was employed (Matzen da Silva et al., 2011; Olson et al., 2009). This represents an average of one mutation each 152,000 years. Ruvolo et al., (1991) determined a mutation rate of 0.85% per million years per lineage for Hominoidea at mt COII. This represents one mutation on average each 199,402 years. This mutation rate was practically identical to that determined by Ruiz-García and Pinedo (2010) in a Lagothrix study (one mutation on average every 191,000 years). Similarly, for Aotus, Ashley and Vaughn (1995) and Ruiz-García et al. (2011) determined one mutation on average every 199,000 years at this same mitochondrial gene. Thus, we have used an average of one mutation each 195,000 years for COII. For mt Cyt-b in mammals, Nabholz et al., (2008, 2009) estimated a mutation rate of 2.5 x 10-2 substitutions/site/million years, which was equal to about one mutation each 120,482 years. We employed this mutation rate for this gene. For the mt D-loop, we employed a mutation rate of 7.05% per million years per linage following Forster et al., (1996), Geraldes et al., (2008), Hardouin et al., (2010), Heyer et al., (2001), Horai et al., (1995), Rajabi-Maham et al., (2008), Savolainen et al., (2002), Tamura and Nei (1993) and Ward et al., (1991), with different mammalian species. This represents one mutation on average each 40,000 years. Henceforth, we used an average of one mutation each 127,000 years for all four mt genes. In this analysis, we have employed only the haplotypes represented by more than one individual, with the exception of those extremely important haplotypes, to more easily view the relationships among the different taxa of capuchins.

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RESULTS In Table 2 we compare the Kimura 2P genetic distance among taxa of Cebus and Sapajus but only for mtCOII. The values between C. albifrons and S. xanthosternos, S. nigritus and S. apella were 6.0%, 7.9% and 6.7% (mean: 6.87%), respectively. C. capucinus showed the following values in regards to the same Sapajus species: 7.1%, 8.9% and 7.8% (mean: 7.93%), respectively. Clearly, all these values are closer to the average genetic distance for species (5.82% ± 1.64%) than to the average genetic distance for genera (15.68% ± 1.73%) for this gene. Within each one of these taxa, C. albifrons and C. capucinus (2.9% and 3.0%, respectively) showed considerably higher internal genetic diversity than Sapajus (S. xanthosternos, 0.1%; S. nigritus, 1.5% and S. apella, 0.4%, respectively). Table 2. Kimura 2P genetic distance (percentage) and standard deviation among the different capuchin taxa studied by means of the mitochondrial COII gene. 1 = Cebus albifrons; 2 = C. capucinus; 3 = C. nigritus; 4 = C. apella; 5 = C. xanthosternos Capuchin taxa 1 2 3 4 5

1 3.9 7.9 6.7 6.0

2 0.4 8.9 7.8 7.1

3 0.8 0.9 3.3 5.1

4 0.7 0.8 0.6 2.8

5 0.8 0.8 0.8 0.6 -

Table 3. Kimura 2P genetic distance (percentage) and standard deviations among the capuchin genera, Cebus and Sapajus, and other Neotropical primate genera (Alouatta, Aotus, Ateles, Lagothrix, Saimiri and Saguinus) studied by means of the mitochondrial COII gene. 1 = Lagothrix; 2 = Alouatta; 3 = Aotus; 4 = Ateles; 5 = Cebus albifrons; 6 = Cebus (= Sapajus) apella; 7 = Saimiri; 8 = Saguinus. In bold, the lowest genetic distance, which corresponds to Cebus and the alleged Sapajus Genera 1 2 3 4 5 6 7 8

1 15.6 14.9 12.4 19.9 18.2 14.9 27.2

2 1.4 15.6 13.1 17.2 15.8 16.2 28.8

3 1.5 1.5 15.1 16.7 16.2 14.5 25.4

4 1.3 1.4 1.6 14.7 13.5 14.3 26.4

5 1.7 1.5 1.5 1.5 5.8 18.2 27.2

6 1.7 1.6 1.7 1.6 0.8 16.4 27.0

7 1.6 1.7 1.7 1.7 1.7 1.8 27.9

8 2.4 2.5 2.3 2.1 2.0 2.1 2.4 -

We analyzed genetic distances with our own sequences for other genera and species of Neotropical primates to compare with the previous results for Cebus and Sapajus (Table 3). The average genetic distance for different species of Alouatta was 7.24% ± 1.38%. For different species of Aotus it was 4.7% ± 1.31%, for different species of Ateles it was 3.20% ± 0.77% and for different species of Saguinus it was 6.87% ± 3.01%. With the exception of the

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Ateles species, all the other values of species within genera were not significantly different from the average value between Cebus and Sapajus (5.8% ± 0.8%). Even the values within Alouatta and within Saguinus were higher than between Cebus and Sapajus. Similarly, the genetic distance between both alleged capuchin monkey genera is significantly lower than the average genetic distance among Alouatta, Lagothrix, Ateles, Aotus, Saimiri and Saguinus (18.64% ± 5.27%; this value is 3.2 times higher than the genetic differences between Cebus and Sapajus). Meanwhile the gracile capuchin group showed the highest genetic diversity of the all the taxa we studied (3.5%; Table 4), the robust capuchin group (0.4%) is even internally less differentiated than the genetic diversity found in single species of restricted distribution such as Saguinus leucopus (1.3%) or in Saguinus oedipus (1.1%). Therefore, the percentages of the genetic distances found did not justify Sapajus as a different genus from Cebus. Additionally, Cebus showed higher genetic diversity than that obtained in Sapajus, which indicates Cebus could be an original taxon and Sapajus deriving from it. The neighbor-joining tree, with the Kimura 2P genetic distance (Figure 1) and four mt genes, showed some extremely interesting features. They do not agree with the previous view of Alfaro et al., (2012a, b), Bobli et al., (2012) and Nascimento et al., (2015). For example, there is not reciprocal monophylia between the two alleged Cebus and Sapajus genera. Meanwhile in the clade of Cebus, only sequences of C. albifrons, C. capucinus and C. olivaceus are present, the Sapajus clade contains sequences of C. albifrons. This result does not support the existence of two well separated genera. Table 4. Mitochondrial nucleotide diversity ( in percentage) and standard deviation (S.D) in capuchin monkey taxa and other Neotropical primate taxa Genera Cebus albifrons Cebus apella Lagothrix Ateles Alouatta Aotus Saimiri Saguinus leucopus Saguinus oedipus

 3.5 0.4 1.5 0.4 1.5 2.8 0.2 1.3 1.1

± S.D

0.4 0.1 0.2 0.1 0.2 0.3 0.1 0.2 0.3

Within the Cebus clade, haplotypes of C. albifrons, C. capucinus and C. olivaceus were intermixed, not conforming clades correlated with the traditional morphological subspecies or species of gracile capuchins. The first branch to diverge within this clade were those corresponding to the Colombian III C. capucinus haplogroup (Ruiz-García et al., 2012; RuizGarcía and Castillo, 2016) together with some C. albifrons haplotypes from the area of Arauca (Eastern Llanos), Guainia (transition area from Eastern Llanos to Amazon) in Colombia (C. a. albifrons) and Negro River (Brazil) (C. a. albifrons-unicolor). The next haplotypes to diverge were C. albifrons from the Arauca region (C. a. albifrons) and from the most western Amazon areas where this species lives (Ecuadorian Amazon; C. a. yuracus). The next cluster to diverge was comprised of C. albifrons individuals from the Western Amazon (Ecuador, Peru and Colombia; the most divergent haplotypes, C. a.

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albifrons/unicolor; C. a. yuracus/cuscinus, within this group were from the Ecuadorian Amazon). This cluster was in the origin of two small sub-clusters within it. One cluster had four animals from the Pacific Ecuadorian coast (traditionally named C. a. aequatorialis) and the other contained two C. a. albifrons from the Acre region (Brazilian Amazon) and the C. olivaceus castaneus individual from French Guiana. Later another large cluster appeared and it was integrated by individuals of C. albifrons from the Colombian departments of Santander, Norte de Santander, Boyaca and Arauca and they were identified following the traditional nomenclature as C. a. adustus and C. a. leucocephalus. Within this cluster there also appeared some exemplars of an Amazon origin. It also contained two individuals of C. capucinus from the vicinity of Buenaventura in the Pacific area of Colombia and that they were not included in the three Colombian haplogroups detected by Ruiz-García et al., (2012, 2016a).

Figure 2. Median Joining Network (MJN) with haplotypes found at the four concatenated mitochondrial genes (control region, COI, COII and Cyt-b) gene for a fraction of the 452 capuchin monkeys analyzed. Yellow circles = all the individuals with Cebus albifrons’s morphotypes; blue circles = all the individuals with Cebus capucinus’s morphotypes; green circles = Sapajus xanthosternos; black circles = Sapajus nigritus; grey circles = all the individuals with Sapajus apella’s morphotypes (including apella, cay, fatuellus, macrocephalus, pallidus, robustus); pink circle = Aotus azarae boliviensis as an outgroup. Red circles indicate missing intermediate haplotypes. Clearly, the haplotypes of C. albifrons and C. capucinus were intermixed and the robust capuchin haplotypes are nested within the gracile capuchin haplotypes. Some C. albifrons’s haplotypes were mixed with some robust capuchin haplotypes. These results disagree with Sapajus as a differentiated genus from Cebus.

The next group to diverge was mainly composed of the Colombian II C. capucinus haplogroup together with two C. albifrons from the Amazon (Ecuador and Colombia; C. a.

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yuracus and C. a. albifrons). The next clade to appear was exclusively integrated by Central American C. capucinus individuals from Panama, Costa Rica and Guatemala. Ruiz-García et al., (2012) and Ruiz-García and Castillo (2016) affirmed that the Colombian II haplogroup seems to be more related to the Central American C. capucinus haplogroup. Later, a group composed by individuals with phenotypes of C. a. versicolor from the Colombian Santander Department appeared. Following this cluster, the next group consisted of mixed individuals from the Colombian Vaupes department (C. a. albifrons) and individuals from the Antioquia and Cordoba departments (C. a versicolor). Later, the main cluster containing animals with phenotypes of C. a. versicolor from the Colombian Tolima, Quindio, Risaralda, Santander, Antioquia, Bolivar and Boyaca Departments formed, including one individual from Vaupes (C. a. albifrons) and another from the Peruvian Amazon (C. a. cuscinus). The last and most recent cluster was formed by individuals of C. a. versicolor (Antioquia, Tolima, Quindio, and Boyaca Departments), C. a. cesarae (Magdalena and Cesar Department), C. a. pleei (Bolivar department), and one natural hybrid individual of C. a. pleei and C. capucinus from the San Jorge River. This last cluster also contained one individual of the Arauca Department, which was not phenotypically classified in either of the recognized morphological subspecies, one animal from the Colombian Amazon (C. a. albifrons) and the C. capuchinus individuals from the Colombian I haplogroup detected by Ruiz-García et al., (2012). Thus, this clade did not contain monophyletic groups which were correlated with the classical morphological species, C. capucinus, C. albifrons and C. olivaceus. As reproductive cohesiveness has been observed in the captivity and in the wild (hybrids) and similar chromosomal rearrangement are found among all these taxa as we will discuss in brief, we believe that this clade could be composed by a unique species or super-species in the geographical area we studied: Cebus capucinus. That is, we consider this clade as a unique species with multiple mitochondrial lineages, where multiple events of migration and mixture occurred and not a genus with multiple species as proposed by Bobli et al., (2012). In the Sapajus clade, the first taxon to diverge was S. xanthosternos, followed by the divergence of S. nigritus. In both cases, these two clusters were highly significant (99% bootstraps). The following divergent cluster was integrated by an individual of C. a. albifrons from the Colombian Amazon. The next cluster to diverge was composed by the four individuals of S. apella cay (Paraguay and southern Brazil). Later, a big cluster appeared integrated by closely related animals we named S. a. macrocephalus (Southern Colombian Amazon, Western Brazilian Amazon, Peru, Bolivia and Northwestern Argentina), with one S. a. robustus (from Eastern Brazil). Interestingly, in this cluster, five C. a. yuracus (four from the Ecuadorian Amazon and one from the Napo River in Peru) were integrated. The last and more recent diverging clade was composed of two sub-clusters, both north of the Amazon River (all the other Sapajus taxa were from the Atlantic coast of Brazil and south of the Amazon River). S. a. fatuellus was from the western area from the Negro River (Colombia), and S. a. apella was from the eastern area of the Negro River (Brazil, Guianas). Also, in this Sapajus clade, seven C. albifrons were enclosed, three from the Napo River in the Ecuadorian Amazon (C. a. yuracus), one from the Colombian Amazon (C. a. albifrons), two from the Colombian Eastern Llanos (C. a. albifrons) and the unique individual of C. a. malitosus (Northern Colombia). Therefore, three well differentiated clades were determined within the Sapajus clade, xanthosternos, nigritus and apella (with different sub-clades in apella: cay, macrocephalus, fatuellus and apella, with very small genetic distances among them). However, some C. albifrons haplotypes were intermixed with them. This agrees quite well

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with the fact that the gracile capuchins showed considerably higher levels of gene diversity than the robust capuchins and that, probably, the first was the origin of the second. In the discussion we offer explanations as to why C. albifrons haplotypes are intermixed with the haplotypes of the tufted capuchins. Note, this does not support Sapajus as a different genus from Cebus.

Figure 3. (Continued).

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Figure 3. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 27 gracile and robust capuchin monkeys studied for 16 concatenated mitochondrial genes. Together the capuchin monkeys, other Neotropical primate species were sequenced for these 16 mitochondrial genes: Ateles geoffroyi (Costa Rica), Lagothrix lagotricha cana (Madeira River, Brazil), Alouatta palliata (Costa Rica), Saimiri cassiquiarensis albigena (Colombia), Aotus nancymaee (Peru), Aotus vociferans (Colombia), Saguinus labiatus (Peru), Saguinus fuscicollis (Peru), Saguinus leucopus (Colombia) and Saguinus oedipus (Colombia). The number in the nodes are bootstrap percentages.

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The MJN procedure correlated very well with that described for the previous tree (Figure 2). The original and the most related capuchin haplotype with the outgroup haplotype (Aotus) corresponded to one haplotype found in two C. albifrons from the Arauca and Negro River areas (H14). This haplotype gave place to the Colombian III C. capucinus haplogroup. Relatively near to these haplotypes appeared a set of haplotypes (H65, H60 and related), which all belonged to the Western Amazon C. albifrons. In the central area of the MJN there appeared a set of haplotypes that belonged to C. a. versicolor, C. a. cesarae-pleei, C. a. albifrons (all from Colombia) together with C. capucinus of the Colombian I haplogroup. Indeed, H1 and H3 were shared by some of these C. albifrons and C. capucinus. In the upper area of the MJN, there are C. capucinus individuals from the Colombian II haplogroup, with H46 shared with two C. albifrons individuals form the Amazon (Ecuador and Colombia). Related with this haplotype set, there are the haplotypes of the Central American C. capucinus. In the lower part of the MJN, there are some dispersed C. albifrons haplotypes, all from the Western Amazon as well as the haplotypes of all the C. a. adustus-C. a. leucocephalus (H12) studied. This was shared with two C. capucinus from the Buenaventura area in Colombia. The most related haplotype (H45) of the gracile capuchins, which seems to give place to the robust capuchin haplotypes, was composed of three C. albifrons expanded in a wide area (Arauca and Guainia in Colombia and the Ecuadorian Amazon). The first robust capuchin haplotypes to appear were from S. xanthosternos, such as was revealed by the previous tree. From a yet undiscovered haplotype coming from S. xanthosternos the haplotypes of S. nigritus appeared. This seems to be an end branch without connections. There are also the haplotypes of S. a. cay, which in turn gave place to the most frequent S. a. macrocephalus haplotype (H93). One S. a. macrocephalus haplotype (H86) was shared with three C. a. yuracus from the Ecuadorian Amazon. The most recent robust capuchin haplotypes were those from S. a. fatuellus and S. a. apella. The most frequent S. a. fatuellus haplotype (H56) was shared with C. albifrons from the Colombian Eastern Llanos and from the Ecuadorian Amazon. Therefore, both analyses, tree and MJN, showed the exact same pattern of evolution in the capuchin monkeys. We estimated some noteworthy temporal splits among some relevant taxa of the capuchin monkeys. The oldest temporal splits within the capuchin monkeys always involved the Colombian III C. capucinus haplogroup and the C. albifrons haplotype (H14). For instance, the beginning of the splits between the Colombian III C. capucinus haplogroup with the Central American C. capucinus haplogroup or the split between the Colombian I C. capucinus haplogroup and the Central American one were 4.87 ± 0.57 MYA and 4.56 ± 0.55 MYA, respectively. The split of H14 and all the remaining Amazon C. albifrons haplotypes was around 4.92 ± 0.79 MYA. The temporal divergence between H14 and one of the main Western Amazon C. albifrons, from which many of the other Cebus lineages were generated, began around 3.76 ± 0.20 MYA. More recently, the temporal split from this Western Amazon C. albifrons with regard to some of the Northern Colombian C. albifrons haplogroups occurred around 3.05 ± 0.17 MYA (main versicolor clade), 2.48 ± 0.19 MYA (main albifrons clade from the Vaupes department), 2.73 ± 0.17 MYA (adustus-leucocephalus clade) and 3.33 ± 0.32 MYA (main cesarae-pleei clade). The temporal divergence from the original C. albifrons’s haplotype and the first diverging haplotype from the Sapajus clade was with one of the haplotypes of S. xanthosternos (around 3.31 ± 0.34 MYA). It was a similar temporal split that we commented about for the diverging process between the Western Amazon C. albifrons haplotypes and the different northern Colombian haplogroups of C. albifrons and C. capucinus. This result emphasizes that the

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origin of Sapajus was throughout Cebus and it appeared more recently than the initial mitochondrial diversification of Cebus capucinus-Cebus albifrons. Indeed, a large fraction of the genetic diversity of Sapajus should be considered nested within Cebus, which does not agree with the view of both taxa as different genera. The temporal splits among some different relevant northern Colombian and Vaupes C. albifrons haplogroups were lower than the previous values and they were as follows: 1.13 ± 0.094 MYA (adustus and leucocephalus vs. versicolor), 1.13 ± 0.15 MYA (adustus vs. cesarae-pleei), 0.57 ± 0.14 MYA (albifrons Vaupes vs. versicolor), 0.88 ± 0.25 MYA (albifrons Vaupes vs. cesarae-pleei) and 0.39 ± 0.15 MYA (versicolor vs. cesarae-pleei). For the Sapajus clade, relevant temporal splits are those between S. xanthosternos and S. a. cay (2.08 ± 0.13 MYA) and S. xanthosternos and S. a. macrocephalus (3.24 ± 0.18 MYA). These values are considerably higher than those estimated among other Sapajus taxa as S. a. cay-S. a. macrocephalus (0.17 ± 0.17 MYA) or S. a. fatuellus-S. a. apella (0.19 ± 0.19 MYA). This agrees with the fact that S. xanthosternos should be differentiated as a species (and the same for S. nigritus) from the other Sapajus taxa. The molecular differentiation among the remaining Sapajus taxa is extremely recent (less than 0.2 MYA) which agrees with the fact that these taxa are, at most, subspecies of a unique species. Within some of these haplogroups, diversification began around 0.78 ± 0.10 MYA (Central American C. capucinus), 0.43 ± 0.14 MYA (Colombian II C. capucinus haplogroup), 0.23 ± 0.086 MYA (versicolor), 0.13 ± 0.084 MYA (cesare-pleei), 0.19 ± 0.12 MYA (S. a. cay), 0.15 ± 0.028 MYA (S. a. apella), 0.15 ± 0.08 MYA (S. a. macrocephalus) and only 3,500 YA in S. a. fatuellus. These temporal estimations clearly showed S. apella as being in the most recent diversification processes within the different groups. Finally, we show a neighbor-joining tree with the Kimura 2P genetic distance (Figure 3) with 27 capuchin monkeys and some specimens of other genera (Alouatta, Ateles, Lagothrix, Aotus, Saimiri and Saguinus) for 16 mitochondrial genes. Although this tree is not as precise as that for the four mitochondrial genes because the number and variety of capuchin taxa is considerably lower, three main ideas are maintained. The first is that there is no reciprocal monophylia between Cebus and Sapajus because the last clade contains some individuals of Cebus, which disagrees with the fact that Cebus and Sapajus are two different genera. The second one is that the Amazon C. albifrons haplotypes originated the northern Colombian C. albifrons. Third, many of the morphologically defined C. albifrons taxa from northern Colombian are intermixed.

DISCUSSION Why the Cebus Genus Should Not Substituted by the Sapajus Genus for the Robust Capuchins Lynch-Alfaro et al., (2012b) proposed the division of capuchin monkeys into two genera, Sapajus, for the robust capuchins, and Cebus, for the gracile capuchins. They based this decision on alleged genetics, morphological, behavioral and ecological evidence. From a genetics point of view, the authors provided three main lines of evidence. 1- Capuchin monkeys were found to contain two well supported reciprocally monophyletic clades, the gracile capuchins (Cebus) and the robust capuchins (Sapajus); 2- The temporal divergence

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between both alleged genera was estimated to have occurred during the late Miocene (around 6.5 MYA) and independent diversification within each of the two genera occurred during the Pliocene; and 3- There is an Amazonian origin for the gracile Cebus clade and an Atlantic Forest or Cerrado origin for the Sapajus clade. However, our genetics results showed that these three lines of evidence for the split of the capuchin monkeys in two different genera are not so clear. In fact, they are incorrect when we include more individuals, localities, and mitochondrial genes sequenced. Here, we provide five findings which firmly contradict all three lines of evidence. 1- There is no reciprocal monophylia between the gracile and the robust capuchin clades. The main gracile capuchin clade did not contain any robust capuchin haplotype, but the robust capuchin clade is mixed with some gracile capuchin haplotypes; 2- The genetic diversity of the robust capuchins is generated from the gracile capuchins and therefore there is no independent diversification of both alleged genera; 3- The gracile capuchin evolution is much more complicated and old than that supposed by Lynch-Alfaro et al., (2012a, b); 4- The temporal split between both capuchin groups is lower than that estimated by Lynch-Alfaro et al., (2012a) ranging, based on our more complete data, from 5.5 to 3.3 MYA, depending on the procedures used. Indeed, as we will discuss later, the split of 3.3 MYA (MNJ procedure) is more probable than the split of 5.5 MYA (Bayesian procedure analyzed elsewhere). Thus, the divergence of both capuchin forms were during the Pliocene rather than in the Miocene as claimed by Lynch-Alfaro et al., (2012a,b) and 5- The genetic distances from relatively neutral molecular markers (which are the relevant characters to determine “real” phylogenetic relationships because are not affected by opportunistic adaptation or positive selection which can distort the “real” phylogenetic relationships between taxa) showed that the differences between gracile and robust capuchins groups are small. In other words, the differences are less than those obtained for different species of Neotropical primates within the same genus. For instance, different species within Alouatta and Saguinus showed higher genetic distances than those of capuchin groups we analyzed. In addition, they were extremely and significantly lower than the values obtained between well differentiated related genera of Neotropical primates. Therefore some of the fundamental genetics arguments put forth by Lynch-Alfaro et al., (2012a, b) for the separation of Cebus and Sapajus are not very consistent. It is also likely that many of the other arguments in favor of Sapajus by Lynch-Alfaro et al., (2012b) are weak and questionable. They employed, at least, five other argument lines for the split of Cebus and Sapajus: A. They argued against Hershkovitz (1949) and Hill (1960), who clustered all robust capuchins as C. apella, and explained the greater genetic and species diversity in the robust group compared to the gracile group. From a genetic point of view, this is incorrect. For instance, only the animals classified “a priori” as C. albifrons showed 3.5% of nucleotide diversity, while the animals classified as S. apella (including cay, macrocephalus, apella and fatuellus) only showed 0.4% of nucleotide diversity (near 9 times higher the nucleotide diversity in the gracile taxa than in the robust taxa). Another related point is that the coat and color characteristics could show more striking and visual differences in the robust clade than in the gracile one, but not in the molecular characteristics. We also highlight specific text of Lynch-Alfaro et al., (2012b) as weak proof for the splitting of capuchin monkeys into two different genera (…“An examination of capuchin monkey diversity, however, reveals far more

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil genetic and morphological difference between robust and gracile species than within either group”…). Perhaps this sentence could be expected between two different genera, but it could also be expected between two different species (with different subspecies) of the same genus. Thus, this sentence “per se” does not constitute any proof in favor of Sapajus as a different genus from Cebus. B. There are many different morphological characteristics between gracile and robust capuchins. For instance, a list of these differences showed by Lynch-Alfaro et al., (2012b) are as follows. The robust capuchins are significantly more robust in terms of cranial and dental characteristics than the gracile capuchins (Ford and Corruccini, 1985). They have both cranial and postcranial specializations for the exploitation of hard and tough foods (Wright et al., 2009). Robust capuchins also have a more pronounced sexual dimorphism in cranial characters compared to gracile capuchins (Silva, 2001). Robust capuchin males have a sagittal crest that is lacking in gracile male capuchins (Silva, 2001). Byron (2009) compared C. apella with gracile capuchins and found that C. apella had a significantly more robust mandibular and temporal fossa morphology. Jungers and Fleagle (1980) showed body proportion differences in early development, in C. albifrons and in S. apella with divergent growth trajectories of limb length as a function of body mass. C. albifrons is longerlimbed in proportion to body mass than S. apella throughout development with this difference increasing as growth progresses. The first problem with these morphological characters is that they could exist between species and they are not necessarily only existing between genera. One magnificent counterexample is that of two wild cats, the puma and the jaguarundi. Traditionally, they were considered two species belonging to two distant genera (Puma concolor and Herpailurus yagouaroundi) because, for instance, their sizes and weights are extremely different (head and body length is 505-645 mm and weight 4-9 kg for the jaguarundi and head and body length is 860-1,540 mm and weight 30-120 kg for the puma). However, molecular analyses (Johnson et al., 2006) showed that, despite the vast differences in size, the ancestors of both diverged around 4-5 MYA and now there is agreement among all the cat specialist to consider these species as part of the same genus (Puma concolor and Puma yagouaroundi). This example (which is absolutely opposite to the philosophy of Lynch-Alfaro et al., 2012b) raises a second question. The major part of these morphological characters do not provide any value from a phylogenetic perspective because they respond to quick adaptative and positive natural selection. There are many examples of this. Masterson (2001) reported that S. nigritus and S. robustus were both significantly larger than, and had significantly larger dental arcades, increased prognathism, and larger in absolute cranium size compared to S. libidinosus. This difference is so important especially in regards to S. cay, with adult crania that are so small, they look like the juvenile ones in S. nigritus and S. robustus. This shows that adaptation via natural selection occurred quickly and also operated within the robust capuchin group. Cáceres et al., (2014) performed an interesting series of geometric morphometric analyses of skull shape (23 homologous landmarks were digitized to describe skull shape) in 228 capuchin monkey individuals belonging to seven taxa of robust capuchins and two taxa of gracile capuchins representing 94 localities in South America. They regressed skull shape against latitude, longitude, skull size and

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environmental variables. The main results of this work clearly showed that many craniometrics characteristics could be not used as phylogenetic characters because they are affected by convergent natural selection by similar environmental pressures. This suggests that there is no clear separation between the two alleged capuchin genera, and overlap occurred in the central area of the RW1/2 plot between species of both Cebus (C. olivaceus and C. albifrons) and Sapajus. The shape variance associated with RW2 described variation in the skull width and zygomatic arch position. Along this axis S. nigritus occupied extreme negative values whereas S. apella had extreme positive values. This shows that there were more differences between two taxa of Sapajus than between these Sapajus taxa with Cebus taxa. A second result showed Hotelling’s pairwise comparisons indicated that C. olivaceus and S. apella were more related to each other and different from many other capuchin taxa in terms of skull shape. A third result showed that some tests resulted in significant differences between S. macrocephalus and S. xanthosternos as compared to S. cay and S. libidinosus. S. macrocephalus and S. xanthosternos were the species with the largest skulls, although they are not directly related as we show with molecular phylogenetic analyses. This is further proof that there is no validity of using size as a phylogenetic tool to separate Cebus and Sapajus into two different genera. A fourth result is related to skull allometry. No slope differences were recorded between genera (P = 0.230) and between species (P = 0.083). Thus, skull allometry has no significant phylogenetic signal. A fifth result showed that latitude has a significant impact on skull shape. Longitude always explained a smaller percentage of shape variance than latitude and its impact was non-significant. Specimens at southern latitudes were generally characterized by more elongated rostrum, and long and narrow skulls and tooth rows. The relative distance between the zygomatic arch and the rostrum tip was also proportionally small. Specimens collected near the equator had relatively shorter muzzles and wider skulls Species distributed in localities with low precipitation, low minimum temperature, and high seasonality exhibited skulls with more elongated muzzles, narrow skulls at the zygomatic arch and relatively larger teeth (S. nigritus and S. cay). Conversely, both C. albifrons, C. olivaceus, S. apella and S. macrocephalus showed positive scores in vector SW1 shape and had more robust skulls, smaller teeth, enlarged neurocrania and wide zygoma. This suggested that both alleged genera vary in skull shape along the same environmental gradient in an identical mode. As explained by Cáceres et al., (2014), during their Pleistocene radiation, new species within the robust capuchin clade moved to the north, and started to resemble (in skull shape) to the Cebus capuchins when they occupied the Amazon rain forest. The pool of Amazonian species includes two gracile capuchins and two robust capuchins. Although differences in skull shape between these species are significant, they all share a wide skull and a narrow muzzle. Interestingly, we find differences not only in skull shape but also in skull size between S. macrocephalus and S. apella, suggesting that these two taxa are morphologically distinct, in spite of the very small genetic distances between them (Ruiz-García et al., 2012b and this work). The same occurred with the two gracile capuchin taxa. It’s apparent in the relative warp and PLS plots. C. albifrons is characterized by a relatively more elongated rostrum than C. olivaceus. Also the genetic distances between C. albifrons and C. olivaceus are very limited

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil (even C. olivaceus is nested within C. albifrons as it was showed in Ruiz-García et al., 2010 and here). In summary, partial least squares between bioclimatic variables and skull shape explained some 98% of the covariation between environment and shape. Species distributed in drier, more seasonal southern localities exhibit a narrow skull with elongated muzzle and relatively larger teeth. Variation partitioning suggests that the difference in skull shape between species is highly correlated with climatic variation but not with skull size. As robust capuchin taxa moved to the north, they adapted to the local environmental conditions, eventually resembling gracile capuchins in skull shape as they reached the Amazon rain forest, in response to their shared environmental conditions. All of these examples indicate that morphometric data are not helpful in determining phylogenetic relationships and systematic differences within capuchin monkeys. Lynch-Alfaro et al., (2012b) claimed that in terms of cranial morphology, there are no characters that allow a researcher to have confidence in determining among Pithecia, Chiropotes, or Cacajao without also having known geographic origins of all samples. Conversely, several cranial characteristics lead to the easy differentiation between the gracile and robust capuchins even for a skull of unknown provenance that lacks any associated pelage data. This argument is misleading and does not bring forth evidence in favor of two different genera of capuchins monkeys. Obviously, natural selection pressures have affected the robust capuchins in their rapid expansion during the late Pleistocene. These varied ecological conditions during colonization are very different to the more homogeneous ecological conditions (and therefore more homogeneous natural selection pressures) in which evolved some genera as those they cited. For instance, we can perfectly discriminate an Asian human skull from human skulls in other areas of the world (Dirkmaat, 2012; Turner, 1976; Wade, 2014), but this doesn’t mean that the Asian humans are from another genus than the rest of the humanity. In another example, no one confounds a skull of a Neanderthal from that of a current human (Homo neanderthalensis and H. sapiens, or, H. s. neanderthalensis and H. s. sapiens; Bilsborough, 1972; Finlayson et al., 2006; Finlayson and Carrión, 2007; Howells, 1974; Jelinek, 1976) because the qualitative and quantitative differences are striking. Yet, no one classifies these human forms in different genera. What is really important is the information we can obtain from neutral or quasi neutral areas of the genome and not from the flexible characters easily affected by adaptation natural selection. Furthermore, we ignore the genetics basis of many of these morphological differences and how much is the phenotypical plasticity of these characters. C. Lynch-Alfaro et al., (2012b) also presented a list of some ecological and some behavioral differences between gracile and robust capuchins to justify them as different genera. Gracile capuchins have larger group sizes and larger home ranges and they are found at lower densities than the robust taxa when the two types are found in sympatry (Terborgh, 1983). Haugaasen and Peres (2005, 2009) found S. macrocephalus as a true habitat generalist, with high population density in all kinds of forest, whilst C. albifrons was found at a lower density than S. macrocephalus in terra firme forest, and rarely observed in the flooded forests, except at the height of the fruiting season. The difference in population density in the two species was considered a result of the heavy reliance of C. albifrons on widely dispersed fruits

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such as figs. S. apella has a more generalized use of forest and edge habitats whilst C. olivaceus prefers high terra firme rainforest, avoiding forest edge in Surinam (Mittermeier and Van Roosmalen, 1981). Territoriality and range behavior also appear different between the two capuchin groups in areas of sympatry. In Manu National Park (Peru), S. macrocephalus groups primarily used a core area, with a relatively small home range, large overlap between groups, and peaceful interactions across groups. In contrast, C. albifrons groups aggressively defended a much larger, nearly exclusive territory and traveled longer distances, spending days in a particular part of the home range until the food sources were exhausted, and then moving on (Terborgh, 1983). Other difference between Cebus and Sapajus is in stone tool use in the wild. It would appear to be ubiquitous across robust capuchin populations in dry habitats (S. flavius, S. libidinosus, S. xanthosternos; Emidio and Ferreira, 2012), but it has not been recorded in other long term studies of Sapajus in rainforest conditions (S. nigritus, S. apella and S. macrocephalus). This can be explained in part because the alluvial floodplain of the central Amazon has no stones available for tool use (Spironello, 1991). However, these differences in ecological traits and behaviors related to ecological characteristics are not related to different genera. These differences could occur between species. Another clear counter-example is again related to wild cats. The ecology and behavior of lions (Panthera leo; for instance a social species) are extremely different to that of the leopards (Panthera pardus; for instance a solitary species) and yet there are no research claims that they form different genera (Ewer, 1973). D. Another list of alleged differences to split Cebus and Sapajus by Lynch-Alfaro et al., (2012b) was that related with courtship, mating, and postcopulatory display. They claimed that Sapajus displays the richest repertoire of sexual and courtship behaviors ever described for nonhuman primates (Fragaszy et al., 2004). In contrast, C. capucinus females in Costa Rica exhibits little proceptive behavior but instead tended to look and behave the same during fertile and nonfertile periods (Carnegie et al., 2005). Identically, C. albifrons females in Ecuador never showed the extended proceptivity displays commonly observed in the robust capuchin taxa (Matthews, 2012). Additionally, in courtship, Sapajus males and females raise their brows up and back and “grin,” pulling their lips back to expose their teeth in a grimace, whilst the gracile capuchins instead protrude their lips in a “duck face,” which continues throughout copulation as well (Matthews, 2012; Perry, 2008). This extreme difference in reproductive signaling suggests reproductive isolation following LynchAlfaro et al., (2012b). However, as in the previous cases, the existence of reproductive isolation is basically one of the properties of what is a species more than what is a genus. They employed typical arguments to differentiate species by using BSC to differentiate genera. Furthermore, the existence of a wide range of sexual and courtship behaviors in the robust capuchins, as well as the use of tools, does not mean they are different genera from Cebus. There are even very different sexual and courtship behaviors in our own species (also submitted to very different environmental conditions in our natural history as in the robust capuchins), yet we don’t claim the existence of different species or genera for humans. Also, no one claims that variability in human sexual behavior is the reason we classify ourselves as a different genus from other Primates.

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil E. Curiously, Lynch-Alfaro et al., (2012b) practically mention nothing about the karyotype evolution of the capuchin monkeys. They literally cited the following: “….. The karyotypes of Cebus and Sapajus differ in fundamental number and diploid number, but the data at present do not allow for a coherent evolutionary analysis because of the blanket use of “C. apella” to describe the samples from the robust group…” As we will show later, there is a notable number of karyological results of the capuchin monkeys, which together the molecular results, allow us to reconstruct the evolutionary history of this group of monkeys. In fact, Nieves et al., (2011) demonstrated that karyological results agree with the capuchin monkeys as a single unit. They employed a heterochromatin probe for chromosome 11 of C. libidinosus (11qHe+ CLI probe), obtained by chromosome microdissection. When FISH experiments were analyzed, in all the capuchin monkeys that they employed, they found six to 22 positive signals among them (both gracile and robust capuchins) located in interstitial and telomeric positions. No hybridization signals were observed when the 11qHe+ probe was applied to over 14 other specimens of Ceboidea (Alouatta, Aotus, Ateles, Saimiri, Callithrix, Callicebus, Leontopithecus and Pithecia). Therefore, this FISH study confirmed the existence of a genus-specific extracentromeric heterochromatin in the capuchin monkeys (both gracile and robust), suggesting that this heterochromatin is the same but with a different amplification pattern among the different taxa of capuchin monkeys. This means that basically the karyological studies agree more with a unique genus of capuchin monkeys than with two genera.

Figure 4. Dendrogram of the phylogenetic relationships of the current Neotropical primate genera extracted from Schneider and Sampaio (2015) with modifications. The figure shows that if Cebus and Sapajus are split, then other genera as Saguinus, Aotus, Alouatta, Ateles and Callicebus should also be split.

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Another interesting question, with practical consequences, is that if we assume the existence of two genera of capuchin monkeys, then, other well resolved Neotropical primate genera must be divided into new genera as well. If we look at a modification of the phylogenetic tree of Schneider and Sampaio (2015) (Figure 4), it’s clear that if Cebus and Sapajus are split, consequently, other genera as Saguinus, Aotus, Alouatta, Ateles, Callicebus and even Callithrix must also be divided into more (new) genera. Additionally, it seems logical that an indiscriminate application of PSC could generate an unmanageably large number of species and even of genera. We would be negligent to forget the great number of problems and confusion caused by using primitive typological classifications in the past (the case of the 86 supposed species of brown bears in North America alone following Merriam, 1918, to only cite one example). The primatologists who follow the PSC, upgrade all the primate subspecies to species, or, even, species to genera (as is the present case) but we consider that this is only moving the problem up one level as it obscures the reality of a real evolutionary unit. The BSC should not be ignored just because for some taxa it is not easily translated into an operational definition. However, in the current case, there are many results on molecular and karyological traits and reproductive natural events to apply the BSC for the capuchin monkeys. Indeed, many researchers, which currently employ molecular markers, however, are still using a typological view of the evolution because they did not understand the basic fundamentals of the Neodarwinism synthesis (Ayala, 1975, 1994a,b, 1995; Dobzhansky, 1937, 1970, 1973; Ford, 1976; Huxley, 1942; Mayr, 1942, 1963, 1977, 1978, 1992; Rensch, 1947, 1950; Simpson, 1944, 1953; Stebbins, 1950; Wright, 1968, 1969, 1977, 1978). For all of these reasons, and from this moment on, we just use the term Cebus for all taxa of capuchin monkeys in this work.

The Evolution of the Cebus Genus The Evolution of the Gracile Capuchins: They Began the History of the Current Capuchins Our analyses showed two possible beginnings for the initial evolution of the capuchin monkeys: 1- The original evolution of the capuchin monkeys began in the area of the Orinoco and Negro River (Orinoco and Northern Amazon; C. albifrons) which quickly generated the first wave of C. capucinus (Colombian III haplogroup) towards what is currently northwestern Colombia, or 2- The oldest capuchin monkey haplotypes were those of the Colombian III C. capucinus haplogroup, which in turn originated the first C. albifrons haplotypes in the Orinoco and Negro River. This molecular result is extremely noteworthy and agrees quite well with the karyological results of the genus Cebus, a fact which was not taken into account by Lynch-Alfaro et al., (2012a, b). Dutrillaux et al., (1986) showed that the ancestral karyotype for Cebus was C. capucinus with 54 chromosomes with 8-9 autosomic pairs being meta or sub-metacentric and with polymorphism in chromosome 19. The C. albifrons’s karyotype is very similar to that C. capucinus, which again agrees quite well with our molecular results to not differentiate C. capucinus and C. albifrons as two clear and well differentiated taxa. A few individuals of both taxa showed 52 chromosomes (Egozcue and Egozcue, 1966; Koiffmann and Saldanha, 1974). C. olivaceus shows 52 chromosomes (with the fusion of chromosomes 10 and 27 of C. capucinus). C. apella also shows 54 chromosomes (as in C. capucinus and many C. albifrons) with 10 autosome pairs as meta or

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sub-metacentric. Three chromosome pairs are different in C. apella relative to C. capucinus (chromosomes 6, 13 and 23). Torres de Caballero et al., (1976) and Amaral et al., (2008) analyzed the interspecies chromosomal comparisons among the capuchin monkey, by using G- and Q-banding patterns. They suggested that C. capucinus, C. albifrons and C. apella share 19 chromosome pairs, C. capucinus and C. albifrons share 25 pairs and C. capucinus and C. apella share 20 pairs. C. capucinus most resembles the putative ancestor, as all chromosomes found in C. capucinus are observed in C. albifrons and C. apella. Both, C. albifrons and C. apella, seem to have been derived from an ancestor with a karyotype similar to C. capucinus. The C. capucinus karyotype is clearly closer to C. albifrons than to C. apella. Figure 5, from Amaral et al., (2008), clearly shows this perception. C. capucinus occupied a more basal position, with a chromosomal composition very similar to the putative ancestral Platyrrhini karyotype, following Richard et al., (1996). The phylogenetic relationships of C. capucinus and C. albifrons, in relation to the ancestral karyotype, are not clearly defined. However, it is clear that the karyotype of some C. albifrons differ from that of some C. capucinus by a pericentric inversion in the 14/15 association, which results in a metacentric association 15/14/15/14. Amaral et al., (2008) identified a tandem fusion, followed by a pericentric inversion involving the homologous human chromosomes HSA15b and HSA8b, in the C. albifrons they studied. Sex chromosomes were similar in all the species of gracile and robust capuchin monkeys, with submetacentric X chromosome and a small acrocentric Y chromosome (Amaral et al., 2008). Within other genera of Neotropical primates, the karyotype diversity is considerably higher than in Cebus (for instance, Aotus, Callicebus, Alouatta, Koiffmann, 1982). Therefore, as it was summarized by De Oliveira et al., (2012), the oldest, and the putative ancestral Neotropical Primate karyotype (2n = 54) is from C. capucinus. The C. apella karyotype can be derived from this by an inversion 14/15/14 (García et al., 2000). It’s not very differentiated from the ancestral karyotype, and therefore provides additional evidence against Sapajus as a differentiated genus from Cebus. Each species-specific fusion explains the reduction in diploid number to 52 in some C. albifrons (12/15) and C. olivaceus (8/15/8), respectively (Amaral et al., 2008).

Figure 5. Chromosomal cladogram showing the relationships among different gracile and robust capuchin species. The tree is from the studies of Amaral et al., (2008) and Neusser et al., (2001).

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The temporal divergence of these first C. capucinus-C. albifrons haplotypes occurred during the beginning of the Pliocene (5-4 MYA). Several important facts occurred in this epoch. For example, the last uplift of the Northern Colombian Andes happened 3-5 MYA reaching its current elevation of 4,000-6,000 meters above sea level (Andriessen et al., 1994; Gregory-Wodzicki, 2000; Van der Hammen, 1995; Van der Hammen et al., 1973). Such an event could be important for the initial fragmentation of the initial C. capucinus-C. albifrons haplotypes. Other larger Amazon changes could have also influenced the initial fragmentation of the original capuchins. Espurt et al., (2007, 2010) demonstrated that the Nazca Ridge subduction imprint had a significant influence on the eastern side of the Andes by means of the Fitzcarrald Arch. This uplift is responsible for the atypical three-dimensional shape of the Amazonian foreland basin. Related to the Nazca Ridge subduction, arc volcanism in the Peruvian Andes ceased around 4 MYA (Rosenbaum et al., 2005). Thus the Fitzcarrald Arch uplift is not older than 4 MYA. Indeed, this arch defines three drainage basins: northern Amazon rivers, eastern Amazon rivers, and southern Amazonian rivers (Madre de Dios River basin and Mamore-Beni River basin). Also at the beginning of the Pliocene (5 MYA), the sea level rose 100 m for a duration of 0.8 MY (Haq et al., 1987). Thus, marine transgressions could have also had a great influence on diversification of these initial capuchin taxa. Later, and independent of either origin hypothesis, a wide array of C. albifrons haplotypes was generated in the Western Amazon which, in turn, generated all the remaining groups of capuchin monkeys. The first major split in the gracile capuchins was detected by Bobli et al., (2012) and they dated this event to around 2.5 MYA (group A in their Figure 2) through the analysis of five specimens (one from Peru, two from Ecuador and two from Brazil). Clearly, the affirmation of Nascimento et al., (2015) about the necessity to sample a larger number of specimens, localities and even other genes to be confident with the origins and evolution of the genus Cebus, is verified here. An artifact of the sampling design of Lynch-Alfaro et al., (2012a), is not being able to detect the oldest diversification process. Therefore, they concluded that the gracile capuchins appear to have radiated rapidly from an albifrons-like ancestor in the Amazon. Additionally, they concluded that dispersal of C. capucinus into Central America (≈1.9 MYA) occurred later than that of Alouatta and Ateles (Collins and Dubach, 2000a,b; Cortés-Ortiz et al., 2003). Based on this chronology, the capuchins were in Central America long after the completion of the Isthmus of Panama (2.8-3.5 MYA) (Nores, 2004; Coates and Obando, 1996). Apparently, this corresponds well with predictions by Ford (2006), who suggested that C. capucinus arrived in Central America in a ‘second wave’ of primate introductions around 2 MYA. However, our data, with many more exemplars from many additional geographical points, do not agree with this hypothesis. The splits between the Colombian C. capucinus haplogroups and the Central American capuchins oscillated from 4.87 to 3.7 MYA. If we take the temporal split between the Colombian II C. capucinus haplogroup and the Central American one as the most probable (3.7 MYA), this coincides with the formation of the Panama land bridge. Or, this coincides with the formation of the Chocó-Panamá island bridge slightly earlier (Galvis, 1980). This bridge could have been used by the ancestors of the current C. capucinus to colonize Central America from Northern South America. During the upper Pliocene orogeny, the present Tuira, Atrato, and Sinú basins as well as nearby lowlands were raised above sea level. Thus, the mountains of Southern Central America and the northern Andes were uplifted to about their present elevation (Van der Hammen, 1961). Even if the divergence splits were around 4.5–5 MYA, the Nicaraguan, Panamanian and Colombian portals remained open (late

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Miocene-Middle Pliocene). Numerous volcanic islands existed from the lower Atrato Valley and the Tuira Basin of Eastern Panama to the Nicaraguan portal. These could have been used by the C. capucinus to migrate northward. The Cuchillo Bridge of the Urabá region, connecting the Tertiary Western Colombian Andes with the Panamanian islands was probably above sea level during this period. Simpson (1950, 1965) claimed that many mammals were ‘‘island hoppers,” and capuchin monkeys are extremely good colonizers. However, for central and Northern Panama, Ruiz-García and Castillo (2016) showed animals from the Colombian II C. capucinus haplogroup and the Central American C. capucinus haplogroup mixed in the same troops with no morphological differences among them. This demonstrates reproductive cohesiveness, which is proof against the claims of Bobli et al., (2012) that the Central American capuchin is another differentiated species of gracile capuchin, C. imitator. We detected at least, five or even six large migration events, and probably many smaller migration events, (Bobli et al., 2012 only detected two of these migration events) related with the Western Amazon C. albifrons array, around 3.7-2.5 MYA (late Pliocene-beginning of Pleistocene): A. One migration towards northern South America generated two or three different groups: 1- One generated a large fraction of the C. albifrons taxa in some areas of the Colombian Amazon and Central and Northern Colombia: C. a. albifrons from Vaupes, C. a. versicolor, C. a. cesarae, C. a. pleei, and the C. capucinus of the Colombian I haplogroup; 2- A second one generated the C. capucinus individuals from the Colombian II haplogroup and 3- directly from a C. albifrons’s wave or from the Colombian II C. capucinus haplogroup appeared the Central American C. capucinus haplogroup as we above explained. B. Another migration towards Northern South America generated C. a. adustus-C. a. leucocephalus and the very limited geographically speaking C. capucinus lineage that we detected in Buenaventura. C. Another migration from the Western Amazon array generated the Pacific Ecuadorian C. albifrons population (C. a. aequatorialis). Our MJN estimation showed that the C. a. aequatorialis haplotype (H7) diverged from an important Western Ecuadorian Amazon C. albifrons haplotype (H65) around 780,000 ± 112,600 YA. This Pacific C. albifrons in Ecuador is intriguing because of the elevation of the Central and Northern Andes 2 MYA. For instance, the Andean chain between Cajamarca and Huancavelica in Peru appeared by volcanism around 2 MYA and the temporal split between Amazon Ecuadorian C. albifrons haplotypes and those from the Pacific Ecuadorian areas is more recent. Certainly, some authors, for instance HernándezCamacho and Cooper (1976) and Ruiz-García et al., (2006), mentioned the existence of a zoological passage east of the eastern Andes cordillera into the upper Magdalena valley in Colombia. Several species of Primates such as Cebus apella, Lagothrix lagotricha lugens, Ateles belzebuth and Saimiri sciureus albigena, as well as other vertebrate species, have used this passage. Maybe a similar passage existed in the Ecuadorian Andes at some point in the past. However, there is another possibility. The current Pacific Ecuadorian C. albifrons population could be related to C. capucinus through the Pacific Coast from the north. Another alternative hypothesis could focus on the fact that this population was created by human action in more recent times.

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D. Another migration route existed from the current Brazilian Amazon Acre going northeast towards Northern South America, generating C. olivaceus. This migration route was also detected by Bobli et al., (2012). They named a group (B) consisting of two parts (B.1 and B.2). B.1 contained two individuals, one from Barcelos on the right bank of the Negro River and the other from the Curanja River, upper Purus River in the Department of Loreto in Peru near the Brazilian border. B.2 contained individuals from the Guiana Shield forming two clades, one clustering the C. albifrons from Negro River and Orinoco, and the other clustering different subspecies of C. olivaceus (except C. o. brunneus of the Venezuela coast). Our results ratified this finding of Bobli et al., (2012), but our results also showed that the mixing of haplotype lineages or migrations are extremely more complex in the area of the Negro River and Orinoco than they detected. E. Finally, there was a migration of C. albifrons haplotypes going east towards South America causing the emergence of the robust capuchins. As we did not study many C. olivaceus specimens or C. kaapori specimens, we don’t know if the first ancestral robust capuchins haplotypes were generated directly from more ancestral C. albifrons haplotypes or if intermediate haplotypes were generated. For instance, ancestors of C. kaapori generated the ancestral haplotypes that generated the origins of the current C. xanthosternos, which is the first differentiated group of the robust capuchins. It’s critical to study a good number of C. kaapori and C. xanthosternos individuals from a molecular perspective to detect shared, or very similar, haplotypes. If the ancestral haplotypes that generated the current C. xantosthernos were not themselves directly generated by ancestral C. albifrons or C. kaapori haplotypes then they should have also generated from C. olivaceus haplotypes. In Figure 5, we can observe that, from a karyotypic perspective, C. olivaceus is closely related to C. apella (apella, cay and robustus), with these species sharing the chromosomal inversion homologous to HSA20. Differentiation between C. olivaceus and C. apella is possible via a pericentric inversion in the association 14/15/14 and a Robertsonian rearrangement in the chromosomes homologous to HSA12 and HSA15b. Related to this hypothesis, Ford and Hobbs (1996) presented preliminary results that C. olivaceus is the gracile capuchin most similar to the robust capuchins in postcranial morphology. However, this morphological similarity does not necessarily show phylogenetic relationships as we discuss in different parts of this work. Also, von Dornum and Ruvolo (1999) showed at the nuclear G6PD locus that C. olivaceus was more related with C. apella than with the clade of C. albifrons-C. capucinus. Independent of these three hypotheses (the ancestors of C. xantosternos were derived from the ancestors of the current C. albifrons, C. kaapori or C. olivaceus), authors have shown that the Amazon and the Atlantic forests were connected for three million years (6 MYA until 3 MYA) allowing the migration of the gracile capuchins towards the Atlantic forests. The palynological studies of Oliveira et al., (1999) showed this fact. Schneider and Sampaio (2015) claimed that the split between the Amazon and the Atlantic Callicebus (C. moloch and C. personatus, respectively) and the divergence between the howler monkey species (Alouatta seniculus and A. fusca) occurred during this period. Similarly, Nascimento et al., (2008) showed different populations of red-handed howler monkeys (Alouatta belzebul) from both forests. This suggests that two morphoclimatic domains with

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Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil predominantly open vegetation biomes (Caatinga and Cerrado) split these populations around 3 MYA (Nascimento et al., 2008).

There is an intriguing question not revealed by previous studies on this topic (the existence of C. albifrons haplotypes within the robust capuchins cluster). There is a Western Amazon C. albifrons haplotype that is the sister clade of all the C. apella haplotypes. Also, there are five Western Amazon individuals of C. albifrons (Napo River area in Ecuador and Peru) mixed with the individuals of C. a. macrocephalus (also Western Amazon). Additionally, there are four Western Amazon individuals of C. albifrons (three from the Napo River area in Ecuador and one from the Colombian Amazon), two Orinoco C. albifrons individuals and the unique specimen analyzed of C. a. malitosus mixed with all the individuals of the C. a. fatuellus (Colombian Amazon and Orinoco). Different hypotheses have come forward from these results. The first one is that this represents a case of incomplete lineage sorting. This phenomenon is due to that some lineages may occur in more than one taxon due to failure of two or more lineages in a population to coalesce in the ancestral population of each species (Ballard and Whitlock, 2004; Degnan and Rosenberg, 2009). Indeed, Nascimento et al., (2015) indicated polyphyletic arrangements for several capuchin taxa, suggesting that incomplete lineage sorting has occurred. If so, this could fundamentally support that the oldest gracile capuchin genetic diversity originated the current robust capuchin gene diversity. Furthermore, this process is relatively recent (Ballard and Whitlock, 2004; if not, incomplete lineage sorting would have disappeared), which is a relevant point against Sapajus as a different genus from Cebus. Taking this into account, different Western C. albifrons directly contributed to the generation of the robust capuchin haplotypes. This does not necessarily mean that C. olivaceus or C. kaapori haplotypes contributed to this fact (only an intensive genetics sampling of the robust capuchins in the Brazilian Atlantic forest could reveal this). Related with this, and if we take only into account the phylogenetic trees, these Western Amazon C. albifrons haplotypes (especially those from the Ecuadorian and the Peruvian Napo area) could be in the origin of the capuchins. However, the MJN analysis showed that these Western Amazon C. albifrons haplotypes were derived for other more ancestral gracile capuchin haplotypes as those from the Orinoco and Negro River and C. capucinus haplogroup III. Networks are more appropriate for intraspecific or very related species phylogenies (as it is the current case) than tree algorithms because they explicitly allow for the coexistence of ancestral and descendant haplotypes, whereas trees treat all sequences as terminal taxa (Posada and Crandall, 2001). A second possibility is that after the robust capuchins returned to the Amazon (LynchAlfaro et al., 2012a), they experienced historic introgression with individuals of C. albifrons. In this case, all the introgression events detected should be between C. apella females and C. albifrons males. The descended hybrids bred over generations with C. albifrons because no C. albifrons individuals presented any morphological character of C. apella. In favor of this hypothesis we note the clade of C. a. macrocephalus which included C. albifrons from the most western area of the Amazon where both species sympatrically live (Ecuador). There were also two C. albifrons, within the C. a. fatuellus clade, also from the Colombian Eastern Llanos where both species live sympatrically. If this second hypothesis is true, we would also expect to detect robust capuchin individuals within the main gracile capuchin clade (robust

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capuchin males x gracile capuchin females). However, we did not detect any such case. Thus, we are inclined to consider the first hypothesis as the most probably of the two. The fact that unique C. a. malitosus individuals were enclosed in the C. apella fatuellus clade together with one Colombian Amazon C. albifrons could be interpreted as supporting the two previous hypotheses. If the first hypothesis was valid, then, C. a. malitosus could represent a sixth migration from the Western Amazon’s C. albifrons array to what is currently Northern Colombia. This is different from the other migrations which generated Northern Colombia’s C. albifrons populations. If the second hypothesis is assured, then C. a. malitosus could be interpreted as a linage formed by the hybridization of C. albifrons and C. apella in the Orinoco (Colombian-Venezuelan Llanos). Later, it migrated to the most northern Colombian areas. The very dark pelage of this taxon could support this last hypothesis. Before discussing the evolution of the robust capuchins, we will comment about the most recent splits in the Central and Northern Colombian C. albifrons. The main splits of the Vaupes C. albifrons population, adustus-leucocephalus, versicolor, cesarae and pleei populations, which have traditionally been considered different subspecies, occurred between 0.4 and 1.1 MYA, during the Pleistocene. The mitochondrial diversification within some of these populations was estimated to have occurred around 0.8-0.13 MYA also during the Pleistocene. The Pleistocene is a fundamental epoch for the proliferation of haplotypes for Cebus and many other species. Shackleton and Opdyke (1973) and Bowen (1978) showed that the Pleistocene is characterized by glacial and inter-glacial cycles of around 100,000 Y due to the eccentricity of the Earth’s orbit, with around 90,000 years of cold temperatures (glacial) and 10,000 years of warm temperatures (inter-glacial). Hays et al., (1976) also demonstrated that changing the Earth’s axis from 22.1 ° to 24.5°, every 41,000 years, and its precession over 22,000 years have some minor influence on the glacial and inter-glacial Pleistocene cycles. Mercer (1984) detected glaciations in the Argentinian Patagonia since 3.5 MYA. Caviedes and Paskoff (1975) and Laugenie (1982) detected, at least, three large glaciations in the last 2 MY in Chile. Clapperton (1981) detected glacial deposits around 3.27 MYA near to La Paz (Bolivia). On the other hand, Bowen (1978) detected at least seven glacial and inter-glacial cycles with glaciations in the last 700,000 Y in the Britain Islands. In Colombia, Hooghiemstra (1984), analyzed palynological and lacustrine sediments and determined 27 large climatic cycles with a periodicity of 100,000 years, although there are only proofs, for Colombia, of the last large glaciation (Van der Hammen et al., 1983; Helmens, 1988). These alternations of cold-dry and warm-humid cycles suggest the possible existence of Plio-Pleistocene refugia following the hypothesis of Haffer (1969, 1997, 2008), provoking population contractions and expansions, which, in turn, could enable the apparition of multiple lineages of gracile capuchins in the Northern Amazon and Northern Colombia. This has had profound consequences, not only in the capuchins, but also in most of the South American fauna (Costa, 2003; Diniz-Filho et al., 2002; Hawkins and Diniz-Filho, 2006; Melo et al., 2009).

The Evolution of the Robust Capuchins: Their Origins in the Atlantic Forests to Again Return to the Amazon It’s clear that C. xanthosternos is the current robust capuchin most closely related to the original robust capuchins which derived from the gracile capuchins. Our results agree extremely well with Nascimiento et al., (2015) and Ruiz-García et al., (2012b), who also detected C. xanthosternos as the first divergent robust capuchin clade. These results also are

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against the point of view of Lynch-Alfaro et al., (2012a), who consider that C. nigritus cucullatus to be the basal divergent taxon of the robust capuchins. Unfortunately, they came to this conclusion because they discarded a divergent sequence of C. xanthosternos (sample 49) because it was downloaded from GenBank of unknown provenance, and they considered a possible captive hybrid or an error resulting from contamination. With the MJN procedure, our split between the ancestral C. albifrons haplotype and C. xanthosternos was around 3.3 MYA during the last phase of the Pliocene. In the case of C. xanthosternos, the striking phenotypic differences of this taxon as described by Rylands et al., (2005), and being restricted to a determined geographical area, agree quite well with the fact that it should be the first divergent species within the robust clade. Seuánez et al., (1986) also determined some differences at the chromosomal level between C. xanthosternos and other lineages of C. apella. C. flavia (also known as C. queirozi; Mendes et al., 2006) is another first robust species candidate to diverge. It would be interesting to analyze it molecularly, since it is a new species of the robust capuchin complex and has been recently rediscovered (Oliveira and Langguth, 2006) from the Pernambuco endemism center (Brazilian states of Alagoas, Pernambuco, Paraíba and Rio Grande do Norte – north of the Sao Francisco River). However, we suggest C. xanthosternos as the best candidate of the current live robust capuchin forms to be most closely related with the original C. capucinus-C. albifrons group. Although C. xanthosternos belongs to the tufted robust group, it is an exception because it lacks tufts. Also, with age, some C. albifrons females (untufted capuchins) tend to develop a certain bushiness on the face and head, which in some cases includes tufts (Hill, 1960; Napier and Napier, 1967). This should be a phenotype link between both gracile and robust capuchin forms. In a parallel study (Ruiz-García et al., 2016a), a Bayesian analysis revealed a temporal divergence between the gracile capuchin clade and the robust + gracile capuchin clade of around 5.52 MYA. The initial diversification in the first clade occurred around 5 MYA. This was followed by the initial diversification in the second clade around 3.68 MYA which split of the ancestors of C. xanthosternos. These temporal splits happened during the last phase of the Miocene and basically during the Pliocene. Casado et al., (2010) estimated the split between these two clades at 4.2 MYA, whilst they estimated the split between C. a. apella and C. a. cay to have occurred around 2.6 MYA (with a Bayesian procedure we obtained a split of 2.42 MYA, but with the MJN the split was around 0.3 MYA). This work together with those from Casado et al., (2010) and Ruiz-García et al., (2016a) detected the split between the gracile and the robust forms of Cebus to have occurred during the Pliocene. Lynch-Alfaro et al., (2012a) estimated an older temporal split between both forms (6.3 MYA; 95% highest posterior density: 4.1–9.4 MYA), to have happened during the Miocene. Nascimento et al., (2015) speculated that the Atlantic Forest was the original place of capuchin ancestors, in agreement with the hypothesis of the African origin of Neotropical primates (Schrago and Russo, 2003). Those capuchin ancestors reached the Atlantic coast of South America and subsequently dispersed to internal regions of this continent. However, this speculation is highly questionable because in the best cases the estimated split between both gracile and robust capuchins occurred 6-7 MYA. Compare this to the first recovered Neotropical primate fossil (Branisella boliviensis) dated to 27 MYA (Feagle, 2002; Takai et al., 2000) and the split between the South American and African Primates estimated at 35 MYA (Harrison, 1987) to 43.9 MYA (Hodgson et al., 2009). Thus, that the ancestral Neotropical primates reached the current Atlantic South American coasts is irrelevant to the

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split of the ancestral capuchin forms. The other alternative speculation of Nascimento et al., (2015) is more probable. They claimed that the ancestral robust capuchins may have originated in the western or central Amazon Forest and expanded their range to the Atlantic Forest, leading to a subsequent and complete speciation in the Amazon Forest. This also led to eliminating the basal robust capuchin form from this location. Then, Cebus xanthosternos represented a remnant distribution of a more widespread basal lineage. As we have commented, this is exactly what our results support. Another interesting difference between this work and that the Lynch-Alfaro et al., (2012a) is that they estimated the robust crown group age, to be around 2.7 MY. This is more than a half a million years older than the gracile crown group (2.1 MY). Additionally, they detected this first split of the gracile capuchin radiation between Amazon Cebus and Venezuelan Coastal/Central American Cebus. Clearly our data are more robust (includes many more individuals and localities and a higher number of genes sequenced) than the work of Lynch-Alfaro et al., (2012a). We also detected more ancestral C. capucinus-C. albifrons haplotypes which show that several hypotheses of LynchAlfaro et al., (2012a) could be refuted or, at least, to be transformed as we show below. Indeed, Lynch-Alfaro et al., (2012a) argued that the temporal split of around 6-7 MYA for both gracile and robust capuchins is similar to that between the genera Lagothrix and Brachyteles (8.0 MYA, Barroso et al., 1997; 9.62 MYA, Meireles et al., 1999; 10.5 MYA, Schrago, 2007). Per se, these temporal comparisons are not very useful to distinguish two different genera in the capuchin monkeys. First, the value of the temporal split between Lagothrix and Brachyteles is considerably higher than the temporal split for the two clades of capuchin monkeys. This is true if it’s compared with the estimate of Lynch-Alfaro et al., (2012a), but especially with our estimate of around 3-4 MYA. Second, several temporal splits between species within genera (and no one has claimed to divide these genera) are similar to the temporal split between the two capuchin clades. For example, consider the cases of Ateles geoffroyi and A. paniscus (4.36 MYA; Meireles et al., 1999) or Callicebus torquatus and C. moloch (5.5 MYA; Schneider et al., 1993). Third, they even argued that some genera of Neotropical primates are older than the split between Sapajus and Cebus. They used the example of Alouatta, where the split between the two main clades (cis- and trans-Andean) was around 6.8 MYA (Cortés-Ortiz et al., 2003), but arguing that for Alouatta there is no large morphological gap/distinction between species. There has been more continuous splitting and speciation through time in Alouatta. Furthermore, ecological niches are more similar across Alouatta, and the species are generally allopatric. However, in our view, Lynch-Alfaro et al., (2012b) failed to mention the main and more determinant point: the presence, or absence of reproductive cohesiveness, which implies the existence or not of pre and/or post reproductive isolation mechanisms. For instance, there would be more morphological homogeneity between the different Alouatta taxa than in the capuchin monkeys, but the extreme and conspicuous karyological differences among many Alouatta taxa show that there is reproductive isolation between many of them, whereas the reproductive cohesiveness is more certain in many capuchin taxa because there are not these conspicuous karyological differences. After the divergence of the ancestors of C. xanthosternos, the next taxon to diverge was C. nigritus (although we did not analyze all the different morphotypes classified within C. nigritus). Mudry et al., (1991) showed that, although this taxon has relatively more similar karyotypes to other robust capuchin taxa than C. xanthosternos did, it has different G banding for chromosome 11 compared to other C. apella populations. Additionally, the C banding in

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this group showed the loss of the large heterochromatic block on chromosome pair 11 present in other C. apella lineages. This taxon also showed eight small and eight large acrocentric chromosomes which disagrees with that determined for other robust capuchin taxa that have seven small and nine large acrocentric chromosomes (Freitas and Seuánez, 1982; Mantecón et al., 1984; Matayoshi et al., 1986). Furthermore, chromosomes 17 and 20 showed inconsistent heterochromatic blocks, which are absent in other C. apella taxa, as well as showing three of the small acrocentrics which correspond to pairs 22 and 23 and show a secondary constriction not present in other forms of C. apella. Indeed, Nieves et al., (2011) used a comparative genome hybridization analysis between C. a. cay and C. nigritus from Argentina where the DNA imbalances involved different genome regions. They were preferentially repetitive in C. a. cay or either coding or very disperse in C. nigritus. Their finding modified the accepted idea that the heterochromatin proportion is the only difference between C. a. cay and C. nigritus. These chromosomal characteristics of C. nigritus clearly contrasted with some of the chromosomal characteristics of C. a. cay found by Seuánez et al., (1983) and Matayoshi et al., (1986). Still, their geographical distribution could overlap in some areas of Eastern Paraguay and Northeastern Argentina. Indeed, Fantini et al., (2011) analyzed the genome of a female fertile hybrid between C. libidinosus (for us, C. a. cay) and C. nigritus using interspecies comparative genomic hybridization (iCGH). Although both taxa have a highly homologous karyotype and can interbreed, they detected different genome sizes supporting the species status for both taxa. The remaining robust capuchin taxa (cay, macrocephalus, fatuellus, apella and one individual of robustus) showed very small genetic distances (average genetic distance of 0.4%) and a very recent diversification process (in the last 0.1-0.3 MYA). This rapid process of diversification was also detected by Lynch-Alfaro et al., (2012a), who estimated that the invasion of the robust capuchins from the Atlantic forest into the Cerrado and the Amazon occurred around 0.4 MYA. Also, Ruiz-García et al., (2012b), detected the split of all the apella populations from C. xanthosternos and C. nigritus to have occurred around 0.3 MYA. Indeed, all apella taxa showed very similar karyotype composition. Nieves et al., (2011) showed chromosome 11 to be the largest acrocentric pair. This chromosome has a heterochromatic block of 75% for the q arm. It is shared by all the robust capuchins, with the exception of C. xanthosternos and C. nigritus. Amaral et al., (2008) showed that C. a. cay presented a very similar karyotype to that of C. a. robustus, both having the same 12 synteny blocks with reference to humans and 18 identical conserved segments with regard to Saguinus oedipus. Additionally, the cladogram of Amaral et al., (2008) supported the monophylia of apella, cay and robustus. They shared two synapomorphic traits, the association 14/15/14, resulting in a submetacentric chromosome, and a pericentric inversion that corresponds to the HSA8b probe. All of these taxa show a migration of the robust clade starting from the Brazilian Atlantic coast and going towards the west—crossing the dry areas of Southern Brazil, Paraguay and Northern Argentina to arrive to the Amazon of Bolivia, Peru, Ecuador, Colombia, Brazil, Venezuela and the area of Guianas. This confirms the point of view of Lynch-Alfaro et al., (2012a) that the robust clade migrated towards the Amazon from the Brazilian Atlantic forest in a very rapid and explosive radiation. This fact explains why the climatic variation seems to have influenced skull shape in robust capuchins to a larger extent than in gracile capuchins. The main reason for this might be that the gracile capuchin ancestor lived in the Amazon rain Forest, which has existed for over 50 million years, ever since the Eocene (Hoorn et al.,

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2010). It is a comparatively more stable biome than others in the Neotropics during the Pleistocene (Mayle, 2004). This agrees quite well with the idea that robust capuchins experienced far more habitat fragmentation than the Amazon gracile capuchins during the Pleistocene. Therefore, this dynamic of strong habitat change and fragmentation outside of the Amazon may have spurred more morphological changes in the robust capuchins than in the gracile ones. Related with this, Casado et al., (2010) showed that C. cay populations suffered a recent demographic expansion during the Pleistocene, a period of repeated glaciation events leading to drastic changes in the vegetation composition of different biomes. This extreme explosive radiation generated many morphotypes highly adapted to the extremely different environments where these robust capuchins colonized (from the dry environment of the Paraguayan Chaco to the Amazon rain forest). But, molecularly and karyologically, these populations are extremely similar and, most likely, no reproductive barriers have been generated. The diversification within cay, macrocephalus, apella and fatuellus occurred in the last phase of the Pleistocene. This period had extreme climate changes. From 128,000 YA to 116,000 YA, there was an inter-glacial cycle. Since then, the last glacial period began and, for instance, in Colombia, the glaciers began to form around 70,000 YA (Wijmstra and Van der Hammen, 1974). Van der Hammen (1985) showed that the large glacier extensions were around 35,000 YA in this country. Later some reduction occurred and then about 25,000 YA the glaciers again extended. Although it was very cold, there was a reduction of the ice in Colombia from 21,000 to 14,000 YA. In contrast, in the Northern Hemisphere the maximum extension of the glaciers occurred around 18,000 YA. Also, the humid conditions in the area of what would become Colombia were insufficient for the advancement of the glaciers. For the last glacial phase, the temperature was 7 C° less than what it is today for the savannah of Bogota (Van der Hammen, 1985). Thus, these rapid and heavy climatic changes could influence the rapid morphological changes in C. apella but with very slight molecular and karyological changes without generating reproductive isolation mechanisms. Lynch-Alfaro et al., (2012a) concluded that “Reinvasion of the Amazon” was the most feasible of the three hypotheses. They claimed that their data strongly supported the predictions of this hypothesis and give no support to the other hypotheses (out of the Amazon or Atlantic versus Amazon). Following these authors, their data upheld all three predictions of the first hypothesis: 1-the initial divergence in capuchin monkeys was between the robust Atlantic Forest capuchins and the gracile Amazon capuchins. They suggested that this process was initiated from vicariance caused by the establishment of the Amazon River around 7 MYA (Hoorn et al., 2010). The ancestral Cebus populations were restricted to the Guiana Shield, whereas the ancestral Sapajus populations were restricted to the Brazilian Shield. 2The robust Amazon capuchins formed a recently evolved subclade nested within the Atlantic Forest robust clade. 3- Robust Amazon capuchins dispersed at a significantly higher rate than either gracile or Atlantic robust capuchins. They concluded that the current wide-ranging sympatry of Cebus and Sapajus across much of the Amazon is best explained by a PliocenePleistocene diversification (2 MYA) of gracile capuchins in the Amazon. It is also explained by a much more recent expansion (0.7 MYA) of the Atlantic Forest S. cay into the southern Cerrado. This was followed by a single explosive Pleistocene invasion (0.4 MYA) of S. apella/macrocephalus across the Amazon, where the robust capuchins are now sympatric with, or have displaced, gracile capuchins across a large portion of their range.

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The current results clearly confirmed the second and third predictions of the “Reinvasion of the Amazon” hypothesis of Lynch-Alfaro et al., (2012a), but not the first prediction. Clearly, the mitochondrial evolution of the gracile capuchins started before the mitochondrial evolution of the robust capuchins. The robust capuchins are derived from some Amazon C. albifrons lineages and this is especially observable in the MNJ analysis. Unfortunately, this fact was never taken into account by Lynch-Alfaro et al., (2012a). Really, the evolution of the robust capuchins is nested within the evolution of the gracile capuchins. Therefore, the first prediction of the “Out of the Amazon” hypothesis (Amazon Basin is the ancestral origin of the capuchin monkeys), or a modification of it, is true. The original capuchin monkeys were gracile and they appeared north of the Amazon River (in the area of the Negro River/Orinoco, or in the northern Pacific area of current Colombia) around 1.5-2 MYA before the robust capuchins appeared in the Atlantic forest area. Later, the second and third predictions of the “Reinvasion of the Amazon” hypothesis of Lynch-Alfaro et al., (2012a) occurred. This new modified hypothesis is called by us the “North Amazon gracile origin, Eastern expansion and subsequent reinvasion of the Amazon.”

The Necessity of a New Systematics for the Cebus Genus We believe that the existence of different definitions of species is conceptually interesting and practically useful. However, there are situations where some species definitions are clearly more acceptable than others. For example, a researcher could be studying a sexual organism and only has limited molecular data, not related to reproductive incompatibility. Perhaps the researcher has some morphological or morphometric data too (not related to sexual characters and thus not considering reproductive isolation). If he/she has no any other information (relevant karyological differences, existence of pre and/or post-zygote reproductive barriers of whatever nature, clear geographic or ecological barriers which might cause allopatric speciation, etc, etc), from his/her desk in an office or in a laboratory, we think that it’s licit to apply the PSC. Nevertheless, if a researcher could obtain (or there are previous studies) some results on the existence of direct, or indirect, reproductive data, the utilization of the BSC is clearly a preferable option over the PSC. In the last few decades, many primatologists, systematics and researchers of other biological sciences have “forgotten” the extreme importance of reproductive data in current sexual organisms, which is directly related with an intuitive idea of what a species is (Ayala, 1994a, b). We recommend to use the BSC over the PSC when direct and indirect reproductive data are presented. Herein, we will apply the BSC to the question of the systematics of Cebus. Different C. capucinus haplogroups evolved in parallel from different C. albifrons groups (and/or vice versa) in the same areas of the Pacific and Northern Colombia, as well as in Central America. There were environmental selection pressures affecting the external morphology of these different lineages. They were helped by an intense gene flow among these original different groups. This hypothesis could very well be possible because in our species (Homo sapiens), this has been clearly documented. The action of natural selection on skin pigmentation reveals itself in several ways. For instance, note the relationship among UV radiation, vitamin D and skin melanin content (Neer, 1975). The geographical areas with high incidence of UV radiation contain human populations with dark or very dark skin color. In this way the incidence of cancer skin is considerably lower (and reproductive fitness is

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higher). There is a higher protection of folic acid and vitamin D amount is enough to build bones with adequate levels of calcium. When humans left Africa and they colonized northern areas (Northern Europe and Northern Asia), the individuals with lighter skin were positively selected because they could more readily absorb small amounts of UV radiation in these latitudes and, therefore, form bones not suffering from rickets (Neer, 1975). It’s easy to observe that European and northern Asians have lighter skin. This has been independently achieved by convergent evolution. In the Europeans, genes were positively selected to lighten the skin. There are around 30 different alleles in Europeans and only a fixed allele found in Africans at the MC1R locus and one allele at the SLC24A5 locus. Those selected in the Europeans were different to those selected in Northern Asians (allele EDARV370A, which also affects other characters such as the hair shape) (Fujimoto et al., 2008; Kamberov et al., 2013; Lamason et al., 2005; Parra, 2007). Thus similar environmental conditions could activate different gene variability to produce similar phenotypes (convergent evolution). Similar convergent effects on color, sizes and morphotypes could have occurred in many other Primates. Therefore, if this hypothesis is supported, the morphological similarity among different groups is the product of convergent natural selection. This would indicate that the traditional color pelage characteristics relied on by primatologists to carry out their systematic classification of many primate species should be not used. They are not good characters for phylogenetic tasks. Another probable hypothesis is that the original C. capucinus lineage (Colombian III haplogroup) was that which originally populated the entire geographical area of Northern Colombia. Then, at least, three C. albifrons’ waves arrived and mixed without any problem with the C. capucinus residents (the group of Vaupes C. a. albifrons lineage-C. a. versicolor-C. a. cesarae-C. a. pleei; some direct Amazon haplotypes; and the C. a. adustus-leucocephalus group) generating the other detected C. capucinus haplogroups. Our results should reflect the introgression from males of C. capucinus to females of C. albifrons. Consider the hybrid we analyzed coming from the Bolivar Department. The specimen was physically more related to C. capucinus but it had the mitochondrial DNA of the versicolor-cesarae-pleei clade. In a second example, we found a different hybrid individual coming from the Sucre Department. It should represent a case of C. a. versicolor’s male introgression into Colombian III C. capucinus haplogroup females. The specimen was more physically related to C. capucinus but it had recognizable C. albifrons characteristics and mitochondrial DNA of the Colombian III C. capucinus haplogroup. Probably, the hybrids basically inherited dominant pelage characteristics (black versus clear colors) from C. capucinus. No matter which of the two hypotheses there were no reproductive barriers. As we previously commented, the karyotypes of C. capucinus and C. albifrons are very similar. Additionally, we showed in Ruiz-García and Castillo (2016), that these different C. capucinus haplogroups were highly affected by gene flow when nuclear DNA microsatellites were analyzed. Even Ruiz-García et al., (2016a) demonstrated that microsatellites results did not discriminate between C. capucinus and some C. albifrons groups. Thus, cohesive reproductive integrity is demonstrable among all of these lineages as we showed with the two hybrid exemplars directly sampled in the wild. Previously, other authors (Hernández-Camacho and Cooper, 1976; Defler, 2010) claimed the existence of hybrids between these two gracile capuchin monkey taxa. Some hybrid animals were obtained in the Barranquilla market decades ago. Their reported origin was the Middle San Jorge Valley. They had intermediate features including a high dark crown, a white bald area on the forehead, and lighter fur on the outside of the arms

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and shoulders (all the features suggesting the predominant pattern of C. capucinus). Very likely, in the Magangue market, there were some hybrid individuals from the Lower Cauca River showing similar characteristics. The exception to this would be the lack of a very high, dark coloration. Additionally, many social conventions such as hand sniffing, sucking body parts, and inserting fingers into another individual’s mouth have been described in C. capucinus as well as in C. albifrons (Defler, 2010), but not in C. apella. The description of vocalizations for C. albifrons by Defler (1979) matches closely to that of C. capucinus. In contrast, Di Bitetti (2002) described several quite different vocalizations for C. nigritus. Indeed, Hernández-Camacho and Cooper (1976) hinted that both, C. capucinus and C. albifrons, should be conspecifics. For all of these reasons, we claim that all the gracile capuchins we studied conformed a unique species or super-species: C. capucinus because it is the oldest of the names for this taxon (C. capucinus, Linnaeus in 1758; C. albifrons, Humboldt in 1812; C. olivaceus, Schomburgk in 1848). We only analyzed one sample of C. olivaceus being placed within the Amazon C. albifrons’s haplotypes. The results of Bobli et al., (2012; see Figure 1), showed several samples of C. olivaceus (C. o. castaneus, C. o. apiculatus, C. o. nigrivittatus and C. o. olivaceus) more related to haplotypes of C. a. albifrons, C. a. unicolor or C. a. yuracus than to C. o. brunneus. The last one was more related to C. albifrons taxa such as C. a. adustus and C. a. leucocephalus. This also indicates that C. olivaceus should be enclosed within C. capucinus. However, our results could not be applied to C. kaapori, because we did not study any samples of that taxon. Thus, additional molecular analyses should be carried out to determine if C. kaapori needs be included within C. capucinus or if it should be considered an individual species. Following our molecular results, we cannot agree with some claims of Bobli et al., (2012). As we previously demonstrated (Ruiz-García et al., 2010, 2012a), they concluded that there is no molecular evidence for subspecific distinction of C. c. limitaneus in Guatemala and Honduras or C. c. imitator in Costa Rica. However, they concluded that the Central America C. capucinus should be differentiated as a new species, C. imitator. However, as we commented above, the new mitochondrial data of Ruiz-García and Castillo (2016) showed that haplotypes of the Colombian II haplogroup and Central American haplogroup were mixed in animals within the same troops. There were with no morphological differences in central and northern Panama and the nuclear DNA microsatellite results showed limited differences among Colombian, Costa Rican and Guatemalan individuals of C. capucinus. Thus, it does not seem realistic to describe, as did Boubli et al., (2012), the Central America capuchins as a new species. The systematics of the different groups of the traditional C. albifrons (if they are not integrated in the superspecies, C. capucinus) from northern Colombia and Venezuela and the central and western Amazon is complex. Following the current molecular results and trying to correlate them with the traditional nomenclature rules and using the previous names from Hershkovitz (1949), Hernández-Camacho and Cooper (1976), Defler and HernándezCamacho (2002) and Defler (2010), we conclude the following: 1- C. a. malitosus (Elliot, 1909) should be maintained although more specimens must be sequenced to strictly determine this taxon; 2- All of the previous taxa named as cesarae (Hershkovitz, 1949), pleei (Hershkovitz, 1949), versicolor (Pucheran, 1845) and albifrons (Humboldt, 1812; herein unicolor was enclosed within albifrons following Defler and Hernández-Camacho, 2002) were intermixed throughout different clades. Thus, we named all of them as C. a. albifrons; 3- C. a. leucocephalus (leucocephalus is an older name than adustus, Gray, 1865 vs.

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Hershkovitz, 1949, respectively) proved to be a monophyletic group and 4- In the Amazon, the exemplars “a priori” classified as albifrons (and unicolor), cuscinus (Thomas, 1901), yuracus (Hershkovitz, 1949) and unspecified subspecies (Hershkovitz, 1949) were intermixed. We name all of them as C. a. albifrons. The question of C. a. aequatorialis and C. a. trinitatus is pending upon the molecular analysis of more samples because our preliminary results did not satisfactory resolve the question. Thus, we tentatively claim the existence of three taxa within C. albifrons: C. a. malitosus, C. a. leucocephalus and C. a. albifrons. Nevertheless, we consider it prudent to consider a unique species, C. capucinus with different lineages intermixed. Or, consider geographical populations with certain main characteristics (ESUs, Moritz, 1994, or other unities that do not necessarily correspond with color coat characteristics), where the traditional typological view of systematics cannot be easily (and really not desirable) applied. In fact, as Fontdevila and Moya (2003) explained, evolutionary biologists should make an effort to rule out the application of a typological view. The gracile capuchin case should be similar to that of human beings. We don’t apply the traditional systematic nomenclature rules for our own species. For all of these reasons, we discard typological views of Bobli et al., (2012), who analyzed a limited number of mitochondrial genes from 50 Cebus individuals and supported the existence of 12 species of gracile capuchins. As we previously commented many of the haplotypes of these alleged taxa are intermixed and these authors never took into account the reproductive cohesiveness of all these forms. This demonstrates how the PSC can obscure our understanding of evolutionary phenomena and the speciation process. In the case of the robust species, with the current molecular data we have, three species should be defined, although some populations and morphotypes have not been not studied by us: C. xanthosternos, C. nigritus and C. apella. The considerations we used to define the first two species were explained above. Our considerations for the third species are as follows. Our molecular analysis included specimens classified “a priori” as different species by morphological characteristics and by geographical origins. Following Groves (2001, 2005), they were: C. apella apella, C. a. fatuellus, C. a. macrocephalus, C. a. peruanus, C. libidinosus pallidus, C. l. paraguayanus, C. l. juruanus and C. nigritus robustus. Following Silva Jr (2001) they were C. apella, C. macrocephalus, C. libidinosus, C. cay and C. robustus. Our molecular results showed that C. a. macrocephalus, C. a. peruanus, C. libidinosus pallidus, C. l. juruanus and C. nigritus robustus (following the nomenclature of Groves, 2001, 2005) and C. macrocephalus, C. libidinosus and C. robustus (following the nomenclature of Silva Jr, 2001) conformed a unique and extremely homogeneous molecular clade we named macrocephalus. Other well defined clusters were cay, apella and fatuellus. However, our results as well as those of Casado et al., (2010) and Ruiz-García et al., (2012b), support limited gene differentiation between these taxa. For instance, macrocephalus-cay showed 0.2% of genetic differentiation, meanwhile macrocephalus-apella showed 0.7% of genetic differentiation and fatuellus-cay also showed the same degree of genetic divergence (0.7%). These values of genetic differentiation are considerably lower than the values obtained for different species within other Neotropical primate genera. Some examples are those from the genetic differentiation between Callicebus lugens and C. torquatus (4.0%; Casado et al., 2007), between C. nigrifrons and C. personatus (6.0%; Bonvicino et al., 2003), between Alouatta pigra and A. palliata (4.0%; Nascimento et al., 2005), between Saguinus midas and S. niger (3.4%; Cropp et al., 1999) or between S. bicolor and S. martinsi (4.1%; Cropp et al., 1999). Similarly, and due to the low level of genetic divergence, Casado et al.,

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(2010) found a lack of definition in different phylogenetic trees for solving the relationship between cay and apella. If we use the BSC, undoubtedly, these robust capuchin taxa should be considered subspecies of a single species, C. apella. Indeed, Ruiz-García et al., (2016b) showed that the DNA microsatellite differences between two “a priori” different species of robust capuchins (Groves 2001, 2005), C. libidinosus pallidus (Bolivia) and C. macrocephalus (Peru and Western Brazilian Amazon) are extremely limited. This agrees with the current mitochondrial data where both robust capuchin taxa are undifferentiated. The highly differentiated morphotypes of these C. apella taxa generated in the last 0.2-0.4 MYA were submitted to very different climatic and ecological environments (due to different natural selection pressures). The explosive radiation is similar to what occurred in our own species (Homo sapiens) endured. Homo sapiens left Africa approximately 0.2-0.1 MYA (Cavalli-Sforza, 1997; Goldstein et al., 1995; Horai et al., 1995; Tishkoff et al., 1996; Vigilant et al., 1991) and was also submitted to very different climatic and ecological environments. This generated the noteworthy phenotypical diversity which we can see in our species. However, no serious scientific claims have stated that the current and different human morphotypes are different species. The molecular differences within our species are extremely limited and the reproductive integrity is a fact (BSC). We propose that the same rules that we apply to ourselves will be applied to the other Primates. Many authors are applying the PSC, in an indiscriminate way, to many different organismal taxa, including mammals and primates. Nevertheless, the large-scale application of this concept has originated many ‘‘crazy’’ new species that are clearly not sustained by a critical analysis. This was named by Isaac et al., (2004) as “taxonomic inflation.” Zachos et al., (2013) suggests that some of the proposed new mammal species are completely unjustifiable (new species of tigers as Panthera sumatrae and P. sondaica, Cracraft et al., 1998; Mazak and Groves, 2006; 12 new species of red deer, Cervus elaphus, Groves and Grubb, 2011; 11 new species of the small rocky antelope, Oreotragus oreotragus or new species of hares, Lepus, Palacios et al., 2008). We agree with Nascimiento et al., (2015) that many cases of paraphyletic or polyphyletic arrangements can be explained by incomplete lineage sorting, as we previously argued. But this means that the split among the groups occurred recently as well (and/or) as the gene flow between these groups. This agrees quite well with the absence of reproductive barriers, which, in turn, is essential for the BSC. Also, we agree with the claim of Nascimiento et al., (2015) that any accurate phylogenetic reconstruction should include sequence data of multiple unlinked nuclear loci in several individuals of any given species. But we must recall that the discrimination power of the nuclear sequences is much lower than that of the mitochondrial DNA as we commented in the introduction for the infra-generic and specific levels. Collins and Dubach (2001) reconstructed the phylogeny of Ateles by means of two mitochondrial genes. However, they did not fully reconstruct it with a nuclear gene. In a similar fashion, Cortés-Ortiz et al., (2003) accurately reconstructed the phylogeny of Alouatta with mitochondrial genes but it was not possible with nuclear gene sequences. Funk and Omland (2003) also suggested that polyphyletic and paraphyletic arrangements might be artifacts resulting from misidentified specimens and species limits, consequently leading to inappropriate taxonomy (McKay and Zink, 2010). However, this was not the case of the current work because all the samples were obtained from wild individuals inhabiting their natural geographic areas.

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What is clear is that we increased the number of individuals and geographical areas sampled and relied on a higher number of mitochondrial genes, than in previous studies. We showed that there is no reciprocal monophylia between gracile and robust capuchins and that the evolution of the gracile capuchins preceded that of the robust capuchins. Furthermore, the first was in the origin of the second, which was not detected in previous studies. Finally, we agree with Zachos (2015) who stated that species are such fundamental units that they should not be introduced carelessly and that species description and splitting based on simple morphometric differences (even significant ones) or phylogenetic relationships derived from very limited molecular datasets should be strongly discouraged. They may serve to support conclusions derived from larger and more complete datasets, but are not enough on their own.

ACKNOWLEDGMENTS Thanks to Dr. Diana Alvarez, Pablo Escobar-Armel, Nicolás Lichilín, Luisa CastellanosMora, Fernando Nassar, Hugo Gálvez, Alan Velarde, Dr. Luís Albuja, Armando Castellanos and Andrés Laguna for their respective help in obtaining wild capuchin monkey samples during the last 20 years. Thanks go also to Dr. Benoit de Thoisy, who provided five samples from French Guiana and to Dr. Alcides Pissinatti, who provided the two samples of C. xanthosternos. Thanks to Instituto von Humboldt (Villa de Leyva; Janeth Muñoz), SDA (Bogotá; Norberto Leguizamón), Peruvian Ministry of Environment, to the PRODUCE (Dirección Nacional de Extracción y Procesamiento Pesquero from Perú), Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales, to the Colección Boliviana de Fauna (Dr. Julieta Vargas), to CITES Bolivia (permissions 01482, 01483, 01737, 01738, 01739, 01740, 01741) and to the Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador) for their role in facilitating the obtainment of the collection permits in Colombia, Peru, Bolivia and Ecuador. Also many thanks to the Brazilian institutions for collaborating with this study (IBAMA). All animal sampling in French Guiana was carried out in accordance with French animal care regulations and laws. The first author also thanks the help of many people of diverse Indian tribes in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana and Alamas), Bolivia (Sirionó, Canichana, Cayubaba and Chacobo), Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna), Brazil (Marubos, Matis, Mayoruna, Kanaimari, Kulina, Maku and Waimiri-Atroari) and Ecuador (Kichwa, Huaorani, Shuar and Achuar).

REFERENCES Andriessen, P. A. M., Helmens, K. F., Hooghiemstra, H., Riezebos, P. A., Van der Hammen, T. (1994). Absolute chronology of the Pliocene-Quaternary sediment sequence of the Bogotá area, Colombia. Quaternary Science Reviews 12: 483-501. Antonovics, J. (2006). Evolution in closely adjacent populations X: long term persistence of pre-reproductive isolation at a mine boundary. Heredity 97: 33-37.

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Ascunce, M. S., Hasson, E., Mudry, M. D. (2003). COII: a useful tool for inferring phylogenetic relationships among New World monkeys (Primates, Platyrrhini). Zoologica Scripta 32: 397-406. Ashley, M. V., Vaughn, T. A. (1995). Owl monkeys (Aotus) are highly divergent in mitochondrial cytochrome c oxidase (COII) sequences. International Journal of Primatology 5: 793–807. Amaral, P. J. S., Finotelo, L. M. F., De Oliveira, E. H. C., Pissinatti, A., Nagamachi, C. Y., Pieczarka, J. C. (2008). Phylogenetic studies of the genus Cebus (Cebidae-Primates) using chromosome painting and G-banding. BMC Evolutionary Biology 8: 169. Ayala, F. J. (1975). Genetic differentiation during the speciation process. Evolutionary Biology 8: 1-78. Ayala, F. J. (1994a). La naturaleza inacabada. Salvat Editores. Barcelona. Ayala, F. J. (1994b). Teoría de la evolución. Ediciones Temas de Hoy. Madrid. Ayala, F. J. (1995). Origen y evolución del hombre. Alianza Editorial. Madrid. Baker, R. J., Bradley, R. D. (2006). Speciation in mammals and the genetic species concept. Journal of Mammalogy 87: 643–662. Ballard, J. W., Whitlock, M. C. (2004). The incomplete natural history of mitochondria. Molecular Ecology 13: 729–744. Bandelt, H-J., Forster, P., Rohl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16: 37-48. Barroso, C. M. L., Schneider, H., Schneider, M. P. C., Sampaio, I., Harada, M. L., Czelusniak, J., Goodman, M. (1997). Update on the phylogenetic systematics of New World monkeys: further DNA evidence for placing the pygmy marmoset (Cebuella) within the genus Callithrix. International Journal of Primatology 18: 651–674. Barton, N., Bengtsson, B. O. (1986). The barrier to genetic exchange between hybridizing populations. Heredity 57: 573-576. Bensasson, D., Zhang, D-X., Hartl, D. L., Hewitt, G. M. (2001). Mitochondrial pseudogenes: evolution’s misplaced witnesses. Trends Ecology and Evolution 16: 314–321. Bilsborough, A. (1972). Cranial morphology of the Neanderthal Man. Nature 237: 351-352. Bonvicino, C. R., Penna-Firme, V., do Nascimento, F. F., Lemos, B., Stanyon, R., Seuánez, H. N. (2003). The lowest diploid number (2n = 16) yet found in any primates: Callicebus lugens (Humboldt, 1881). Folia Primatologica 74:141–149. Boubli, J. P., Rylands, A. B., Farias, I. P., Alfaro, M. E., Lynch-Alfaro, J. W. (2012). Cebus phylogenetic relationships: A preliminary reassessment of the diversity of the untufted capuchin monkeys. American Journal of Primatology 74: 381-393. Bowen, D. Q. (1978). Quaternary geology. A stratigraphic framework for multidisciplinary work. Pergamon Press. Pp. 1-221. Byron, C. D. (2009). Cranial suture morphology and its relationship to diet in Cebus. Journal of Human Evolution 57:649–655. Cabrera, A. (1957). Catálogo de los mamíferos de América del Sur. I (Metatheria, Unguiculata, Carnívora). Revista del Museo Argentino de Ciencias Naturales “Bernardo Rivadavia,” Zoología 4:1-307. Cabrera, A., Yepes, J. (1940). Historia Natural Ediar. Mamíferos Sud-Americanos. Compañía Argentina de Editores, Buenos Aires, Argentina.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

257

Cáceres, N., Meloro, C., Carotenuto, F., Passaro, F., Sponchiado, J., Melo, G. L., and Raia, P. (2014). Ecogeographical variation in skull shape of capuchin monkeys. Journal of Biogeography 41: 501.512. Carnegie, S. D., Fedigan, L. M., Ziegler, T. E. (2005). Behavioral indicators of ovarian phase in white-faced capuchins (Cebus capucinus). American Journal of Primatologica 67: 51– 68. Casado, F., Bonvicino, C. R., Seuánez, H. N. (2007). Phylogeographic analysis of Callicebus lugens (Platyrrhini, Primates). Journal of Heredity 98: 88–92. Casado, F., Bonvicino, C. R., Nagle, C., Comas, B., Manzur, T. D., Lahoz, M. M., Seuánez, H. N. (2010). Mitochondrial divergence between 2 populations of the Hooded Capuchin, Cebus (Sapajus) cay (Platyrrhini, Primates). Journal of Heredity 101: 261–269. Cavalli-Sforza, L. L. (1997). Genes, peoples and languages. Proceedings of the National Academy of Sciences USA 94: 7719-7724. Caviedes, C. N., Paskoff, R. (1975). Quaternary glaciations in the Andes of north-central Chilean Journal of Glaciology 14: 155-170. Clapperton, Ch. M. (1981). Quaternary glaciation in the Cordillera Blanca, Perú and the Cordillera Real, Bolivia. Rev. CIAF 6: 93-111. Coates, A. G., Obando, J. A. (1996). The geologic evolution of the Central American isthmus. In: Jackson, J. B. C., Budd, A. F., Coates, A. G., (Eds.). Evolution and environment in tropical America. (pp. 21-56)Chicago: The University of Chicago Press. Collins, A. C., Dubach, J. M. (2000a). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology 21: 381– 420. Collins, A. C., Dubach, J. M. (2000b) Biogeographic and ecological forces responsible for speciation in Ateles. International Journal of Primatology 21: 421-444. Collins, A. C., Dubach, J. M. (2001). Nuclear DNA variation in spider monkeys (Ateles). Molecular Phylogenetics and Evolution 19: 67–75. Cortés-Ortiz, L., Bermingham, E., Rico, C., Rodriguez-Luna, E., Sampaio, I., Ruiz-García, M. (2003). Molecular systematics and biogeography of the Neotropical monkey genus Alouatta. Molecular Phylogenetics and Evolution 26: 64–81. Costa, L. P. (2003). The historical bridge between the Amazon and the Atlantic forest of Brazil: a study of molecular phylogeography with small mammals. Journal of Biogeography 30: 71–86. Coyne, J., Mah, K., Cristianshen, A. (1994). Genetics of a pheromonal difference contributing to reproductive isolation in Drosophila. Science 265: 1461-1464. Cracraft, J. (1983). Species concepts and speciation analysis. In Johnston RJ (Ed.). Current ornithology. Vol I. p. 159-187. Plenum Press, New York. Cracraft, J., Feinstein, J., Vaughn, J., Helm-Bychowski, K. (1998). Sorting out tigers (Panthera tigris): mitochondrial sequences, nuclear inserts, systematics, and conservation genetics. Animal Conservation 1: 139–150. Cropp, S. J., Larson, A., Cheverud, J. M. (1999). Historical biogeography of tamarins, Saguinus: the molecular phylogenetic evidence. American Journal of Physical Anthropology 108:65–89. Defler, T. R. (1979). On the ecology and behavior of Cebus albifrons in eastern Colombia: I. Ecology. Primates 20:475–490. Defler, T. R. (2003). Primates de Colombia. Colombia, Bogotá: Conservación Internacional.

258

Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil

Defler, T. R. (2010). Historia natural de los primates colombianos. Editorial Universidad Nacional de Colombia. Pp. 1-609. Defler, T. R., Hernández-Camacho, J. I. (2002). The true identity and characteristics of Simia albifrons Humboldt, 1812: description of neotype. Neotropical Primates 10: 49-64. De Oliveira, E. H. C., Neusser, M., Müller, S. (2012). Chromosome Evolution in New World Monkeys (Platyrrhini). Cytogenettic and Genome Research 137: 259-272. De Oliveira, P. E., Barreto, A. M. F., Suguio, K. (1999). Late Pleistocene/Holocene climatic and vegetational history of the Brazilian caatinga: the fossil dunes of the middle Sao Francisco River. Palaeogeography, Palaeoclimatology, Palaeoecology 152: 319–337. Degnan, J. H., Rosenberg, N. A. (2009). Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends in Ecology and Evolution 24: 332–340. Di Bitetti, M. S. (2002). Food-associated calls in the tufted capuchin monkey (Cebus apella). Dissertation Abstr Int B62:3883. Diniz-Filho, J. A. F., de Sant’Ana, C. E. R., de Souza, M. C., Rangel, T. F. L. V. B. (2002) Null models and spatial patterns of species richness in South American birds of prey. Ecology Letters 5: 47–55. Dirkmaat, D. (2012). A companion to forensic anthropology. Wiley-Blackwell. Pp. 1-752. Dobzhansky, Th. (1937). Genetics and the origin of species. Columbia University Press, New York. Dobzhansky, Th. (1970). Genetics of the evolutionary process. Columbia University Press, New York. Dobzhansky, Th. (1973). Nothing in Biology Makes Sense Except in the Light of Evolution. American Biology Teacher 35: 125–129. Dutrillaux, B., Couturier, J., Viegas-Pequinot, E. (1986). Evolution chromosomique des Plathyrriniens. Mammalia 50: 56-81. Egozcue, J., Egozcue, M. V. de. (1966). The chromosomes of Cebus capucinus. Mamm. Achrom. Newsl. 20: 71-72. Elliot, D. G. (1913). A Review of Primates. Monograph Series, American Museum of Natural History, New York. Emidio, R. A., Ferreira, R. G. (2012). Tool use by capuchin monkeys in the Brazilian Caatinga: variation in energy gain by season and by habitat type. American Journal of Primatology 74: 332-343. Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W., Regard, V., Antoine, P. O., Salas-Gismondi, R., Bolanos, R. (2007). How does the Nazca Ridge subduction influence the modern Amazonian foreland basin? Geology 35: 515–518. Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W., Barbarand, J. (2010). The Nazca Ridge and uplift of the Fitzcarrald Arch: implications for regional geology in northern South America. In: Hoorn, C., Wesselingh, F. (Eds.). Amazonia, Landscape and Species Evolution: A Look into the Past. Wiley-Blackwell, Oxford, Pp. 89-102. Ewer, R. F. (1973). The Carnivores. Cornell University Press, New York. Pp. 1-500. Fantini, L., Mudry, M. D., Nieves, M. (2011). Genome size of two Cebus species (primates: Platyrrhini) with a fertile hybrid and their quantitative genomic differences. Cytogenetic and Genome Research 135: 33–41. Feagle, J. (2002). Early platyrrhines of southern South America. In Tejedor, M. (Ed.). The Primate Fossil Record. Cambridge University Press. Pp. 1-170.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

259

Finlayson, C., Giles Pacheco, F., Rodríguez-Vidal, J., Fa, D. A., Gutierrez López, J. M. et al. (2006). Late survival of Neanderthals at the southernmost extreme of Europe. Nature 443: 850–853. Finlayson, C., Carrión, J. S. (2007). Rapid ecological turnover and its impact on Neanderthal and other human populations. Trends in Ecology and Evolution 22: 213–222. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299. Fontdevila, A., Moya, A. (2003). Evolución: Origen, adaptación y divergencia de las especies. Editorial Síntesis, S. A. Madrid, Spain. Pp. 1-591. Ford E. B. (1976). Genetics and adaptation. Institute of Biology studies, Edward Arnold, London. Ford, S. M. (2006). The biogeographic history of Mesoamerican primates. In Estrada, A., Garber, P. A., Pavelka, M., and Luecke, L. (Eds.). New perspectives in the study of Mesoamerican primates. (pp. 81-114). Springer, New York. Ford, S. M., Corruccini, R. S. (1985). Intraspecific, interspecific, metabolic, and phylogenetic scaling in platyrrhine primates. In Jungers, W. L. (Ed.). Size and scaling in primate biology. (pp. 401-435). New York: Plenum Press. Ford, S. M., Hobbs, D. G. (1996). Species definition and differentiation as seen in the postcranial skeleton of Cebus. In Norconk, M. A., Rosenberger, A. L., and Garber, P. A., (Eds.). Adaptive radiations of Neotropical primates. (pp. 229-249). New York: Plenum Press. Forster, P., Harding, R., Torroni, A., Bandelt, H-J. (1996). Origin and evolution of Native American mtDNA variation: a reappraisal. American Journal of Human Genetics 59:935–945. Fragaszy, D. M., Visalberghi, E., Fedigan, L., Rylands, A. B. (2004). Taxonomy, distribution and conservation: Where and what are they, and how did get there? In: Fragaszy, D. M., Fedigan, L., and Visalberghi, E. (Eds). The Complete Capuchin: The Biology of the Genus Cebus. (pp. 13-35). Cambridge University Press, Cambridge. Freitas, L., Seuánez, H. (1982), Chromosome heteromorphisms in Cebus apella. Journal of Human Evolution 10: 173-180. Fujimoto, A., Kimura, R., Ohashi, J., et al. (2008). A Scan for Genetic Determinants of Human hair morphology: EDAR is Associated with Asian Hair Thickness. Human Molecular Genetics 17: 835–843. Funk, D. J., Omland, K. E. (2003). Species level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics 34: 397–423. Galvi, J. (1980). Un arco de islas Terciario en el Occidente colombiano. Geología Colombiana 11:7–43. García, F., Nogués, C., Ponsà, M., Ruiz-Herrera, A., Egozcue, J., Garcia Caldés, M. (2000). Chromosomal homologies between humans and Cebus apella (primates) revealed by ZOO-FISH. Mammalian Genome 11: 399–401. Geraldes, A., Basset, P., Gibson, B., Smith, K. L., Harr, B., Yu, H. T., Bulatova, N., Ziv, Y., Nachman, M. W. (2008). Inferring the history of speciation in house mice from autosomal, X-linked, Y-linked and mitochondrial genes. Molecular Ecology 17:5349– 5363.

260

Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil

Goldstein, D. B., Ruiz Linares, A., Cavalli-Sforza, L. L., Feldman, M. W. (1995). Genetic absolute dating based on microsatellites and the origin of modern humans. Proceedings of the National Academy of Sciences USA 92: 6723-6727. Gregory-Wodzicki, K. M. (2000). Uplift history of the central and northern Andes: a review. Geological Society of America Bulletin 112: 1091–1105. Groves, C. P. (2001). Primate Taxonomy. Smithsonian Institution Press, Washington, DC. Groves, C. P. (2005). Order primates. In: Wilson, D. E., Reeder, D. M. (Eds.). Mammal species of the world: a taxonomic and geographic reference. 3rd ed. (pp. 111-184). Baltimore: Johns Hopkins Univ Press. Groves, C., Grubb, P. (2011). Ungulate Taxonomy. The Johns Hopkins University Press, Baltimore. Haffer, J. (1969). Speciation in Amazonian forest birds. Science 165: 131-137. Haffer, J. (1997). Alternative models of vertebrate speciation in Amazonia: an overview. Biodiversity Conservation 6: 451-476. Haffer, J. (2008). Hypotheses to explain the origin of species in Amazonia. Braz. J. Biol. 68: 917-947. Haq, B. U., Hardenbol, J., Vail, P. R. (1987). Chronology of fluctuating sea levels since the Triassic. Science 235: 1156–1167. Hardouin, E. A., Chapuis, J-L., Stevens, M. I., van Vuuren, B. J., Quillfeldt, P., Scavetta, R. J., Teschke, and M., Tautz, D. (2010). House mouse colonization patterns on the subAntarctic Kerguelen Archipelago suggest singular primary invasions and resilience against re-invasion. BMC Evolutionary Biology 10: 325–339. Harrison, T. (1987). The phyletic relationships of the early catarrhine primates: a review of the current evidence. Journal of Human Evolution 16:41–80. Haugaasen, T., Peres, C. A. (2005). Primate assemblage structure in Amazonian flooded and unflooded forests. American Journal Primatology 67: 243–258. Haugaasen, T., Peres, C. A. (2009). Interspecific primate associations in Amazonian flooded and unflooded forests. Primates 50:239–251. Hawkins, B. A., Diniz-Filho, J. A. F. (2006) Beyond Rapoport’s rule: evaluating range size patterns of New World birds in a two-dimensional framework. Global Ecology and Biogeography 15: 461–469. Hays, J. D., Imbrie, J., Shackleton, N. J. (1976). Variation in the Earth’s orbit: Pacemaker of the Ice Ages. Science 94: 1121-1132. Hebert, P. D. N., Ratnasingham, S., de Waard, J. R. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B 270 (Suppl.): S96-9. Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T., Francis, C. M. (2004). Identification of birds through DNA barcodes. PLoS Biology 2: 1657-63. Helmens, K. F. (1988). Late Pleistocene glacial sequence in the area of the high plain of Bogota (Eastern cordillera, Colombia). Palaeogeogr. Palaeoclimatology and Palaeoecology, 67: 263-283. Hernández-Camacho, J., Cooper, R. W. (1976). The nonhuman primates of Colombia. In Thorington Jr RW, Heltne PG (Eds.), Neotrop. Primates: Field Studies and Conservation (pp. 35-69). Washington D. C.: National Academy of Sciences.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

261

Hershkovitz, P. (1949). Mammals of northern Colombia. Preliminary report n° 4: Monkeys (Primates), with taxonomic revisions of some forms. Proceedings United States National Museum 98: 323-427. Heyer, E., Zietkiewicz, E., Rochowski, A., Yotova, V., Puymirat, J., Labuda, D. (2001). Phylogenetic and familial estimates of mitochondrial substitution rates: study of control region mutations in deep-rooting pedigrees. American Journal of Human Genetics 69: 1113–1126. Hill, W. C. O. (1960). Primates: Comparative Anatomy and Taxonomy. IV. Cebidae, Part A. Edinburgh University Press, Edinburgh. Hooghiemstra, H. (1984). Vegetational and climatic history of the high plain of Bogota, Colombia: A continuous record of the last 3. 5 Millions years. In Van der Hammen, T. (Ed.). El Cuaternario de Colombia 10. Ed. (pp. 1-368). Cramer. Hodgson, J. A., Sterner, K. N., Matthews, L. J., Burrell, A. S., Jani, R. A., Raaum, R. L., Stewart, C. B., Disotell, T. R. (2009). Successive radiations, not stasis, in the South American primate fauna. Proceedings of the National Academy of Sciences USA 106:5534–5539. Hoorn, C., Wesselingh, F. P. (2010). Amazonia: landscape and species evolution. A look into the past. Wiley-Blackwell Publishing Ltd. Pp. 1-446. Horai, S., Hayasaka, K., Kondo, R., Tsugane, K., Takahata, N. (1995). Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial. Proceedings of the National Academy of Sciences USA 92: 532-536. Howells, W. (1974). L’Homme de Néanderthal. La Recherche 47: 634-642. Huxley, J. S. (1942). Evolution: the modern synthesis. Allen and Unwin, London. Isaac, J. B., Mallet, J., Mace, G. M. (2004). Taxonomic inflation: its influence on macroecology and conservation. Trends Ecology and Evolution 19: 464–469. Jelinek, J. (1976). The Homo sapiens neanderthalensis and Homo sapiens sapiens relationships in Central Europe. Anthropologie XIV, 1/2: 79-81. Johnson, W. E., Eizirik, E., Pecon-Slattery, J., Murphy, W. J., Antunes, A., Teeling, E., O’Brien, S. J. (2006). The Late Miocene radiation of the modern Felidae: a genetic assessment. Science 311: 73–77. Jungers, W. L., Fleagle, J. G. (1980). Postnatal growth allometry of the extremities in Cebus albifrons and Cebus apella: a longitudinal and comparative study. American Journal of Physical Anthropology 53:471–478. Kamberov, Y. G., Wang, S., Tan, J., Gerbaul, P., et al. (2013). Modeling recent human evolution in mice by expression of a selected EDAR variant. Cell 152: 691–702. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120. Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X., Wilson, A. C. (1989). Dynamics of Mitochondrial DNA Evolution in Animals: Amplification and Sequencing with Conserved Primers. Proceedings of the Natinoal Academy of Sciences USA 86: 6196-6200. Koiffmann, C. P. (1982). Variabilidade cromossomica em macacos da familia Cebidae. In Saldanha, P. H. (Ed.). Genética comparada de primatas brasileiros. Sociedade Brasileira de Genética, Ribeirao Preto, Sao Paulo, Brazil. Pp. 1-172.

262

Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil

Koiffmann, C. P., Saldhana, P. H. (1974). Cytogenetics of Brazilian monkeys. Journal of Human Evolution 3: 275–282. Lamason, R. L., Mohideen, M., Mest, J. R. et al. (2005). SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310: 1782-1786. Laugenie, G. (1982). La región des Lacs: Recherche sur l’evolution géomorphologique d’un piedmont glaciaire andin. Tome I-II. CNRS. Lynch Alfaro, J. W., Boubli, J. P., Olson, L. E., Di Fiore, A., Wilson, B., Gutiérrez-Espeleta, G. A., Chiou, K. L., Schulte, M., Neitzel, S., Ross, V., Schwochow, D., Nguyen, M. T. T., Farias, I., Janson, C. H., Alfaro, M. E. (2012a). Explosive Pleistocene range expansion leads to widespread Amazonian sympatry between robust and gracile capuchin monkeys. Journal of Biogeography 39: 272–288. Lynch-Alfaro, J. W., De Souza, E., Silva Jr., J, Rylands, A. B. (2012b). How different are robust and gracile capuchin monkeys? An argument for the use of Sapajus and Cebus. American Journal of Primatology 74: 273-286. Mantecón, M. A. F. de, Mudry, M. D., Brown, A. D. (1984). Cebus apella de Argentina, distribución geográfica, fenotipo y cariotipo. Revista del Museo Argentino de Ciencias Naturales “Bernadino Rivadavia” (Zoología) 13: 399-408. Masterson, T. J. (2001). Geographic cranial variation among three subspecies of Cebus apella. American Journal of Primatology 52:46–47. Matayoshi, T., Howlin, E., Nasazzi, N., Nagle, C., Gadow, E., Seuánez, H. (1986). Chromosome studies of Cebus apella. The standard karyotype of Cebus apella paraguayanus, Fisher 1829. American Journal of Primatology 10: 185-193. Matthews, L. J. (2012). Variations in sexual behavior among capuchin monkeys function for conspecific mate recognition: a phylogenetic analysis and a new hypothesis for female proceptivity in tufted capuchins. American Journal of Primatology 74: 287-298. Matzen da Silva, J., Creer, S., dos Santos, A., Costa, A. C., Cunha, M. R., Costa, F. O., Carvalho, G. R. (2011). Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Crustacea: Malacostraca). Plos ONE 6: e19449. Mayle, F. E. (2004). Assessment of the Neotropical dry forest refugia hypothesis in the light of palaeoecological data and vegetation model simulations. Journal Quaternary Science 19:713–720. Mayr, E. (1942). Systematics and the Origin of Species. Columbia University Press. New York, USA. pp. 1-334. Mayr, E. (1963). Animal Species and Evolution. Harvard University Press: Cambridge, Massachusetts. Mayr, E. (1977). Darwin and natural selection. How Darwin may have discovered his highly unconventional theory. American Scientist 65: 321-327. Mayr, E. (1978). Origin and history of some terms in systematic and evolutionary biology. Systematic Zoology 27: 83-88. Mayr, E. (1992). Darwin's Principle of Divergence. Journal of the History of Biology 25:343359. Mazak, J. H., Groves, C. P. (2006). A taxonomic revision of tigers (Panthera tigris) of Southeast Asia. Mammalian Biology 71, 268–287. McKay, B. D., Zink, R. M. (2010). The causes of mitochondrial DNA gene tree paraphyly in birds. Molecular Phylogenetics and Evolution 54: 647–650.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

263

Meireles, C. M., Czelusniak, J., Schneider, M. P., Muniz, J. A. P. C., Brigido, M. C., Ferreira, H. S., Goodman, M. (1999). Molecular phylogeny of Ateline New World Monkeys (Platyrrhini, Atelinae) -globin gene sequences: evidence that Brachyteles is the sister group of Lagothrix. Molecular Phylogenetics and Evolution 12: 10–30. Melo, A. S., Rangel, T. F. L. V. B., Diniz-Filho, J. A. F. (2009) Environmental drivers of beta-diversity patterns in New-World birds and mammals. Ecography 32: 226–236. Mendes Pontes, A. R., Malta, A., Asfora, P. H. (2006). A new species of capuchin monkey, genus Cebus Erxleben (Cebidae, Primates): found at the very brink of extinction in the Pernambuco Endemism Centre. Zootaxa 1200: 1-12. Mercer, J. H. (1984). Changes in the Ice cover of temporate and tropical South America during the last 25. 000 years. Zbl. Geo. Palaont. Teil I, H11/12: 1661-1665. Merriam, C. H. (1918). Review of the grizzly and big brown bears of North America (genus Ursus) with description of a new genus Vetularctos. North American Fauna 41: 1–136. Mittermeier, R. A., Coimbra-Filho, A. F. (1981). Systematics: species and subspecies. In Coimbra-Filho and A. F., Mittermeier, R. A. (Eds.). Ecology and behavior of Neotropical Primates Vol 1. (pp. 29-109). Mittermeier, R. A., Van Roosmalen, M. G. M. (1981). Preliminary observations on habitat utilization and diet in eight Surinam monkeys. Folia Primatologica 36:1–39. Montgelard, C., Catzeflis, F. M., Douzery E. (1997). Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Molecular Biology and Evolution 14:550–559. Moritz, C. (1994). Defining evolutionary significant units for conservation. Trends in Ecology and Evolution 9: 373-375. Morral, N., Bertrantpetit, J., Estivill, X. et al. (1994). The origin of the major cystic fibrosis mutation (delta F508) in European populations. Nature Genetics 7: 169-175. Mudry, M. D., Slavutsky, I., Zunino, G., Delprat, A., Brown, A. (1991). The chromosomes of Cebus apella from Argentina. Revista Brasileira de Genetica 14: 729-738. Nabholz, B., Glemin, S., Galtier, N. (2008). Strong variations of mitochondrial mutation rate across mammals - the longevity hypothesis. Molecular Biology and Evolution 25: 120130. Nabholz, B., Glémin, S., Galtier, N. (2009). (The erratic mitochondrial clock: variations of mutations rate, not population size, affect mtDNA diversity across birds and mammals. BMC Evolutionary Biology 9: 54 doi:10. 1186/1471-2148-9-54. Napier, J. R., Napier, P. H. (1967). A handbook of living primates. London: Academic Press. Nascimento, F. F., Bonvicino, C. R., da Silva, F. C. D., Schneider, M. P. C., Seuánez, N. H. (2005). Cytochrome b polymorphisms and population structure of two species of Alouatta (Primates: Alouattinae). Cytogenetics Genome Research 108:78–86. Nascimento, F. F., Bonvicino, C. R., de Oliveira, M. M., Schneider, M. P. C., Seuánez, H. N. (2008). Population genetic studies of Alouatta belzebul from the Amazonian and Atlantic forests. American Journal of Primatology 70:423–431. Nascimento, F. F., Lazar, A., Seuánez, H. N., Bonvicino, C. R. (2015). Reanalysis of the biogeographical hypothesis of range expansion between robust and gracile capuchin monkeys. Journal of Biogeography. http://wileyonlinelibrary. com/journal/jbi doi:10. 1111/jbi. 12448. Neer, R. M. (1975). The evolutionary significance of vitamin D, skin pigment and ultraviolet light. American Journal of Physical Anthropology 43: 409-416.

264

Manuel Ruiz-García, María Ignacia Castillo and Kelly Luengas-Villamil

Neusser, M., Stanyon, R., Bigoni, F., Wienberg, J., Müller, S. (2001). Molecular cytotaxonomy of New World monkeys (Platyrrhini) – comparative analysis of five species by multi-color chromosome painting gives evidence for a classification of Callimico goeldii within the family of Callitrichidae. Cytogenetics and Cell Genetics 94: 206–215. Nieves, M., De Oliveira, E. H., Amaral, P. J., Nagamachi, C. Y., Pieczarka, J. C., et al. (2011). Analysis of the heterochromatin of Cebus (primates, Platyrrhini) by micro-FISH and banding pattern comparisons. Journal of Genetics 90: 111–117. Nores, M. (2004). The implications of Tertiary and Quaternary sea level rise events for avian distribution patterns in the lowlands of northern South America. Global Ecology and Biogeography 13: 149-162. Oliveira, M. M., Langguth, A. (2006). Rediscovery of Marcgrave’s capuchin monkey and designation of a neotype for Simia flavia Schreber, 1774 (Primates, Cebidae). Boletim do Museu Nacional, nova série, Rio de Janeiro 523: 1-16. Olson, M. A., Zajac, R. N., Russello, M. A. (2009). Estuarine-scale genetic variation in the polychaete Hobsonia florida (Ampharetidae; Annelida) in long island sound and relationships to Pleistocene glaciations. Biological Bulletin 217: 86-94. Palacios, F., Angelone, C., German, A., Reig, S. (2008). Morphological evidence of species differentiation within Lepus capensis Linnaeus, 1758 (Leporidae, Lagomorpha) in Cape Province, South Africa. Mammalian Biology 73: 358–370. Parra, E. J. (2007). Human pigmentation variation: Evolution, genetic basis, and implications for public health. Yearbook of Physical Anthropology 50: 85-105. Patton, J. L., da Silva, M. N. F., Malcolm, J. R. (2000). Mammals of the Rio Jurua and the evolutionary and ecological diversification of Amazonia. Bulletin of The American Museum of Natural History 244: 1-306. Perry, S. (2008). Manipulative monkeys: the capuchins of Lomas Barbudal. Cambridge: Harvard University Press. Pp. 1-368. Posada, D., Crandall, K. A. (2001). Intraspecific gene genealogies: trees grafting into networks. Trends in Ecology and Evolution 16:37–45. Queiroz, H. L. (1992). A new species of capuchin monkey, genus Cebus Erxleben, 1777 (Cebidae: Primates) from eastern Brazilian Amazonia. Goeldiana Zoologia 15:1–13. Rajabi-Maham, H., Orth, A., Bonhomme, F. (2008). Phylogeography and postglacial expansion of Mus musculus domesticus inferred from mitochondrial DNA coalescent, from Iran to Europe. Molecular Ecology 17:627–641. Rensch, B. (1947). Evolution above the species level. Columbia University Press, NewYork. Rensch, B. (1950). Die Abhangigkeit der relativen Sexualdifferenz von der Korpergrosse. Bonner Zoologische Beitrage 1: 58–69. Richard, F., Lombard, M., Dutrillaux, B. (1996). ZOOFISH suggests a complete homology between human and capuchin monkey (Platyrrhini) euchromatin. Genomics 36: 417–423. Rosenbaum, G., Giles, D., Saxon, M., Betts, P. G., Weinberg, R. F., Duboz, C. (2005). Subduction of the Nazca Ridge and the Inca Plateau: Insights into the formation of ore deposits in Peru. Earth and Planetary Science Letters 239: 18–32. Ruiz-García, M., Pinedo-Castro, M. (2010). Molecular systematics and phylogeography of the genus Lagothrix (Atelidae, Primates) by means of mitochondrial COII gene. Folia Primatologica 81: 109–128.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

265

Ruiz-García, M., Castillo, M. I. (2016). Genetic structure, spatial patterns and historical demographic evolution of white-throated capuchin (Cebus capucinus, Cebidae, Primates) populations of Colombia and Central America by means of DNA microsatellites. In RuizGarcía, M., and Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P., and Shostell, J. M. (2006). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report 73: 3–47. Ruiz-García, M., Castillo, M. I., Vásquez, C., Rodriguez, K., Pinedo-Castro, M., et al. (2010). Molecular phylogenetics and phylogeography of the white-fronted capuchin (Cebus albifrons; Cebidae, Primates) by means of mtCOII gene sequences. Molecular Phylogenetics and Evolution 57: 1049-1061. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L. F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). Internation Journal of Primatology 32:1218–1241. Ruiz-Garcia, M., Castillo, M. I., Ledezma, A., Leguizamon, N., Sánchez, R., et al. (2012a). Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of Primatology 74: 366-380. Ruiz-García, M., Castillo, M. I., Lichilin, N., Pinedo-Castro, M. (2012b). Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, C. xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene sequences. Folia Primatologica 83: 100-125. Ruiz-García, M., Sánchez-Castillo, J. S., Castillo, M. I., Luengas-Villamil, K. (2016a). Mitogenomics of the capuchin monkeys (Cebus): one unique genus and less species than currently claimed by primatologists. Molecular Phylogenetics and Evolution (submitted). Ruiz-García, M., Castillo, M. I., Luengas-Villamil, K., Leguizamón, N. (2016b). Invalidation of three robust capuchin species (Cebus libidinosus pallidus, C. macrocephalus and C. fatuellus; Cebidae, Primates) in the Western Amazon and Orinoco by analyzing DNA microsatellites. In: Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Ruiz-García, M., and Shostell, J. M. (Eds.). Nova Science Publishers, Inc. New York, USA. Ruvolo, M., Disotell, T. R., Allard, M. W., Brown, W. M., Honeycutt, R. L. (1991). Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence. Proceedings of the National Academy of Sciences of the USA 88: 1571–1574. Rylands, A. B., Schneider, H., Mittermeier, R. A., Groves, C. P., Rodriguez-Luna, E. (2000). An assessment of the diversity of New World Primates. Neotropical Primates 8: 61-93. Rylands, A. B., Kierulff, M. C. M., Mittermeier, R. A. (2005). Notes on the taxonomy and distributions of the tufted capuchin monkeys (Cebus, Cebidae) of South America. Lundiana 6: 97-110. Saitou, N., Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:405–425. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular cloning: A laboratory manual. 2nd Ed. New York: Cold Spring Harbor Laboratory Press.

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Savolainen, P., Zhang, Y. P., Luo, J., Lundeberg, J., Leitner, T. (2002). Genetic evidence for an East Asian origin of domestic dogs. Science 298: 1610-1613. Schneider, H., Sampaio, I. (2015). The systematics and evolution of New World primates – A review. Molecular Phylogenetics and Evolution 82: 348–357. Schneider, H., Schneider, M. P., Sampaio, I., Harada, M. L., Stanhope, M., Czelusniak, J., Goodman, M. (1993). Molecular phylogeny of the New World monkeys (Platyrrhini, primates). Molecular Phylogenetics and Evolution 2: 225–242. Schrago, C. G. (2007). On the time scale of New World primate diversification. American Journal of Physical Anthropology 132: 344–354. Schrago, C. G., Russo, C. A. (2003). Timing the origin of New World monkeys. Molecular Biology and Evolution 20: 1620–1625. Seuánez, H. N., Armada, J. L., Barroso, C., Rezende, C., Da Silva, V. F. (1983). The meiotic chromosomes of Cebus apella (Cebidae, Platyrrhini). Cytogenetic and Cell Genetics 36: 517-524. Seuánez, H. N., Armada, J. L., Freitas, L., Rocha e Silva, R. da., Pissinatti, A., CoimbraFilho, A. F. (1986). Intraspecific chromosome variation in Cebus apella (Cebidae, Platyrrhini): the chromosomes of the yellow-breasted capuchin Cebus apella xanthosternos Wied, 1820. American Journal of Primatology 10: 237-247. Shackleton, N., D Opdyke, N. (1973). Oxygen isotop and paleomagmetic stratigraphic of Equatorial Pacific core 128-238. Oxygen isotopes temperatures and ice volumes on a 5 to 6. 10 year scale. Quaternary Research 3: 39-55. Silva, Jr J de S. (2001). Especiacao nos macacos-pregos e caiararas, género Cebus Erxleben, 1777 (Primates, Cebidae). Doctoral Thesis. Pp. 377. Universidade Federal do Rio de Janeiro, Rio de Janeiro. Simpson, G. G. (1944). Tempo and Mode in Evolution. Columbia University Press, New York. Simpson, G. G. (1950). History of the fauna of Latin America. American Scientists 38:361– 389. Simpson, G. G. (1953). The Major Features of Evolution. Columbia University Press, New York. Pp. 1-434. Simpson, G. G. (1965). The geography of evolution. Collected essays. Philadelphia and New York: Chilton Books. Spironello, W. R. (1991). Importancia dos frutos de palmeiras (Palmae) na dieta de um grupo de Cebus apella (Cebidae, Primates) na Amazonia Central. In Rylands, A., Bernardes, A. T., (Eds.). A primatologia no Brasil. Volume 3. (pp. 285-296). Belo Horizonte, Brazil: Sociedade Brasileira de Primatologia. Stebbins, G. L. (1950). Variation and evolution in plants. Columbia University Press. New York. pp. 1-634. Tagliaro, C. H., Schneider, M. P. C., Schneider, H., Sampaio, I. C., Stanhope M. J. (1997). Marmoset phylogenetics, conservation perspectives, and evolution of the mtDNA control region. Molecular Biology and Evolution 14:674–684. Takai, M., Anaya, F., Shigehara, N., Setoguchi, T. (2000). New fossil materials of the earliest new world monkey, Branisella boliviana, and the problem of platyrrhine origins. American Journal of Physical Anthropology 111: 263–281.

It Is Misleading to Use Sapajus (Robust Capuchins) as a Genus?

267

Tamura, K., Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512-526. Terborgh, J. (1983). Five New World primates: a study in comparative ecology. Princeton: Princeton University Press. Thalmann, O., Hebler, J., Poinar, H. N., Paabo, S., Vigilant, L. (2004). Unreliable mtDNA data due to nuclear insertions: A cautionary tale from analysis of humans and other apes. Molecular Ecology 13: 321–335. Tishkoff, S. A., Dietzsch, E., Speed, W., Pakstis, A. J., Kidd, J. R., Cheung, K. Bonné-Tamir, B., Santachiara-Benerecetti, A. S. Moral, P., and Krings, M. (1996). Global patterns of linkage disequilibrium at the CD4 locus and modern human origins. Science 271: 13801387. Torres de Caballero, O. M., Ramírez, C., Yunis, E. (1976). Genus Cebus Q and G-band karyotypes and natural hybrids. Folia Primatologica 26: 310-321. Turner, C. G. (1976). Dental evidence on the origins of the Ainu and Japanese. Science 193: 911-913. Vaidya, G., Lohman, D. J., Meier, R. (2011). SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27: 171–180. Van der Hammen, T. (1961). The Quaternary climatic changes of Northern South America. Annals of the New York Academy of Science 95: 676-683. Van der Hammen, T. (1985). The Plio-Pleistocene climatic record of the tropical Andes. Journal Geological Society London 142: 483-489. Van der Hammen, T. (1995). El estudio del Plioceno y Cuaternario de la Sabana de Bogotá: Introducción histórica. Análisis Geográficos 24: 13-32. Van der Hammen, T., Werner, J. H., Van Dommelen, H. (1973). Palynological record of the upheaval of the Northern Andes: a study of the Pliocene and Lower Quaternary of the Colombian Eastern Cordillera and the early evolution of its high-Andean biota. Review of Palaeobotany and Palynology 16: 1-122. Van der Hammen, T., Wermeer, J. H., Van Dommelen, H. (1983). Palynological record of the up-heaval of the northern Andes: A study of the Pliocene and Lower Quaternary of the Colombian Eastern Cordillera and the early evolution of its High-Andean biota. Rev. Palaeobotany and Palynology 16: 1-122. Vigilant, L., Stoneking, M., Harpending, H., Hawkes, K., Wilson, A. C. (1991). African populations and the evolution of human mitochondrial DNA. Science 253: 1503-1507. Von Dornum, M., Ruvolo, M. (1999). Phylogenetic relationships of the New World monkeys (Primates, Platyrrhini) based on nuclear G6PD DNA sequences. Molecular Phylogenetics Evolution 11: 459–476. Wade, N. (2014). A Troublesome Inheritance: Genes, Race and Human History. The Penguin Press, USA. Pp. 1-295. Walsh, P. S., Metzger, D. A., Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506513. Ward, R. H., Frazier, B. L., Dew-Jager, K., Paabo, S. (1991). Extensive mitochondrial diversity within a single Amerindian tribe. Proceedings of the National Academy of Sciences USA 88: 8720–8724.

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Wijmstra, T. A., Van der Hammen, T. (1974). The last interglacial cycle: state of affairs of correlation between data obtained from the land and from the ocean. Geologie en Mijnbouw 53: 386-392. Wright, B. W., Wright, K. A., Chalk, J., Verderane, M. P., Fragaszy, D., Visalberghi E, Izar, P., Ottoni, E. B., Constantino, P., Vinyard, C. (2009). Fallback foraging as a way of life: using dietary toughness to compare the fallback signal among capuchins and implications for interpreting morphological variation. American Journal of Physical Anthropology 140: 687–699. Wright, S. (1968). Evolution and the Genetics of Populations: Genetics and Biometric Foundations volume 1 (Genetic and Biometric Foundations). University of Chicago Press, Chicago. Wright, S. (1969). Evolution and the Genetics of Populations: Genetics and Biometric Foundations volume 2 (Theory of Gene Frequencies). University of Chicago Press, Chicago. Wright, S. (1977). Evolution and the Genetics of Populations: Genetics and Biometric Foundations volume 3 (Experimental Results and Evolutionary Deductions). University of Chicago Press, Chicago. Wright, S. (1978). Evolution and the Genetics of Populations: Genetics and Biometric Foundations volume 4 (Variability within and Among Natural Populations). University of Chicago Press, Chicago. Zachos, F. E. (2015). Tree thinking and species delimitation: Guidelines for taxonomy and phylogenetic terminology. Mammalian Biology, (in press), http://dx. doi. org/10. 1016/j. mambio. 2015. 10. 002. Zachos, F. E., Apollonio, M., Barmann, E. V., Festa-Bianchet, M., Gohlich, U., Habel, J. C., Haring E., Kruckenhauser, L., Lovari, S., McDevith, A. D., Pertoldi, C., Rossner, G, E., Sánchez-Villagra, M. R., Scandura, M., Suchentrunk, F. (2013). Species inflation and taxonomic artefacts—A critical comment on recent trends in mammalian classification. Mammalian Biology 78: 1-6.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 8

SEX CHROMOSOMES AND SEX DETERMINATION IN PLATYRRHINI Eliana R. Steinberg and Marta D. Mudry Grupo de Investigación en Biología Evolutiva (GIBE) – Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina IEGEBA, CONICET

ABSTRACT In the order Primates the most widespread sex chromosome system is the XX/XY. The X chromosome is highly conserved both at a structural as well as at gene level. By contrast, the Y chromosome shows a wide spectrum of morphological and genetic species-specific divergence. In Platyrrhini, modifications of this ancestral XX/XY sexual system were observed. Multiple sex chromosome systems have been described in different genera of neotropical primates, originating from Y-autosome translocations. Other features that makes the Y chromosome of neotropical primates different from the human Y chromosome is the absence of chromosomal homeology between them and the different meiotic behavior when compared to the human Y. These differences in the Y chromosomes can also be observed at the molecular genetic level. The presence of the gene homeologous to human SRY was detected, showing species-specific sizes for the SRY HMG box sequence, both in Platyrrhini and Catarrhini. However, the regions adjacent to the human SRY gene are not conserved in neotropical primates, in contrast with the conservation observed in old world primates. All these findings show a differential evolutionary history of the Platyrrhine Y chromosome compared to Catarrhine Y at the genic and chromosome levels.

Keywords: Platyrrhini, Y chromosome, multiple sex chromosome systems, meiotic studies, SRY

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[email protected].

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SEX DETERMINATION Since ancient times, numerous scientists have attempted to elucidate how sex is determined in the offspring. Over the years, as knowledge about the matter expanded, the answer to this question has undergone radical changes. Aristotle claimed that sex was determined by the “heat” of the male during mating. The more “heated” the passion, the greater the probability of male offspring. In this light, females were considered males whose development was interrupted prematurely because the “cold” womb countered the “heat” of the father’s semen. This idea was accepted by the anatomist Galen (200 AD), who described females as underdeveloped males, whose genitals were equal to those of men, being only internal representations of these (turned outside-in) (Gilbert, 2010). Until the 20th century it was believed that the environment (temperature and nutrition in particular) was important in sex determination. In 1980, Geddes and Thomson in their review of the subject argued that factors favoring the storage of energy and nutrients predispose female offspring, while factors that favor the use of these resources predispose male offspring (Gilbert, 2010). In 1891 H. Henking published the morphological description of the spermatogenesis of the Heteroptera Pyrrhocoris apterus, in which he described a chromatin body as “Chromosome X” due to its unusual staining during the meiotic prophase. These observations were interpreted by McClung and Wilson, who assumed that the presence of one or two “X chromosomes” determined the binary decision to male or female in the X0-XX systems (reviewed in Solari, 1993). From that moment on, more and more evidence of sex determination by nuclear inheritance accumulated. Today we know that in different vertebrate species, both environmental and internal mechanisms may operate in this process.

SEX DETERMINING MECHANISMS Sex determination in mammals, and among them in primates, can be analyzed considering the different stages of embryonic development: primary or gonadal sex determination and secondary or extra-gonadal sex determination. The first implies the differentiation of the ovary or testicle from an undifferentiated gonad. The extra-gonadal sex determination involves the development of the male and female phenotypes in response to hormones secreted by the differentiated gonads. In the absence of gonads, a female phenotype will develop (Gilbert, 2010). In this contribution we will only refer to primary sex determination. In Primates, the taxon subject of this book, the only primary sex determination system observed is male heterogamy. Heterogamy refers to systems of sex chromosomes (also called gonosomes) in which the male or the female have heteromorphic sex chromosomes, i.e., where morphological differences are observed in a chromosome pair that constitutes the sex chromosome system in the heterogametic sex. The heteromorphic sex chromosomes are the inherited basis of sex determination and it is determined at conception (Bull, 1983; Charlesworth and Mank, 2010). Mammals are an example of male heterogamy, in which

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males have a XY sexual system and females are XX. In birds, however, sex chromosomes are called Z and W (to differentiate them from the X and Y chromosomes of mammals, respectively) and have female heterogamy, i.e., ZZ are males and ZW are females (reviewed in Solari, 1993). In this chapter we will concentrate on the mechanisms of primary sex determination in primates and, within them, the different types of sex chromosome systems. Some of the hypotheses on the evolution of these sex chromosome systems in this group of mammals will be discussed.

SEX CHROMOSOMES IN PRIMATES Primates are a remarkably diverse group of mammals both for its phenotypical diversity as well as for its wide geographical distribution and habits. Current forms of the Order Primates comprise two sub-orders, Strepsirrhini (lemurs, quirogaleides, lorisiforms, aye-ayes, indriids and bush babies) and Haplorrhini (Tarsiformes, new world primates or Platyrrhini and old world primates or Catarrhini), including in the latter group the Hominoidea (Groves, 2001). This heterogeneity finds its correlation at the karyological level. In non-human primates a wide chromosomal diversity has been observed, with diploid number (2n) ranging from 2n = 16 in the black marmoset Callicebus lugens (Bonvicino et al., 2003) to 2n = 80 in the Horsfield’s tarsier, Tarsius bancanus (Eberle, 1975). The immense variety of karyotypes described above provides important evidence about the possible role that chromosomal rearrangements could play in the evolutionary changes experienced by primates. Chromosomal rearrangements (inversions, translocations, fusions and fissions, etc.) contribute to the reorganization of the mammalian genome generating new chromosomal forms and a source of variability on which natural selection can act (King, 1993; Robinson and Ruiz Herrera, 2010). Under the biological species concept (Mayr, 1942) chromosomal differences between closely related species (karyotype specificity) could act as reproductive isolation mechanisms. Thus, these cytogenetic differences could be related to decreased fertility in preventing interspecific hybrid crosses (Reig, 1984), therefore acting as a reproductive isolation mechanism. These chromosomal changes could affect both autosomes and sex chromosomes, with an effect that may or not be visible at phenotypic level and that may or may not contribute to the speciogenesis processes of a particular group of organisms. Thus, in the last 20 years, new techniques have been developed to perform karyological comparisons with evolutionary interpretation purposes. One is the Fluorescent In Situ Hybridization technique (FISH), which has proven to be a fast and reliable method to establish homeologies (understood as recognition of pairs of homologous chromosomes between organisms of different species and valid for phylogenetic analysis) between different taxa and in particular within the Primates Order (Wienberg et al., 1990; Consigliere et al., 1996, 1998; Morescalchi et al., 1997; de Oliveira et al., 2002, 2012; Stanyon et al., 2004, 2008; Dumas et al., 2007; Amaral et al., 2008; Steinberg et al., 2014a). FISH technique is based on the use of fluorescent probes (small sequences of DNA labeled with a fluorescent molecule) that, contacted with another unlabeled sequence, bind to those regions in which a

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chromosomal region is shared with a high degree of similarity. Thanks to its precision, it was successfully used to verify the homeologies previously hypothesized using methodologies based on chromosome morphology and differential staining techniques such as G and C banding (Steinemann et al., 1984). In mammals, comparative FISH analysis has allowed researchers to determine a high degree of genomic conservation in this taxon. Only a few changes were fixed in some taxa, such as fissions, fusions and translocations of chromosome arms, which are considered “landmark rearrangements”, useful as characters to be considered in phylogenetic analyses (Wienberg et al., 1990; O’Brien et al., 1999; Wienberg 2004, 2005; Müller 2006; Stanyon et al., 2004, 2008; de Oliveira et al., 2012). Thus, using this approach it was observed that for the sex chromosomes the X chromosome is highly conserved in placental mammals both at a structural as well as at gene level (Solari, 1993; Graves and Westerman, 2002; Graves and Peichel, 2010). In 1967, Ohno proposed two hypotheses to explain the high genomic conservation observed in the X chromosome and the absence of conservation of the Y chromosome. The first hypothesis indicates that the X chromosome in mammals would be highly conserved because rearrangements in this chromosome would alter the dosage compensation phenomenon. Dosage compensation is a process that inactivates gene expression in one of the two X chromosomes in females to match gene expression with males, which have a single X chromosome (Lyon, 1961). Any change in this process would cause an imbalance in gene expression between males and females and, therefore, would be selected against. The second hypothesis postulates a mechanism for the evolution of sex chromosomes. According to this second hypothesis the heteromorphic sex chromosome pair would originally have emerged from an autosomal pair, in which one of the homologs acquired a mutation originating a locus with sex determining function in male mammals. To preserve this sex-determining gene from any modification or elimination by recombination between the X and the Y, the emergence of a suppression of recombination in the Y chromosome would have been selected. This suppression of recombination allows mutations occurring in the Y chromosome to be fixed in the population and therefore reduce the likelihood that these mutations are repaired, leading to the eventual degradation of the Y chromosome and causing the differentiation of the heteromorphic XY pair (reviewed in Ayling and Griffin, 2002). The human X chromosome represents about 5% of the total haploid genome (Ohno, 1967; Graves, 1998, 2000). By contrast, the Y chromosome shows a wide spectrum of morphological and genetic species-specific divergence (Solari, 1993; Wimmer et al., 2005). This chromosome is usually much smaller, accounting for only 2-3% of the haploid genome. It has also been discovered that it has a reduced number of functional genes (about 78 in humans) and in about 95% of its length recombination with the X chromosome does not occur, a region called Male Specific Region of the Y or MSY (Skaletsky et al., 2003). MSY sequences in the Y chromosome can be grouped in two major classes: the ones with homologous sequences to the X chromosome (degenerate copies of the X sequences) and the ones without these homologous sequences (Lahn and Page, 1999; Bellott et al., 2014). The latest are denominated ampliconic sequences and contain coding and non-coding sequences organized in multicopy gene families that are expressed only in testicles and have an important role in spermatogenesis (Li et al., 2013). These multigenic families contain palindromic sequences (two similar repeats that point to opposite directions, separated by a spacer sequence). Genic conversion between these palindromes allows recombination within

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these genic families (and within the Y chromosome) thus delaying the effects of the molecular degeneration of the Y chromosome (Steinemann and Steinemann, 2005; Cortéz et al., 2014). Therefore, the accumulation of these ampliconic sequences has a stabilization effect on the Y chromosome’s genic content (Li et al., 2013; Cortéz et al., 2014). Unlike autosomes, in which both members of each chromosome pair share homology throughout its length, sex chromosomes have a reduced homology region, called PseudoAutosomal Region (PAR) (Burgoyne, 1982). It is in this region that sex chromosomes synapse and recombine. Humans have two PARs. The PAR1 occupies a 2.6 megabase region on the end of the short arms of both the X and Y chromosomes. Deletions within this region cause infertility. PAR2 occupies approximately 500 kb and recombination occurs in this region in approximately 4% of the meiotic products, suggesting that it may not be essential for the proper segregation of chromosomes, but their presence would stabilize the XY bivalent during the meiotic process (Solari, 1999). In other great apes and old world monkeys, the presence of a PAR2 was not observed, so it is considered that its appearance in humans is recent, after their divergence from the rest of the old world monkeys, more than 35 million years ago (Graves, 2000).

PLATYRRHINI SEX CHROMOSOMES In the order Primates the most widespread sex chromosome system is the XX/XY. However, in neotropical primates (new world primates or Platyrrhini) modifications of this ancestral sexual system are observed. Y-autosome translocations that generate multiple sex chromosome systems have been described in this group (Figure 1). Multiple sex chromosome systems have been observed in different genera of neotropical primates (Ma et al., 1975; Armada et al., 1987; Seuánez et al., 1989; Lima and Seuánez, 1991; Rahn et al., 1996; Mudry et al., 1998, 2001; Moura-Pensin et al., 2001; Solari and Rahn, 2005; Steinberg et al., 2008, 2014a; 2014b). Given this diversity, it is proposed that the sexual system could be a character of diagnostic value in primates and particularly in Platyrrhini. To this end, mitotic parameters have been widely used in past decades. Now it is known that only meiotic data allows to identify and unambiguously confirm the sex chromosome system; however such studies are notoriously scarce and in some species, null (Egozcue, 1969; Ma et al., 1976; Seuánez et al., 1983; Armada et al., 1987; Lima and Seuánez, 1991; Rahn et al., 1996; Mudry et al., 1998, 2001; Nieves et al., 2005; Steinberg et al., 2007, 2008, 2014b; García Cruz et al., 2009, 2011). Meiotic characterization of neotropical primates species provides information relevant to possible evolutionary interpretations, contributing in turn to solve taxonomic questions. Non-human primates are one of the mammals phylogenetically closest to Homo sapiens, constituting for that reason one of the most widely used experimental models after rodents, for all types of biological studies. In this context the study of meiotic behavior in nonhuman primates is not only important to confirm the sex chromosome system but it is also useful for a better understanding of the characteristics conserved between primate species at the karyological level. Regarding sexual systems, among neotropical primates Alouatta is a genus that can be taken as an example of these multiple sex chromosome systems (Revised in Mudry al., 2015).

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The presence of multiple sex chromosome systems has only been confirmed to date by meiotic studies in male howlers in a few species, showing three different conformations: X1X1X2X2/X1X2Y1Y2 type (forming in males a chain of 4 items or quadrivalent in metaphase I) in Alouatta seniculus (Lima and Seuánez, 1991), A. caraya (Mudry et al., 1998, 2001) and A. pigra (Steinberg et al., 2008); X1X1X2X2/X1X2Y type (forming in males a chain of 3 elements or trivalent in Metaphase I) in A. belzebul (Armada et al., 1987) and A. palliatta (Solari and Rahn, 2005); and a X1X1X2X2X3X3/X1X2X3Y1Y2 conformation (forming in males a chain of 5 items or pentavalent in metaphase I) in A. guariba clamitans (Steinberg et al., 2014b).

Figure 1. a) Possible origin for the multiple male sex determination system X1X2Y1Y2 in Alouatta caraya (mod. from Rahn et al. 1996). In this species the autosomal chromosomal pair involved in the Y-autosome translocation is the ACA7 chromosome pair. The ancestral X chromosome is shown in white color, the ancestral Y in light gray and the autosomal pair (A7) in dark gray. From an XY sexual system, two simultaneous breaks in A7p prox and in the distal region of Yq, followed by a reciprocal translocation between them, would have originated the chromosomes Y1 and Y2. The homologous chromosome to the A7 pair, not involved in the translocation, would constitute the X 2 chromosome and the ancestral X would now be denominated X1. b) A. caraya G-banded sex chromosomes. c) A. caraya Diakinesis/Metaphase I spermatocyte. The black arrow indicates the sexual quadrivalent (Bar = 5 μm).

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FISH analysis using human chromosome probes in A. caraya, A. macconnelli, A. guariba, A. sara and A. seniculus arctoidea showed that the autosomes involved in Yautosome translocation, which led to multiple sex chromosome systems in South American howlers, exhibited a hybridization signal for human chromosomes 3 and 15 (Consigliere et al., 1996, 1998; Oliveira et al., 2002; Mudry et al., 2001) (Figure 2). However, the Mesoamerican howlers, A. pigra and A. palliata, do not have this association, denominated as 3/15 synteny, in their sex chromosomes (Steinberg et al., 2014a).

Figure 2. Fluorescent in situ Hybridization pattern with the probes for human chromosomes X, 3 and 15 in the South American species of Alouatta sp. with male X1X2Y1Y2 sex chromosome systems. On the right, the hybridization pattern of human chromosomes X, 3 and 15.

The 3/15 synteny was described in the autosomes of two Atelidae species: Ateles geoffroyi and A. belzebuth hibridus (Morescalchi et al., 1997). But this synteny was not observed either in Cebus cay (ex C. libidinosus) or in the squirrel monkey Saimiri boliviensis (Mudry et al., 2001). This 3/15 synteny could be regarded as the ancestral condition for the Atelidae family, where the association with the multiple sex chromosome systems constitutes an evolutionary novelty (apomorphy) in howlers and the loss of the association an apomorphy for the Lagothrix and Brachyteles group. Another possible interpretation is that it could have appeared independently in Alouatta, and have been involved in the Y-autosome translocation, whereas it was not involved in the sex chromosome system of Ateles (de Oliveira et al., 2005). However, the HSA3/15 syntenic association is not an ancestral condition for Alouatta, since the most basal species A. pigra, A. palliata and A. belzebul (Consigliere et al. 1996; Steinberg et al., 2014a) do not possess this association. This may suggest that at least two independent events of Y-autosome translocations would have occurred in the genus Alouatta that would result in different multiple sex chromosome systems for the Mesoamerican and South American species (Steinberg et al., 2014a). Considering this data it has been suggested that, in a taxon with such noticeable chromosome variability as the one observed in howlers, the presence of these systems should have a selective advantage. One hypothesis is that the acquisition of new chromatin mass through the rearrangement between an autosome and the Y chromosome might have added certain dynamic stability to the small and ancient Y in meiosis (it is known that small chromosomes are more prone to segregation errors) contributing to the fixation of these multiple sex chromosome systems. Another feature of the autosomal chromatin added to the

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Y chromosome may be related to male fertility, accumulating genes related to male phenotype, which will form a beneficial linkage block (Mudry et al., 2001). Another hypothesis is that these Y-autosome translocations are a way of delaying the molecular degeneration of the Y chromosome, since the addition of the autosomal sequences will “rejuvenate” this chromosome (Steinemann and Steinemann, 2005). Another feature that makes the Y chromosome of neotropical primates different from the human Y chromosome is the absence of chromosomal homeology between them. In previous FISH studies with human Y chromosome probes on metaphases of neotropical primates, the human probe showed no hybridization signal on any of the different neotropical primates analyzed, in contrast to old world monkeys where the conservation of the Y chromosome was described (Archidiacono et al., 1998). For the X chromosome, however, a marked conservation was described in the literature in both groups of primates (Consigliere et al., 1996, 1998; Mudry et al., 2001; de Oliveira et al., 2002). Furthermore, the Y chromosome of Platyrrhini has a different meiotic behavior from the human Y chromosome. Previous meiotic studies in C. cay using a silver nitrate staining technique described the behavior of the XY pair as “human like,” that is, a behavior during meiotic division similar to the human XY pair (Seuánez et al., 1983; Mudry et al., 2001). The use of the immunofluorescence technique (Roig et al., 2004) allowed the visualization of specific proteins involved in the recombination process in this species using antibodies against them (García-Cruz et al., 2009, 2011). As a remarkable feature, it was observed a region with positive mark for SYCP1 (synaptonemal complex protein that indicates regions of synapsis) in the XY pair in the non-recombinant portion of these chromosomes. This situation was not previously observed in human spermatocytes (R. Garcia-Cruz and M. Garcia-Caldés, personal communication), although it has been described in other species of mammals such as the mouse Mus musculus (Page et al., 2006) suggesting that the behavior of the XY pair in Cebus does not correspond entirely with that of Homo sapiens, and thus it is not a “human like” sex chromosome system. These differences in the Y chromosomes of old world primates and new world primates can also be observed at the molecular genetic level. In the next section of this chapter we will focus on studies performed in Platyrrhini on the gene responsible for sex determination in mammals, the SRY.

GENES INVOLVED IN SEX DETERMINATION IN PRIMATES In 1973, Jost and his colleagues demonstrated that sexual differentiation in mammals begins in the fetal testes. These authors suggested the existence of a sex determination factor on the Y chromosome, which they called TDF (Testis Determining Factor). They based their conclusion on the fact that, regardless of the number of X chromosomes present, when the individual has a Y chromosome the phenotype is male. Exceptions occur (such as the XY females) when this factor is deleted or mutated (Jost et al., 1973). Studying these aneuploidies, in 1990 the SRY gene (Sex-determining Region of the Y chromosome) located on the short arm of the human Y chromosome was identified (Sinclair et al., 1990). This sequence is considered the determining factor in testis and encodes a 204 amino acid peptide in humans which has a DNA-binding domain called HGM box (High

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Mobility Group). The presence of this DNA binding domain has been described in numerous transcription factors and their role is to induce a twist in the region of DNA to which it binds. The alteration in the geometry of DNA (a 80° twist in the mouse DNA and 65º twist in human) suggest that, besides acting as a transcription factor, the SRY protein coordinates local chromatin structure, approaching and recruiting the transcription machinery necessary to mediate the activation or repression of target genes (Graves, 1998). The gene encoding the SRY protein is almost on the edge (5 kb) of the pseudoautosomal region, so it is not uncommon that sometimes an erroneous crossing over occurs between the X chromosome and the Y chromosome in that area and the gene is transferred to the X chromosome. When this happens you can obtain hermaphrodite individuals (46, XX mosaics). In this case the SRY will be inactivated at random by the dosage compensation phenomenon and incomplete masculinization would be observed. This may indicate the need for a threshold level of SRY to ensure normal testis determination (Veitia et al., 2001). Several testis-specific genes contain binding sites for SRY, and it is considered that binding to these sites begins the route of testicular development (Graves, 1998). SRY acts indirectly on sex determination, since it induces the secretion of a chemotactic compound in the genital ridge that allows migration of mesonephric cells to the XY gonad. These mesonephric epithelial cells induce the gonadal cells to become Sertoli cells with typically masculine expression patterns. The mesonephric cells are essential for the formation of testis cords (Gilbert, 2010). Individuals with mutations in SRY are generally infertile, so almost all mutations are de novo. Because most mutations causing sex reversal are within the HMG box, it is believed that the main activity of this protein would lie in its ability to bind and alter DNA geometry to allow gene transcription (Ayling and Griffin, 2002). As previously mentioned, the Y chromosome of primates is cytogenetically characterized by its high structural variability and their commitment in various patterns of sex determination (reviewed in Solari, 1993). In old world primates the conservation of both the human Y chromosome and SRY gene were described (Archidiacono et al., 1998); in neotropical primates, however, the absence of Y-chromosome homeology with the human Y chromosome was observed (Consigliere et al., 1996, 1998; Mudry et al., 2001; de Oliveira et al., 2002; Steinberg et al., 2014a). Regarding SRY, Moreira (2002) studied its gene evolution by molecular biology techniques (PCR, “Polymerase Chain Reaction”) in 7 genera of Platyrrhini, observing 7 substitutions corresponding to the HMG box region, comparing humans and new world primates. Considering this background, this issue was addressed in our research group. Using PCR amplification the presence of the SRY in 5 ceboidea was detected observing speciesspecific sizes for the HMG box sequence (Ateles chamek 400-300 bp; Ateles paniscus 150 bp, Alouatta caraya 250-200 bp and 200-150 bp, Cebus cay 150 bp, Saimiri boliviensis 200 bp), different from the 299 bp length observed for the human HMG box (Nieves et al., 2003). Previous FISH studies using a probe of the human SRY gene showed that in old world primates, the SRY is close to the pseudoautosomal region (Gläser et al., 1998). In this study species of new world primates of the genera Callithrix and Ateles were analyzed and the SRY was also observed close to the PAR.

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Figure 3. Chromosomal segment of the human Y chromosome that contains the sequences comprised in the SRY probe used (Modified from http://www.abbottmolecular.com/us/products/analyte-specificreagent/fish/vysis-sry-probe-lsi-sry-spectrumorange.html).

With the aim of studying gene conservation of this gene in Ceboidea, the commercial probe (Vysis LSI-SRY) that comprises the Yp11.3 region of the human Y chromosome (and contains the SRY as well as other genes, as illustrated in Figure 3) was used (Steinberg, 2011). When this probe was applied in FISH studies analyzing the howler species A. caraya, A. pigra, A. guariba clamitans and A. palliata no hybridization signal was observed (Figure 4). The same result was obtained in Cebus cay, C. nigritus, Aotus azarae and Saimiri boliviensis boliviensis (Figure 5). Metaphases used as controls for application of FISH (Pan troglodytes and Homo sapiens) showed a clear positive hybridization signal in an acrocentric chromosome corresponding to the Y element (Figure 6). A possible interpretation is that the probe did not hybridize in Platyrrhini metaphases because in these species of neotropical primates the regions adjacent to the human SRY gene are probably not conserved. The probe used by Gläser et al., (1998) was compiled from cosmids whose sequence covered a narrower region than the probe used in our work, allowing them to visualize hybridization. The sequences comprised in the Vysis LSI-SRY probe are microsatellite loci (STR or Short Tandem Repeats) that in humans are deleted in certain infertility cases (Chandley and Cooke, 1994). These are not the only sequences related with human infertility that are absent in Platyrrhini. The DAZ (“Deleted in AZoospermia”) gene family is also absent in this group of primates but present in old world monkeys (Gläser et al., 1998). The absence of these gene families might indicate that these genes are not involved in Platyrrhini spermatogenesis or are not as important to it as they are in humans. Reproductive biology studies in this group of primates are still scarce and this subject has not been addressed, so more studies are needed in order to test this hypothesis.

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Figure 4. Conservation analysis of the SRY gene in howler monkeys (Bar = 10 μm). Metaphases hybridized with the HSASRY probe (red) and the control probe HSA21 (green). Arrows indicate the chromosome that showed a positive signal of hybridization. a) Homo sapiens (positive control of hybridization, signal both in HSAY and HSA21). b) Alouatta caraya (positive signal in ACA21 for HSA21). c) A. guariba clamitans (positive signal in AGU18 for HSA21). d) A. palliata (positive signal in APA1 for HSA21). e) A. pigra (positive signal in API4 for HSA21).

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Figure 5. Conservation analysis of the SRY gene in other Ceboidea (Bar = 10 μm). Metaphases hybridized with the HSASRY probe (red) and the control probe HSA21 (green). Arrows indicate the chromosome that showed a positive signal of hybridization. a) Homo sapiens (positive control of hybridization, signal both in HSAY and HSA21) b) Cebus libidinosus=Cebus cay (positive signal in CLI11 for HSA21). b) Cebus nigritus (positive signal in CNI11 for HSA21). c) Saimiri boliviensis (positive signal in SBOC7 for HSA21). e) Aotus azarae (positive signal in AAZ5 for HSA21).

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Figure 6: Metaphases hybridized with the HSASRY probe (red) and the control probe HSA21 (green) in Hominoidea (Bar=10 μm). Arrows indicate the chromosome that showed positive signal of hybridization. a) Homo sapiens (positive signal both in HSAY and HSA21). b) Pan troglodytes (signal both in PTRY and PTR22).

With the advent of the genomic age in the XXI century, the genome of several primate species has been sequenced. The majority of the non-human primates sequenced genomes are old world primates: Pan troglodytes (Hughes et al., 2010), Gorilla gorilla (Scally et al., 2012), Pongo pygmaeus (Locke et al., 2011) and Macaca mulatta (Hughes et al., 2012). So far only one Platyrrini species, the marmoset Callithrix jacchus, has its genome fully sequenced (Worley et al., 2014). All these genome projects cover the autosomal genome. However, the presence of highly repetitive sequences in the Y chromosome has hindered the efforts for fully sequencing it. To date only the euchromatic regions of the Y chromosomes are sequenced. Still noticeable genomic differences were found among these Y chromosome (Cortéz et al., 2014) that confirm the cytogenetic evidence previously described. All of these findings show a differential evolutionary history of the Platyrrhine Y chromosome compared to the Catarrhine Y ones, both at the genic and chromosome levels. This emphasizes the need for more genetic studies to be performed in this primate group, both at the molecular and cytogenetic levels, and more Platyrrhini species need to be included in the priorities of the genome consortiums.

REFERENCES Amaral, P. J. S., Finotelo, L. F. M, de Oliveira, E. H. C., Pissinatti, A., Nagamachi, C. Y., Pieczarka J. C. (2008). Phylogenetic studies of the genus Cebus (Cebidae-Primates) using chromosome painting and G-banding. BMC Evolutionary Biology 8: 169. Archidiacono, N., Storlazzi, C. T., Spalluto, C., Ricco, A. S., Marzella, R., Rocchi, M. (1998). Evolution of chromosome Y in primates. Chromosoma 107 (9): 241-246. Armada, J. L. A., Barroso, C. M. L., Lima, M. M. C., Muniz, J. A. P. C., Seuánez, H. (1987). Chromosome Studies in Alouatta belzebul. American Journal of Primatology 13: 283296.

282

Eliana R. Steinberg and Marta D. Mudry

Ayling, L. J. and Griffin, D. K. (2002). The evolution of sex chromosomes. Cytogenetic and Genome Research 99: 125-140. Bellott, D. W., Hughes, J. F., Skaletsky, H., Brown, L. G., Pyntikova, T., Cho, T. J., Koutseva, N., Zaghlul, S., Graves, T., Rock, S., Kremitzki, C., Fulton, R. S., Dugan, S., Ding, Y., Morton, D., Khan, Z., Lewis, L., Buhay, C., Wang, Q., Watt, J., Holder, M., Lee, S., Nazareth, L., Alföldi, J., Rozen, S., Muzny, DM., Warren, W. C., Gibbs, R. A., Wilson, R. K., and Page, D. C. (2014). Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators. Nature 508: 494–499. Bonvicino, C. R., Penna-Firme, V., do Nascimento, F. F., Lemosa, B., Stanyon, R., Seuánez, H. N. (2003). The Lowest Diploid Number (2n = 16) yet Found in Any Primate: Callicebus lugens (Humboldt, 1811). Folia Primatologica 74: 141-149. Bull, J. J. (1983). Evolution of Sex Determining Mechanisms. Benjamin-Cummings Pub Co. Burgoyne, P. S. (1982). Genetic homology and crossing over in the X and Y chromosomes of mammals. Human Genetics 61 (2): 85-90. Chandley, A. C. and Cooke, H. J. (1994). Human male fertility-Y-linked genes and spermatogenesis. Human Molecular Genetics 3: 1449-1452. Chowdhary, B. P., Raudsepp, T., Frönicke, L., Scherthan, H. (1998). Emerging Patterns of Comparative Genome Organization in Some Mammalian Species as Revealed by ZooFISH. Genome Research 8: 577-589. Consigliere, S., Stanyon, R., Koehler, G., Agoramoorthy, G., Wienberg, J. (1996). Chromosome painting defines genomic rearrangements between red howler monkey subspecies. Chromosome Research 4: 264-270. Consigliere, S., Stanyon, R., Koehler, U., Arnold, N., Wienberg, J. (1998). In situ hybridization (FISH) maps chromosomal homologies between Alouatta belzebul (Platyrrhini, Cebidae) and other primates and reveals extensive interchromosomal rearrangements between howler monkey genomes. American Journal of Primatology 46: 119-133. Cortez, D., Marin, R., Toledo-Flores, D., et al. (2014). Origins and functional evolution of Y chromosomes across mammals. Nature 508: 488–493. de Oliveira, E. H. C., Neusser, M., Figueiredo, W. B., Nagamachi, C., Pieczarka, J.C., Sbalqueiro, I.J., Wienberg, J., Müller, S. (2002). The phylogeny of howler monkeys (Alouatta, Platyrrhini): Reconstruction by multicolor cross-species chromosome painting. Chromosome Research 10: 669-683. de Oliveira, E. H. C., Neusser, M., Pieczarka, J. C., Nagamachi, C., Sbalqueiro, I. J., Müller, S. (2005). Phylogenetic inferences of Atelinae (Platyrrhini) based on multi-directional chromosome painting in Brachyteles arachnoides, Ateles paniscus paniscus and Ateles belzebul marginatus. Cytogenetic and Genome Research 108: 183-90. de Oliveira, E. H. C., Neusser, M., Müller, S. (2012) Chromosome Evolution in New World Monkeys (Platyrrhini). Cytogenetics and Genome Research 137: 259–272. Dobzhansky, T. (1937). Genetics and the Origin of Species. Columbia University Press, New York, USA. Dumas, F., Stanyon, R., Sineo, L., Stone, G., Bigoni, F. (2007). Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting. BMC Evolutionary Biology 7: Suppl 2: S11. Eberle, P. (1975). Chromosome finds in primates and cytogenetical aspects in the evolution of man. Journal of Human Evolution 4 (6): 435-439.

Sex Chromosomes and Sex Determination in Platyrrhini

283

Egozcue, J. (1969). Aneuploidía cromosómica: nuevas observaciones sobre la separación precoz de bivalentes en meiosis I. Sangre XIV (4): 442-452. García-Cruz, R., Robles, P., Steinberg, E. R., Camats, N., Brieño, M. A., Garcia-Caldés, M., Mudry, M. D. (2009). Pairing and recombination features during meiosis in Cebus paraguayanus (Primates: Platyrrhini). BMC Genetics 10: 25. García-Cruz, R., Pacheco, S., Brieño, M. A., Steinberg, E. R., Mudry, M. D., Ruiz-Herrera, A., Garcia Caldés, M. (2011). A comparative study of the recombination pattern in three species of Platyrrhini monkeys (Primates). Chromosoma 120 (5): 521-530. Gilbert, S. F. (2010). Developmental Biology, Ninth Ed. Sinauer Associates Inc., Sunderlan, MA, USA. Gläser, B., Grützner, F., Willmann, U., Stanyon, R., Arnold, N., Taylor, K., Rietschel, W., Zeitler, S., Toder, R., Schempp, W. (1998). Simian Y Chromosomes: species-specific rearrangements of DAZ, RBM, and TSPY versus contiguity of PAR and SRY. Mammalian Genome 9 (3): 226-231. Graves, J. A. M. (1998). Evolution of the mammalian Y chromosome and sex-determining genes. Journal of Experimental Zoology 281: 472–481. Graves, J. A. M. (2000). Human Y Chromosome, Sex Determination and Spermatogenesis – A Feminist View. Biology of Reproduction 63: 667-676. Graves, J. A. M. and Westerman, M. (2002). Marsupial genetics and genomics. Trends in Genetics 18: 517-521. Graves, J. A. M. and Peichel, C. L. (2010). Are homologies in vertebrate sex determination due to shared ancestry or to limited options? Genome Biology 11: 205. Groves, C. P. (2001). Primate Taxonomy. USA and England: Smithsonian Institution Press. Hughes, J. F., Skaletsky, H., Pyntikova, T., Graves, T. A., van Daalen, S. K., Minx, P. J., Fulton, R. S., McGrath, S. D., Locke, D. P., Friedman, C., Trask, B. J., Mardis, E. R., Warren, W. C., Repping, S., Rozen, S., Wilson, R. K., and Page, D. C. (2010). Chimpanzee and human Y chromosomes are remarkably divergent in structure and gene content. Nature 463: 536-539. Hughes, J. F., Skaletsky, H., Brown, L. G. et al. (2012). Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes. Nature 483: 82-86. Jost, A., Vigier, B., Prépin, J., Perchellet, J. P. (1973). Studies on sex differentiation in mammals. Recent Programs in Hormone Research 29: 1-41. King, M. (1993). Species Evolution: The Role of Chromosome Change. Cambridge University Press. Lahn, B. T. and Page, D. C. (1999). Four evolutionary strata on the human X chromosome. Science 286: 964-967, Li, G., Davis, B. W., Raudsepp, T., Pearks Wilkerson, A. J., Mason, V. C., Ferguson-Smith, M., O’Brien, P. C., Waters, P. D., Murphy, W. J. (2013). Comparative analysis of mammalian Y chromosomes illuminates ancestral structure and lineage-specific evolution. Genome Research 23: 1486–1495. Lima, M. M. C. and Seuánez, H. (1991). Chromosome studies in the red Howler Monkey, Alouatta seniculus stramineus (Platyrrhini, Primates): description of an X1X2Y1Y2/X1X1X2X2 sex chromosome system and karyological comparisons with other subspecies. Cytogenetics and Cell Genetics 57: 151-156. Locke, D. P., Hillier, L. W.; Warren, W.C. et al. (2011). Comparative and demographic analysis of orangutan genomes. Nature 469: 529-533.

284

Eliana R. Steinberg and Marta D. Mudry

Lyon, M. F. (1961). Gene action in the X-chromosome in the mouse (Mus musculus). Nature 190: 372-373. Ma, N. S. F., Jones, T. C., Thorignton, R. W., Miller, A., Morgan L. (1975). Y-Autosome Translocation in the Howler Monkey, Alouatta palliata. Journal of Medical Primatology 4: 299-307. Ma, N. S. F., Elliott, M. W., Morgan, L., Miller, A., Jones, T. C. (1976). Translocation of Y chromosome to an autosome in the Bolivian Owl Monkey, Aotus. American Journal of Physical Anthropology 45 (2): 191-202. Mayr, E. (1942). Systematics and the origin of species. New York: Columbia University Press. Moreira, M. A. M. (2002). SRY evolution in Cebidae (Platyrrhini: Primates). Journal of Molecular Evolution 55: 92-103. Morescalchi, M., Schempp, W., Consigliere, S., Bigoni, F., Wienberg, J., Stanyon, R. (1997). Mapping chromosomal homology between humans and the black-handed spider monkey by fluorescence in situ hybridization. Chromosome Research 5: 527-536. Moura-Pensin, C., Pieczarka, J. C., Nagamachi, C. Y., Pereira Carneiro Muniz, J. A., do Carmo de Oliveira Brigido, M., Pissinati A., Marinho, A., de Souza Barros, R. M. (2001). Cytogenetic relations among the genera of the subfamily Pitheciinae (Cebidae, Primates). Caryologia 54: 385-391. Mudry, M. D., Rahn, M. I., Gorostiaga, M., Hick, A., Merani, M. S., Solari, A.J. (1998). Revised Karyotype of Alouatta caraya (Primates: Platyrrhini) Based on Synaptonemal Complex and Banding Analyses. Hereditas 128: 9-6. Mudry, M. D., Rahn, M. I., Solari, A. J. (2001). Meiosis and chromosome painting of sex chromosome systems in Ceboidea. American Journal of Primatology 54: 65-78. Mudry, M. D., Nieves, M., Steinberg, E. R. (2015). Cytogenetics of Howler Monkeys. Chapter 4 in Howler Monkeys: Adaptive Radiation, Systematics, and Morphology. (Ed.). M. Kowalewski, P. Garber, L. Cortés Ortiz, B. Urbani and D. Youlatos. Springer Verlag, Berlín, Alemania. Müller, S. (2006). Primate Chromosome Evolution. In Genomic Disorders, James R. Lupski and Pawel Stankiewicz eds. Totowa, NJ: Humana Press. Nieves, M., Mudry, M. D., Copelli, S. (2003). Conservación del cromosoma Y humano en Ceboidea (Primates: Platyrrhini). Journal of Basic and Applied Genetics 15 (S2): 46. Nieves, M., Ascunce, M. S., Rahn, M. I., Mudry, M. D. (2005). Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters. Primates 46: 155–164. O’Brien, S. J., Menotti-Raymond, M., Murphy, W. J., Nash, W. G., Wienberg, J., Stanyon, R., Copeland, N. G., Jenkins, N. A., Womack, J. E., Marshall Graves, J. A. (1999). The Promise of Comparative Genomics in Mammals. Science 286: 458-481. Ohno, S. (1967). Sex Chromosomes and Sex-Linked Genes. Springer-Verlag. Page, J., Fuente, R., Gómez, R., Calvente, A., Viera, A., Parra, M. T., Santos, J. L., Berríos, S., Fernández-Donoso, R., Suja, J. A., Rufas, J. S. (2006). Sex chromosomes, synapsis, and cohesins: a complex affair. Chromosoma 115 (3): 250-259. Rahn, M. I., Mudry, M. D., Merani, M. S., Solari, A. J. (1996). Meiotic behavior of the X1X2Y1Y2 quadrivalent of the primate Alouatta caraya. Chromosome Research 4: 350356.

Sex Chromosomes and Sex Determination in Platyrrhini

285

Reig, O. A. (1984). Significado de los métodos citogenéticos para la distinción y la interpretación de las especies, con especial referencia a los mamíferos. Zoología XIII (3): 19-44. Robinson, T. J. and Ruiz-Herrera, A. (2010). Mammalian Chromosomal Evolution: From Ancestral States to Evolutionary Regions. In Evolutionary Biology - Concepts, Molecular and Morphological Evolution, Pierre Pontarotti (Ed.) Berlin, Heidelberg: SpringerVerlag. Roig, I., Liebe, B., Egozcue, J., Cabero, Ll., García, M., Scherthan, H. (2004). Femalespecific features of recombinational double-stranded DNA repair in relation to synapsis and telomere dynamics in human oocytes. Chromosoma 113 (7): 22-33. Scally, A., Dutheil, J. Y., Hillier, L. W. et al. (2012). Insights into hominid evolution from the gorilla genome sequence. Nature 483: 169-175. Seuánez, H. N., Armada, J. L., Barroso, C., Rezende, C., da Silva, V. F. (1983). The meiotic chromosomes of Cebus apella (Cebidae, Platyrrhini). Cytogenetic and Genome Research 36: 517-524. Seuánez, H. N., Forman, L., Matayoshi, T.; Fanning, T. G. (1989). The Callimico goeldii (Primates, Platyrrhini) genome: Karyology and middle repetitive (LINE-1) DNA sequences. Chromosoma 98: 389-395. Sinclair, A. H., Berta, P., Palmer, M. S., Ross Hawkins, J., Griffiths, B. L., Smith, M. J., Foster, J. W., Frischauf, A. M., Lovell-Badge, R., Goodfellow, P. N. (1990). A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346: 240-244. Skaletsky, H., Kuroda-Kawaguchi, T.; Minx, P. J. et al. (2003). The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423: 825-837. Solari, A. J. (1993). Sex chromosomes and sex determination in vertebrates. CRC Press. Solari, A. J. (1999). Genética Humana. Fundamentos y aplicaciones en medicina. 2nd edition. Editorial Médica Panamericana. Solari, A. J. and Rahn, M. I. (2005). Fine structure and meiotic behaviour of the male multiple sex chromosomes in the genus Alouatta. Cytogenetic and Genome Research 108: 262-267. Stanyon, R., Bigoni, F., Slaby, T., Muller, S.; Stone, G., Bonvicino, C. R., Neusser, M., Seuánez, H. N. (2004). Multi-directional chromosome painting maps homologies between species belonging to three genera of New World monkeys and humans. Chromosoma 113 (6) 305-315. Stanyon, R., Rocchi, M., Capozzi, O., Roberto, R., Misceo, D., Ventura, M., Cardone, M. F., Bigoni, F., Archidiacono, N. (2008). Primate chromosome evolution: ancestral karyotypes, marker order and neocentromeres. Chromosome Research 16: 17-39. Steinberg, E. R., Nieves, M., Mudry, M. D. (2007). Meiotic characterization and sex determination system of neotropical primates: Bolivian squirrel monkey Saimiri boliviensis (Primates: Cebidae). American Journal of Primatology 69: 1236-1241. Steinberg, E. R., Cortés-Ortiz, L., Nieves, M., Bolzán, A. D., García-Orduña, F., HermidaLagunes, J., Canales-Espinosa, D., Mudry, M. D. (2008). The karyotype of Alouatta pigra (Primates: Platyrrhini): mitotic and meiotic analyses. Cytogenetic and Genome Research 122: 103–109. Steinberg, E. R. (2011). Determinación sexual en Primates Neotropicales: el caso de los monos aulladores. PhD Thesis. Buenos Aires University. Argentina. 241 pp.

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Steinberg, E. R., Nieves, M., Mudry, M. D. (2014a). Multiple sex chromosome systems in howler monkeys (Alouatta: Platyrrhini). Comparative Cytogenetics 8(1): 43–69. Steinberg, E. R., Fortes, V. B., Rossi, L. F., Murer, L., Lovato, M., Merani, M. S., Mudry, M. D. (2014b). Cytogenetic characterization of brown howler monkeys, Alouatta guariba clamitans (Atelidae, Platyrrhini): confirmation of an X1X2X3Y1Y2 sex chromosome system. In Anais do 60º Congreso Brasilero de Genética, Sociedade Brasileira de Genética, Guarujá, SP, Brasil. Steinemann, M., Pinsker, W., Sperlich, D. (1984). Chromosome homologies within the Drosophila obscura group probed by in situ hybridization. Chromosoma 91 (12): 46-53. Steinemann, S. and Steinemann, M. (2005). Retroelements: tools for sex chromosome evolution. Cytogenetics and Genome Research 110: 134-143 Veitia, R. A., Salas-Cortés, L., Ottolenghi, C., Pailhoux, E, Cotinot, C., Fellous, M. (2001). Testis determination in mammals: more questions than answers. Molecular and Cellular Endocrinology 179: 3-16. Wienberg, J. (2004). The evolution of eutherian chromosomes. Current Opinion in Genetics and Development 14: 657-666. Wienberg, J. (2005). Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution. Cytogenetic and Genome Research 108: 139-60. Wienberg, J., Jauch, A., Stanyon, R., Cremer, T. (1990). Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8: 347-350. Wimmer, R., Kirsch, S., Rappold, G. A.; Schempp, W. (2005). Evolutionary breakpoint analysis on Y chromosomes of higher primates provides insight into human Y evolution. Cytogenetic and Genome Research 108: 1-3. Worley, K. C., Warren, W. C., Rogers, J., Locke, D. et al. (2014). The common marmoset genome provides insight into primate biology and evolution. The Marmoset Genome Sequencing and Analysis Consortium. Nature Genetics 46: 850-857.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 9

CAN MITOCHONDRIAL DNA, NUCLEAR MICROSATELLITE DNA AND CRANIAL MORPHOMETRICS ACCURATELY DISCRIMINATE DIFFERENT AOTUS SPECIES (CEBIDAE)? SOME INSIGHTS ON POPULATION GENETICS PARAMETERS AND THE PHYLOGENY OF THE NIGHT MONKEYS Manuel Ruiz-García1,, Adriana Vallejo1, Emily Camargo1, Diana Alvarez1, Norberto Leguizamon2 and Hugo Gálvez3 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética. Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC., Colombia 2 Secretaría Distrital Ambiental (SDA), Bogotá DC., Colombia 3 Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA), Estación Experimental, Iquitos, Perú

ABSTRACT We investigated the use of diverse procedures to discriminate among Aotus taxa (Aotus; Cebidae, Platyrrhini) and provide new insights about the systematics and phylogenetic relationships of Aotus taxa. To carry out these tasks we measured 38 craniometric characters in 80 individuals representing eight Aotus taxa. We sequenced 190 specimens at the mitochondrial COII gene for all 13 Aotus taxa recognized to date as well as genotyped (12 nuclear DNA microsatellites) 143 individuals belonging to seven Aotus taxa. The use of skull morphometrics allowed us to differentiate the most southern of the taxa analyzed, Aotus azarae boliviensis. Microsatellite analyses in combination with the FST statistic, gene flow and AMOVA analyses also significantly differentiated A. a. boliviensis. The use of morphometrics and microsatellites did not clearly differentiate A. nancymaae (a red-necked taxon) from all of the other gray-necked Aotus taxa at the 

Correspondence: [email protected], [email protected].

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Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al. northern side of the Amazon River. However, the other more southern Aotus taxa were not included (A. nigriceps, A. infulatus and A. a. azarae) in these analyses. Microsatellites did not detect any significant bottleneck in the three taxa with the highest sample sizes (A. nancymaae, A. vociferans and A. lemurinus griseimembra). The mitochondrial analyses revealed a higher discrimination power than the other two procedures. Two large clusters were found with two relevant sub-clusters inside each one of them: A. nancymaae + A. miconax and all the Northern Amazon taxa (excluding A. trivirgatus) on one side, and A. trivirgatus and all the other Southern Amazon taxa on the other side. In the Northern Aotus group, the morphological and molecular differentiation is very small but the chromosome differentiation is outstanding. This indicates that the fundamental processes for diversification are parapatric chromosomal events (more than peripatric or stasipatric chromosomal events) following fision processes from 2n = 46, 47, 48 (A. vociferans) to 2n = 58 (A. l. lemurinus). Taking all of the data into account we propose that within Aotus, four superspecies could be defined: 1- A. vociferans with six karyomorphs, including vociferans, brumbacki, jorgehernandezi, griseimembra, lemurinus and zonalis; 2- A. trivirgatus, although we are fairly ignorant about this taxon; 3- A. miconax, including A. miconax and A. nancymaae, although we don’t know the karyotype of the original A. miconax and 4- A. azarae with diverse karyomorphs (less differentiated than in A. vociferans), including nigriceps, azarae, boliviensis and infulatus.

Keywords: Aotus, skull morphometrics, nuclear DNA microsatellites, mitochondrial genes, mt COII, parapatric chromosomal speciation, superspecies, phylogenetic inferences

INTRODUCTION The night monkeys are in the genus Aotus, a controversial Neotropical primate taxon in reference to systematics as well as in regard to other Platyrrhini genera. Aotus was initially considered to be a monotypic genus with a single species, Aotus trivirgatus (Hershkovitz, 1949). The genus was originally described by Humboldt in 1812. However, starting in the 1970’s, many authors identified considerable karyotypic variation (Brumback et al., 1971; Brumback 1973, 1974, 1976; Brumback and Willenborg, 1973; Ma 1981a, b, 1983; Ma et al., 1976a, b, 1977, 1978, 1980, 1982, 1985; Thorington and Vorek, 1976). These studies led to the proposal of a number of new species. These pioneer works, such as those of Brumback et al., (1971), Brumback (1973) and Ma et al., (1976a) discovered polymorphic karyotypes in one area of Colombia (2n = 52, 53 and 54) and 2n = 54 in Peru. Indeed, Brumback (1974) reported the finding of a karyotype 2n = 50 for an alleged individual coming from Paraguay. Following these initial cytogenetic discoveries, Hershkovitz (1983) revised the genus and proposed a scheme encompassing nine night monkey species and four subspecies, arranged in two main groups, based on the coloration of the pelage on the sides of the neck. The “graynecked” group is distributed north of the Amazon-Solimoes River (the Northern group), whereas the “red-necked” group occurs south of this River (the Southern group). The first group north of the Amazon River contained four species (Aotus lemurinus [with two subspecies, A. l. lemurinus and A. l. griseimembra], A. brumbacki, A. trivirgatus and A. vociferans). The second group contained five species (Aotus nancymaae, A. miconax, A. nigriceps, A. infulatus and A. azarae with two subspecies, A. azarae azarae and A. a. boliviensis) located mostly south of the Amazon River. In fact, Hershkovitz (1983) studied

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the alleged Paraguayan individual of Brumback (1974), determined its origin to be in the Meta Department in Colombia, and named it A. brumbacki. However, Hershkovitz’s (1983) scheme was challenged by subsequent studies based on cytogenetics, morphology and molecular data. Ford (1994), for example, using multivariate analysis on both craniodental metrical data of 193 Aotus skulls and data on pelage characteristics of 105 adult Aotus skins, questioned the Hershkovitz’s classification. This author concluded that there were only seven species, two Northern species, Aotus trivirgatus and A. vociferans (which included A. lemurinus and A. brumbacki) and five Southern species, A. nancymaae, A. miconax (although both taxa could be a single species), A. nigriceps, A. azarae (only A. a. azarae), and A. infulatus (including infulatus and azarae boliviensis). After consulting with Hershkovitz, Ramírez-Cerqueira (1983) included A. hershkovitzi as an additional fifth northern species based on an inadequate description and considering, particularly, its diagnostic diploid number of 58 and fundamental number of 76, the highest known for the genus. Groves (2001) listed four subspecies of Aotus lemurinus (A. lemurinus lemurinus, A. l. griseimembra, A. l. zonalis, and A. l. brumbacki) in his influential book. He also listed Aotus hershkovitzi, A. trivirgatus, and A. vociferans in the Northern group and A. miconax, A. nancymaae, A. nigriceps, and A. azarae (with three subspecies: A. azarae azarae, A. a. boliviensis and A. a. infulatus) in the Southern group. Groves (2001) accepted brumbacki as a form of lemurinus, whereas infulatus, azarae and boliviensis formed a complex with overlapping characteristics, close to Aotus nigriceps but specifically different. However, earlier, Thorington and Vorek (1976) had criticized the establishment of subspecies for Aotus on the grounds that it would complicate the recognition of discrete populations and mosaic evolution, and that there did not seem to be species-wide phenotypes. Indeed, many authors have emphasized that the morphological similarity of several Aotus taxa has led to frequent misidentifications, mainly at the boundaries of their distribution (Menezes et al., 2010). At least, 18 distinct karyotypes are now known, with 2n varying between 46 and 58 chromosomes (Ma, 1981a, 1983; Ma et al., 1976a, 1985; Galbreath, 1983; Pieczarka et al., 1992, 1993; Torres et al., 1998; Defler et al., 2001; Defler and Bueno, 2007; Menezes et al., 2010). Some karyotype studies do not necessarily support the above taxonomies. For example, Defler et al., (2001) showed that A. hershkovitzi is not differentiated from A. lemurinus lemurinus and that the taxon described by Hershkovitz as A. l. lemurinus was in fact A. l. zonalis. Defler and Bueno (2007) studied a specimen karyotyped by Torres et al., (1998), as 2n = 50, and sampled in the Quindio region of Colombia, and defined this as a new species called A. jorgehernandezi. Ma (1981a) compared the karyotypes of A. nigriceps and A. azarae boliviensis and showed that a fusion of the Y chromosome occurred with the short arm of a medium-size subtelocentric autosome in both taxa. G-banding data demonstrated that this autosome was the same in both taxa (Ma 1981a; Ma et al., 1980), although these taxa differ by three autosomic rearrangements. Similarly, Pieczarka and Nagamachi (1988) and Pieczarka et al., (1993) found that the karyotypes of A. infulatus and A. azarae boliviensis are very similar, suggesting that these are the same species. On the other hand, the sufficient chromosomal differences needed to isolate Aotus individuals should be investigated. For instance, Giraldo et al., (1986) examined 288 Colombian A. l. griseimembra from the lower Río San Jorge (Bolívar Department, Northern Colombia) with 2n = 52, 53 and 54 and they found a balanced polymorphism with no problems for interbreeding among the three karyotypes.

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In consideration of these facts, we have studied three large Aotus samples from three different perspectives (one morphometrics and two molecular ones). The first one was a sample of 80 Aotus skulls, representing eight Aotus taxa: A. l. griseimembra, A. l. zonalis, A. l. lemurinus, A. brumbacki, A. vociferans, A. trivirgatus, A. nancymaae and A. a. boliviensis for a total of 38 cranial, mandibular and teeth traits. In this chapter, we use a mixed nomenclature of Hershkovitz (1983) and Groves (2001) up until we discuss our phylogenetic results. The second one was the largest sample size to date for studying sequences of the mitochondrial cytochrome oxidase subunit II (mt COII). This included 190 Aotus individuals representing, for first time, all the Aotus taxa recognized to date. The mt COII gene has been used to infer phylogenetic relationships in primates (within the Hominoidea, Adkins and Honeycutt, 1991; Ruvolo et al., 1991; within the Cercopithecoidea, Disotell et al., 1992; within the Strepsirrhini, Adkins and Honeycutt, 1994, and within several Neotropical primate genera, including Aotus (Ashley and Vaughn, 1995; Plautz et al., 2009; Menezes et al., 2010; Ruiz-García et al., 2011, 2013), Alouatta (Figueiredo et al., 1998), Ateles (Collins and Dubach, 2000a, b; Ruiz-García et al., 2016a, this volume), Lagothrix (Ruiz-García and Pinedo-Castro, 2010; Ruiz-García et al., 2014a), Saimiri (Ruiz-García et al., 2015), Cebus (Ruiz-García et al., 2010; 2012a,b; 2016b,c,d, this volume) or Saguinus and other Callitrichinae (Sena et al., 2002; Ruiz-García et al., 2014b)). The use of the COII gene (and of mitochondrial coding regions) for inferring primate phylogeny can be problematic because of the rapid rate of molecular evolution at mitochondrial loci and the saturation problem regarding a phylogenetically informative signal at the 3rd position within codons at the intergeneric level. Nonetheless, Ascunce et al., (2003) have shown that the gene can be very informative at the intrageneric level. However, we cannot completely exclude the possibility that some sequences obtained in this study represent numts (mitochondrial DNA fragments inserted into the nuclear genome) rather than true mtDNA (Chung and Steiper, 2008) as we will show. Therefore, special care must be taken to use this marker for phylogenetic inferences in Aotus. The third sample consisted of 143 Aotus individuals representing seven taxa (A. l. griseimembra, A. l. zonalis, A. brumbacki, A. vociferans, A. trivirgatus, A. nancymaae and A. a. boliviensis). It was analyzed for 12 nuclear DNA microsatellites, which are composed of tandem repetitive units of 2–6 base pairs in length (Weber and May, 1989). Microsatellites are randomly distributed, highly polymorphic and frequently found inside eukaryotic genomes. The small amount of DNA needed for these molecular analyses has helped researchers to use noninvasive procedures when sampling wild animals which has in turn led to a strong incorporation of molecular techniques in the study of population biology dynamics (Bruford and Wayne, 1993). All the samples for mt COII gene sequences and microsatellites were obtained from wild organisms. Morphological descriptions and geographical references were recorded to avoid incorrect classifications of samples. We can see many incorrect individuals and sequences in some articles as well as in the GenBank because they do not correspond to their real taxa. Here we highlight some of these cases. For instance, we detected: the sample of an alleged A. a. boliviensis from Ashley and Vaughn (1995) is really A. a. azarae; a sample of an alleged A. trivirgatus of Collura et al., (unpublished) is really A. l. griseimembra; the sample of an alleged A. nigriceps of Plautz et al., (2009) is really A. trivirgatus; the sample of an alleged A. a. azarae of Plautz et al., (2009) is really A. nigriceps; the alleged A. trivirgatus from GenBank (AY250707) is really A. l. griseimembra; two alleged A. nancymaae from GenBank (AJ489745 and AJ489746) are really two A. l.

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griseimembra and even one alleged A. a. azarae from GenBank (AF181085) is really a Saimiri. There are even some mistakes of the Aotus 2n karyotype in the bibliography (for example, Menezes et al., 2010, cited 2n = 49m/50h for A. nigriceps or 2n = 50 for A. vociferans). Thus it’s extremely important to include the correct geographical origins and morphotypes of Aotus individuals for molecular and karyotype studies. As discussed above, the topic of night monkey systematics presents a very interesting problem but these monkeys are also extremely interesting for another reason. The Aotus species have been used in the search for vaccines against malaria and other tropical diseases. However, not all of the Aotus species are useful for this task. This is due to the fact that different Aotus taxa have different susceptibility to the malaria parasite (Schmidt, 1973). This has generated an intense legal and illegal traffic of these monkeys to diverse laboratories of the world. Especially important seems to be the intense illegal traffic of individuals of different Aotus taxa (especially, A. nancymaae) from Peru with destination to Colombia (Maldonado, 2011; Maldonado and Peck, 2014; Maldonado et al., 2009; Ruiz-García et al., 2012a). The main aims of the current work are therefore to determine the power of discrimination of different characters (craniometrics, mitochondrial DNA and nuclear DNA microsatellites) to differentiate different Aotus taxa and to bring forward new insights of the systematics of Aotus. We especially focus on the Northern group (sensu Hershkovitz 1983) with reference to its number of species, which ranges from only two species (Ford, 1994) to seven species (Defler and Bueno, 2007) depending on authors.

MATERIAL AND METHODS Morphological Samples and Procedures We applied different multivariate techniques to analyze relationships among 80 Aotus skulls. These 80 samples were distributed as follows: 21 skulls of A. l. griseimembra (Colombia), 20 skulls of A. a. boliviensis (Bolivia), nine skulls of A. nancymaae (Peru and Brazil), eight skulls of A. vociferans (Colombia), seven skulls of A. brumbacki (Colombia), six skulls of A. l. zonalis (Colombia), four skulls of A. l. lemurinus (Colombia), four skulls of Aotus sp (Colombia) and one skull of A. trivirgatus (Venezuela). A total of 38 cranial, mandibular and teeth traits were measured (Table 1). In the first analysis we did not use any type of standardization or transformation to determine the simultaneous impact of size and shape among the individuals analyzed. Different distance matrices (correlation, variance-covariance, Euclidean and Manhattan distances; Sneath and Sokal, 1973; Marcus, 1990) were calculated among the individuals analyzed. Each one of these procedures has different mathematical properties, which must be evaluated to see the effects on the results. To establish possible relationships among the 80 Aotus skulls, a nonmetric multidimensional scaling analysis (MDS) was applied to the distance matrices following Kruskal (1964a,b). The stress statistic was estimated to measure the goodness of fit of the distances in the configuration space to the monotone function of the original distances. We choose the analyses with the best values for the stress statistic following Spence (1972) and Spence and Ogilvie (1973). A graphic matrix (“Minimum

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Spanning tree”) was superimposed (Gower and Ross, 1969; Rohlf, 1970) in order to see the probable local distortion generated by the process of dimensional reduction. These analyses were carried out with the program NTSYS 2.02g (Rohlf, 2000). Table 1. Cranial, mandible and dental measurements (38) analyzed in 80 skulls from eight different Aotus taxa 1-Maximum Transversal Width 2-Zygomatic Width 3-Superior Facial Height 4-Total Facial Height 5-Nasal Width 6-Bigonian Width 7-Auricular Height 8-Greatest Skull Length 9-Nasal Height 10-Minimum Postorbital Width 11-Maximum Postorbital Width 12-Lower Face Length 13-Base Face Length 14-Basal Height 15-Palate Length 16-Palate Width 17-Foramen Magnum Length 18-Foramen Magnum Width 19-Symphisis Height 20-Maximum Length of Mandible 21-Mandibular body Height between P1 and P2 22-Mandibular body Height between M1 and M2 23-Mandibular body Height between M2 and M3 24-Mandibular Branch Width 25-Mandibular Branch Height 26-Biauricular Breadth 27-Upper Canine Length 28-Lower Canine Length 29-Upper Canine Breadth 30-Lower Canine Breadth 31-Upper Molar Length 32-Lower Molar Length 33-Upper Molar Breadth 34-Lower Molar Breadth 35-Maximum Biorbital Width 36-Orbital Height 37-Opistion-Nasal Spine-Opistion Distance (subnasal prognathisme) 38-Ectoconion-Nasion-Ectoconion Distance

In order to detect differences among the different Aotus taxa, a Canonical Population analysis (PCA) was performed. This method separates groups along axes with high discrimination power (canonic axes) using the Mahalanobis distance (Mahalanobis, 1936).

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This analysis is based on two hypotheses: there is homogeneity between all covariance matrices corresponding to the population groups (this was verified with a maximumlikelihood test) and the means of the k groups must be significantly different. To contrast this hypotheses, the Wilks’s  and the Fisher-Snedecor F associate value test by means of the Rao (1951) approximation was used. Subsequently, a canonical transformation was made and the eigenvalues and the significance of the first canonical axes with Barlett’s test were calculated. Additionally, the factorial structure of the canonic variables, the canonical representation and the confidence region radius at 90% level, were determined. The expression for the radius is R/N1/2, where R2 = F (N – k) n / (N – k- n + 1), with P (F > F) = 1 -  for the FisherSnedecor distribution with n and N – k – n – 1 degrees of freedom (N = total population number, k = number of population groups, and n = number of variables). This analysis was performed using the MULTICUA Software created by Cuadras (1991). Burnaby’s size adjustment method was also applied (Burnaby, 1966). This is useful because this procedure determines the relationships among the individuals exclusively by shape using the program NTSYS 2.02g (Rohlf, 2000). The relationships among the individuals were undertaken with an UPGMA tree. These three sets of analyses were performed for all the morphometric characters studied (38), the cranial characters (24), and the mandible and teeth characters (14).

Molecular and Sample Procedures In the wild, we directly sampled 190 Aotus specimens for the mt COII gene as follows: 1A. vociferans: 43 individuals (32 from Colombia, two from Ecuador, two from Brazil and seven from Peru); 2- A. l. zonalis: two individuals from Colombia; 3- A. l. griseimembra: 44 individuals from Colombia; 4- A. brumbacki: seven individuals from Colombia; 5- A. jorgehernandezi: one individual from the Quindio Department (Colombia); 6- A. l. lemurinus: five individuals from Colombia; 7- A. nancymaae: 48 individuals, all from different areas of Peru (Tapiche, Ucayali, Huallaga, Nanay, Amazon rivers); 8- A. miconax: two individuals from Peru (Bongaro and another individual confiscated in Chiclayo); 9- A. trivirgatus: four individuals from Northern Brazil (Negro River and affluent); 10- A. nigriceps: 10 individuals (six from Brazil and four from Peru); 11- A. infulatus: eight individuals from Brazil; 12- A. a. boliviensis: five individuals (two from Rondonia, Brazil and three from the Santa Cruz Department in Bolivia); 13- A. a. azarae: nine individuals from Argentina (Corrientes and Formosa) and two individuals whose specific designations are dubious (one albine individual obtained in Iquitos, Peru [A. nancymaae or A. vociferans] and one individual from the Madre de Dios River in Southern Peru [A. nigriceps or A. a. boliviensis]). The following species were used as outgroups: seven individuals of different subspecies of Cebus albifrons (Colombia and Ecuador), seven individuals of C. capucinus (Colombia and Costa Rica), three individuals of Saimiri oerstedii citrinellus (Costa Rica), five individuals of S. cassiquiarensis albigena (Colombia), two S. sciureus (French Guiana) and four individuals of S. ustus (Brazil). For the microsatellite analysis, 143 Aotus specimens were analyzed: 48 individuals of A. l. griseimembra (Colombia), 10 individuals of A. l. zonalis (Colombia), 12 individuals of A. brumbacki (Colombia), 28 individuals of A. vociferans (Colombia and Peru), four individuals

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of A. trivirgatus (Brazil), 32 individuals of A. nancymaae (Peru) and nine individuals of A. a. boliviensis (Bolivia). Our sampling procedures complied with all the protocols approved by the Ethical Committee of the Pontificia Universidad Javeriana (No. 45677) and the laws of the Ministerio de Ambiente, Vivienda y Desarrollo Territorial (R. 1252) from Colombia. This research also adhered to the American Society of Primatologists’ Principles for the Ethical Treatment of Primates.

Mitochondrial Sequences Samples of blood (one ml) from animals captured in Peru and Colombia as well as samples of hair (with follicles) from individuals kept as pets by Indian communities throughout Colombia, Peru, Ecuador, Bolivia, Brazil and Paraguay were preserved in disodic EDTA. We obtained blood DNA using phenol-chloroform (Sambrook et al., 1989), and hair DNA using 10% Chelex resin (Walsh et al., 1991). We used the primers L6955 (5’AACCATTTCATAACTTTGTCAA-3’) and H7766 (5’-CTCTTAATCTTTAACTTAAAAG3’) to amplify (Polymerase Chain Reaction, PCR) the mt COII gene (located in the lysine and asparagines tRNAs) (Ashley and Vaughn, 1995; Collins and Dubach, 2000a). We performed each PCR in a 50-l volume with reaction mixtures including 4 l of 10x Buffer, 6 l of 3mM MgCl2, 2 l of 1 mM dNTPs, 2 l (8pmol) of each primer, 2 units of Taq DNA polymerase, 13.5 l of H20 and 2 l of DNA. PCR reactions were carried out in a Geneamp PCR system 9600 (Perkin Elmer) and in a Bio-Rad thermocycler. We used the following temperatures and cycles: 95°C for 5 minutes, 35 cycles of 45 s at 95°C, 30 s at 50°C and 30 s at 72°C and a final extension time for 5 minutes at 72°C. Using the molecular weight marker X174 DNA digested with Hind III and Hinf I, we checked all the amplifications, including positive and negative controls, in 2% agarose gels. We purified the amplified samples with membrane-binding spin columns (Qiagen), directly sequenced the double-stranded DNA in a 377A (ABI) automated DNA sequencer in both directions and then repeated the sequencing of each sample to ensure accuracy.

Microsatellite Markers We used 12 microsatellite markers (AP40, AP68, AP74, D5S111, D5S117, D6S260, D8S165, D14S51, D17S804, PEPC3, PEPC8 and PEPC59). The AP74, AP68 and AP74 markers were designed for Alouatta palliata and PEPC3, PEPC8 and PEPC59 for Cebus apella, while the remaining markers were designed for humans (Ellesworth and Hoelzer, 1998). These microsatellites have been successfully used in other Neotropical primates such as Alouatta, Ateles, Lagothrix, Cebus, Saimiri, Callicebus and Saguinus (Ruiz-García, 2005; Ruiz-García et al., 2006, 2007 and in this book). Our final PCR volume and reagent concentrations for the DNA extraction from blood were 25 μl, with 3 μl of 3 mM MgCl2, 2.5 μl of buffer 10×, 1 μl of 0.4 mM dNTP, 1 μl of each primer (forward and reverse; 4 pmol), 13.5 μl of H2O, 2 μl of DNA, and 1 Taq polymerase unit per reaction (1 μl). For the PCR reactions with hair, the overall volume was 50 μl, with 20 μl of DNA and twofold amounts of

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MgCl2, buffer, dNTPs, primers, and Taq polymerase. We performed all PCR reactions in a PerkinElmer Geneamp PCR System 9600 thermocycler for 5 min at 95°C, 30 1-min cycles at 95°C, 1 min at the most accurate annealing temperature (57°C for AP40, 50°C for AP68, and 52°C for the remaining markers), 1 minute at 72°C, and 5 min at 72°C. We kept amplification products at 4°C until they were used in a denatured 6% polyacrylamide gel in a Hoefer SQ3 sequencer vertical chamber. Depending upon the size of the markers analyzed, and the presence of 35 W as a constant, we stained the gels with AgNO3 (silver nitrate) after 2–3 h of migration. We used the molecular markers HinfI and ϕ174 (cut with HindIII). We repeated the PCR reactions three times for DNA extracted from hair. Thus, allelic dropout was highly improbable, but we cannot completely exclude the existence of null alleles, which could increase the number of false homozygous genotypes. Nevertheless, it is improbable that all loci were affected in the same way.

Mitochondrial Phylogenetics Procedures The Akaike information criterion (AIC; Akaike, 1974; Posada and Buckley, 2004) and the Bayesian information criterion (BIC; Schwarz, 1978) were used to determine the best evolutionary nucleotide model for the mt COII gene sequences. Additionally, we obtained maximum likelihood estimates of transition/transversion bias (Tamura et al., 2013). The Kimura (1980) 2P genetic distance matrix among all the Aotus taxa (11) pairs was estimated with their standard errors by means of a 10,000 maximum likelihood permutation. Phylogenetic trees were constructed by using two procedures: 1- a Maximum Likelihood tree (ML) obtained with RAxML v.7.2.6 Software (Stamatakis, 2006) and 2- a Minimum Evolution tree (ME) with the Kimura (1980) 2P genetic distance obtained with Mega 6.05 Software (Tamura et al., 2011). The significance of the tree nodes was measured with 100 bootstraps.

Microsatellite Statistical Analyses The coalescence theory generated by Beaumont and Nichols (1996) was used to detect whether the microsatellites used were effected by constrictive or diversifying natural selection within the Aotus genus. We used the fdist program and we obtained the observed and expected FST statistical values for each marker used throughout the samples. Both the infinite allele (IAM) and the step-wise (SMM) mutation models were considered. A total of 5,000 iterations were completed to calculate the values that represented the relationship between the FST statistic and the expected heterozygosity of the markers. The iterations were grouped into batches of 200 from which the medians and the 2.5% and the 97.5% quartiles were calculated. The observed FST and the heterozygosity values were superimposed under this distribution and distributed by the median and the quartiles. Values that are outside of this theoretical distribution indicate that the microsatellites in question are being affected by natural selection. The genetic heterogeneity among the seven Aotus taxa analyzed with microsatellites was studied for each marker. For this, we used the gene frequencies of the 12 microsatellites relying on exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches,

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and 5,000 iterations per batch. A FST population comparison pair analysis with exact tests with Markov chains and the same parameters as above were also undertaken. A Wright Fstatistics analysis (Wright, 1951) with the procedure of Michalakis and Excoffier (1996) was carried out. The standard deviations of the F-statistics were calculated using a jackknifing over loci and the 99% confidence intervals were measured by means of bootstrapping over loci. The following procedure was used to measure the significance of FST. It used 10,000 randomizations of genotypes among populations and did not assume random mating within populations by means of the log-likelihood G test (Goudet et al., 1996). The significance of FIS and FIT was also found by using 10,000 randomizations of alleles within samples and in the overall sample. Additionally, the gene diversity analysis of Nei (1973) was also undertaken. Possible theoretical gene flow estimates among all the Aotus taxa studied taken together were measured using the private allele model (Slatkin, 1985; Barton and Slatkin, 1986) as well as by taxa pairs from FST. All these analyses were carried out by means of the Genepop v. 4.2.1 Software (Raymond and Rousset, 1995), Arlequin 3.5.1.2 Software (Excoffier et al., 2005) and FSTAT.2.9.1 Software (Goudet, 1995). We used an AMOVA analysis of the Aotus taxa studied to determine the distribution of gene diversity at different hierarchical geographical levels (Excoffier et al., 1992). Two analyses were done, one considering the Amazon River dividing the Northern Aotus population (A. l. zonalis, A. l. griseimembra, A. brumbacki, A. vociferans and A. trivirgatus) and the Southern Aotus population (A. nancymaae and A. a. boliviensis) following the Hershkovitz’s (1983) hypothesis and a second one with all the Aotus taxa in a group and A. a. boliviensis in another group (hypothesis where this taxon is the most differentiated of all those studied for microsatellites). The fixation indices of Wright (1951) were estimated in the AMOVA analysis: sc (variation of populations within the groups), ct (variation among groups) and st (variation among individuals). This analysis was carried out by means of the Arlequin 3.5.1.2 Software (Excoffier et al., 2005). To determine if the degree of microsatellite differentiation was extreme among the Aotus taxa, we developed two assignment analyses by using the GENECLASS 2 Program (Piry et al., 2004). We performed two strategies, one Bayesian method (Cornuet et al., 1999) with the “leave one out” procedure and one genetic distance method (standard genetic distance, Nei, 1972) with the “as is” procedure. The assignation analyses were carried out without simulations and served to estimate the probabilities of individuals belonging or being excluded from the original populations where they were “a priori” assigned (P < 0.05). Another assignment analysis was applied using Structure 2.3 (Falush et al., 2007). It employs Markov Chain Monte Carlo procedures and the Gibbs sampler and uses multilocus genotypes to infer population structure, and simultaneously assigns individuals to specific populations. The model considers K populations, where K may be unknown, and the individuals are assigned tentatively to one population or jointly to ≥ 2 populations (if their genotypes are considered admixed). Two analysis groups were carried out. First, we considered the admixture model, wherein the individuals may have mixed ancestry and the no-admixture model, both with no prior population information to assist with clustering (USEPOPINF = 0). In addition,  was inferred (Dirichlet parameter for degree of admixture; with an initial value of  = 1) using uniform priors (its value was the same for all populations). The maximum value of this parameter was 10. Allele frequencies were uncorrelated among populations, assuming different values of FST for each population. We revealed the presence of the most

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probable number of gene pools by using the increasing likelihood method. The second analysis was undertaken with a model that incorporates informative geographic origin individual priors to assist with the clustering of weakly structured data in order to determine migrants or detect slightly different populations (USEPOPINF = 1) with both admixture and no admixture models. Furthermore, in this case, in order to apply the same conditions to that of the previous case, we introduced LocPrior = 1, Gensback = 2, and Migprior = 0.05. The program was run with 1,000,000 iterations after a burn period of 100,000 iterations for each analysis. Each analysis was performed twice with convergent results. The last analyses were focused on the detection of recent bottleneck events using the theory generated by Cornuet and Luikart (1996) and Luikart et al., (1998). The population, which experienced a recent bottleneck, simultaneously decreases the allele number and the expected levels of heterozygosity. Nevertheless, the allele number (ko) is reduced faster than the expected heterozygosity. Therefore, the value of the expected heterozygosity calculated throughout the allele number (Heq) is lower than the obtained expected heterozygosity (He). For neutral markers, in a population in gene mutation drift equilibrium, there is an equal probability that a given locus has a slight excess or deficit of heterozygosity in regard to the heterozygosity calculated from the number of alleles. In contrast, in a bottlenecked population, a large fraction of the loci analyzed will exhibit a significant excess of the expected heterozygosity. To measure this probability, four diverse procedures were used as follows: sign test, standardized difference test, Wilcoxon´s signed rank test and graphical descriptor of the shape of the allele frequency distribution. A population, which did not suffer a recent bottleneck event, will yield an L-shape distribution (such as expected in a stable population in mutation-gene drift equilibrium), whereas a recently bottlenecked population will show a mode-shift distribution. The Wilcoxon´s signed rank test probably has its greatest power when the number of loci analyzed is low, such as in the current case. The BOTTLENECK Software (Piry et al., 1999) was used for this task. Another procedure used to detect any possible bottleneck was that created by Garza and Williamson (2001). This procedure is based on the ratio M = k/r, where k is the total number of alleles detected in a locus given and r is the spatial diversity; that is, the distance between alleles in number of repeats and the overall range in allele size. When a population is reduced in size, this ratio will be smaller than in equilibrium populations. To calculate this M value, the program will simulate an equilibrium distribution of M and give assumed values for three parameters of the two phase mutation model ( = 4Ne, ps = mean percentage of mutations that add or delete only one repeat unit, and g = mean size of larger mutations). Once M is obtained, it is ranked relative to the equilibrium distribution. Using conventional criteria, there is evidence of a significant reduction in population size if less than 5% of the replicates are below the observed value. The average values used in this analysis were obtained from the MISAT program by Nielsen (1997). This analysis was carried out with the M-P-Val and Critical-M programs from Garza and Williamson (2001).

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Figure 1. Multidimensional Scaling Analysis (MDS) by using correlations with all 38 cranial, mandible and teeth characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was superimposed to connect the different specimens studied. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

RESULTS Morphological Results Different morphological analyses are shown in Figures 1 (MDS analysis with correlations with all the characters measured), 2 (MDS analysis with the Euclidean distance only for cranial characters), 3 (MDS analysis with the Euclidean distance only for mandible and teeth characters), 4 (PCA with cranial characters), 5 (PCA with mandible characters), 6 (UPGMA with all the characters measured with the Euclidean distance with the Burnaby’s procedure), 7 (UPGMA with the cranial characters with correlations and the Burnaby’s procedure) and 8 (UPGMA with the mandible and teeth characters with correlations and the Burnaby’s procedure). All the analyses, independent of the effects of size + shape or only shape in the cranial or mandible characters (or both together), yielded the same results. All the individuals

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of the different Aotus taxa were intermixed. Only a large fraction of A. a. boliviensis individuals were differentiated from the other Aotus taxa. This was especially detected in the PCA analyses as well as in the UPGMA trees with the Burnaby’s procedure, where a nested cluster with a large fraction of the individuals of A. a. boliviensis was observed. Therefore, with the exception of A. a. boliviensis, morphometrics of skulls and mandibles did not differentiate Aotus taxa.

Figure 2. Multidimensional Scaling Analysis (MDS) by using the Euclidean distance with only the 24 cranial characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was superimposed to connect the different specimens studied. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

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Figure 3. Multidimensional Scaling Analysis (MDS) by using the Euclidean distance with only the 14 mandible and teeth characters measured in different Aotus taxa. A Minimum Spanning Tree (MST) was superimposed to connect the different specimens studied. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

Figure 4. Population Canonic Analysis (PCA) with only the 24 cranial characters measured in different Aotus taxa. Ab = A. brumbacki; Alg = A. l. griseimembra; Alz = A. l. zonalis; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.

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Figure 5. Population Canonic Analysis (PCA) with only the 14 mandible and teeth characters measured in different Aotus taxa. Ab = A. brumbacki; Alg = A. l. griseimembra; Alz = A. l. zonalis; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.

Mitochondrial Analyses With BIC, the best substitution model for the mt COII sequences was Tamura 92 + G (24,792.431), whereas for the AIC it was General Time Reversible + G (20,396.182). The estimated transition/transversion bias was 4.19 (maximum Log likelihood = -9,883.810). Table 2. Kimura (1980) 2P genetic distances at the mitochondrial COII gene among the different Aotus taxa considered. Below, genetic distance values in percentages (%). Above, standard errors in percentages. 1 = A. l. griseimembra-A. l. zonalis; 2 = A. l. lemurinus; 3 = A. jorgehernandezi; 4 = A. brumbacki; 5 = A. vociferans; 6 = A. nancymaae; 7 = A. miconax; 8 = A. trivirgatus; 9 = A. nigriceps; 10 = A. azarae; 11 = A. infulatus-A. a. boliviensis Taxa 1 2 3 4 5 6 7 8 9 10 11

1 1.8 3 2.7 4.3 5.8 4.4 5.6 4.7 5.7 4.8

2 0.3 2.9 2.5 4.2 5.1 3.7 4.7 3.9 5.1 4.1

3 0.4 0.5 2.2 3.4 5.8 4.4 6.6 4.7 5.8 5.0

4 0.3 0.4 0.3 3.5 5.6 4.3 5.8 4.5 5.4 4.7

5 0.5 0.6 0.4 0.4 5.7 4.4 6.1 5.2 6.1 5.6

6 0.7 0.7 0.6 0.6 0.5 2.0 6.6 6.2 7.3 6.6

7 0.6 0.7 0.7 0.6 0.5 0.3 5.2 4.7 5.8 5.1

8 0.7 0.7 0.8 0.8 0.8 0.8 0.8 4.3 6.2 4.6

9 0.7 0.7 0.7 0.7 0.7 0.8 0.8 0.7 3.8 1.7

10 0.7 0.7 0.8 0.8 0.8 0.9 0.9 0.9 0.6 3.0

11 0.7 0.6 0.7 0.7 0.7 0.7 0.7 0.7 0.4 0.5 -

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Figure 6. UPGMA phenogram with all 38 cranial, mandible and teeth characters measured in different Aotus taxa by using the Euclidean distance with the Burnaby’s procedure to delete the effect of the size. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

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Figure 7. UPGMA phenogram with only the 24 cranial characters measured in different Aotus taxa using correlations with the Burnaby’s procedure to delete size effect. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

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Figure 8. UPGMA phenogram with only the 14 mandible and teeth characters measured in different Aotus taxa using correlations with the Burnaby’s procedure to delete size effect. All = Aotus l. lemurinus; Alz = A. l. zonalis; Alg = A. l. griseimembra; Ab = A. brumbacki; Av = A. vociferans; An = A. nancymaae; At = A. trivirgatus; Aa, Aspb, Atb = A. azarae boliviensis; Asp = A. sp.

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Table 2 shows the Kimura 2P genetic distance among Aotus taxa pairs. The lowest genetic distance pairs were for A. nigriceps-A. infulatus (1.7%), A. l. griseimembra-A. l. lemurinus (1.8%), A. nancymaae-A. miconax (2.0%), A. brumbacki-A. jorgehernandezi (2.2%), A. brumbacki-A. l. lemurinus (2.5%), A. l. griseimembra-A. brumbacki (2.7%), A. jorgehernandezi-A. l. lemurinus (2.9%), A. l. griseimembra-A. jorgehernandezi (3.0%), A. a. azarae-A. infulatus and bolivianus (3.0%), A. vociferans-A. jorgehernandezi (3.4%) and A. vociferans-A. brumbacki (3.5%). The highest values of genetic distance pairs were for A. vociferans-A. a. azarae (6.1%), A. vociferans-A. trivirgatus (6.1%), A. nancymaae-A. nigriceps (6.2%), A. a. azarae-A. trivirgatus (6.2%), A. jorgehernandezi-A. infulatus and bolivianus (6.6%), A. jorgehernandezi-A. trivirgatus (6.6%) and A. jorgehernandezi-A. a. azarae (7.3%). The ML tree (Figure 9a) and the ME tree with the Kimura 2P genetic distance (Figure 9b) basically showed the same trends. The most differentiated cluster was composed of 22 sequences where mixed Aotus taxa were represented (A. vociferans, A. l. griseimembra, A. nigriceps, A. a. boliviensis, A. brumbacki, A. l. lemurinus and A. nancymaae). The most striking fact is that this cluster is outside of the relationship between the designed outgroup (Cebus and Saimiri) and the remaining Aotus. The fact, that this cluster was constituted by animals of different Aotus taxa and that they were more differentiated from the other Aotus individuals than the Cebus and Saimiri were, suggests that the amplifications of these 22 Aotus individuals should correspond to numts (mitochondrial DNA fragments inserted into the nuclear genome). This is extremely interesting because we don’t detect numts for the mt COII gene in many other genera of Neotropical primates that we have studied (Alouatta, Ateles, Callicebus, Cebus, Lagothrix, Pithecia, Saguinus and Saimiri). If so, the sequencing of the mt COII gene in Aotus should be done very carefully to not enclose numts sequences which could affect phylogenetic inferences. Within the Aotus cluster, the first big cluster [99 (99)%, these numbers are the bootstraps for the two commented trees] was comprised of four well defined subclusters: trivirgatus (99 [99]%), azarae (79 [73]%), nigriceps (77 [71]%) and infulatus with boliviensis (97 [93]%). The second big cluster was integrated by all the A. nancymaae’s individuals sampled plus the two A. miconax’s individuals sampled [99 (99)%]. The third large cluster contained mixed haplotypes of A. vociferans, A. l. griseimembra, A. l. lemurinus, A. l. zonalis, A. brumbacki and A. jorgehernandezi [75 (73)%]. The outgroups, Cebus (integrated by C. capucinus and different traditional subspecies of C. albifrons: pleei, versicolor, cesarae, albifrons, aequatoralis and malitosus) and Saimiri (integrated by the traditional oerstedii citrinellus, albigena, sciureus and ustus) showed elevated percentages of bootstraps (99 [99] and 99 [99]%, respectively). Therefore, the mitochondrial gene sequences differentiated more clearly many taxa of Aotus with reference to the discriminatory power of the cranial biometric characters, showing three or four large groups of Aotus.

Microsatellite Analyses The application of the fdist program showed that, independently of the mutational model (IAM and SMM), the markers D5S111, D5S117, PEPC59 and D6S269 could be influenced by constrictive natural selection. The remaining microsatellites didn’t deviate from a neutral model (Kimura, 1983) (Figure 10a, b).

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The genic differentiation of the seven Aotus taxa (with microsatellites and 12 markers) is displayed in Table 3. All the markers, with the exception of AP40, significantly discriminated the seven taxa when they were taken as a whole (Bonferroni’s correction). Table 4 shows the gene differentiation by Aotus taxa pairs by using the FST statistic and exact probabilities. The significant pairs were A. brumbacki-A. vociferans, A. l. zonalis-A.a. boliviensis, A. brumbacki-A. a. boliviensis, A. vociferans-A. a. boliviensis and A. nancymaae-A. a. boliviensis. Clearly, A. a. boliviensis was the most differentiated Aotus taxon of all those analyzed with microsatellites, similar to the morphometric analysis.

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Figure 9. Maximum likelihood (ML) tree for 190 Aotus individuals representing the 13 Aotus taxa recognized to date by using mitochondrial COII gene sequences. Number in the nodes are bootstrap percentages (A). Minimum Evolution (ME) tree with the Kimura (1980) 2P genetic distance for 190 Aotus individuals representing the 13 Aotus taxa recognized to date by using mitochondrial COII gene sequences. Number in the nodes are bootstrap percentages (B).

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Figure 10. Fdist analysis to detect possible natural selection on the 12 nuclear DNA microsatellites applied to seven Aotus taxa. For an infinite allele mutation model (IAM) (A). For a step-wise mutation model (SMM) (B).

Globally, the gene flow estimate with the private allele method for all seven Aotus taxa taken simultaneously was Nm = 0.305. This indicates very low gene flow values for the existence of a unique Aotus taxon. Table 5 shows gene flow estimation pairs for all the Aotus taxa analyzed. All the gene flow estimation pairs were higher than Nm = 1, with the exception of the pairs, A. l. zonalis-A.a. boliviensis (Nm = 0.302), A. brumbacki-A. a. boliviensis (Nm = 0.446) and A. vociferans-A. a. boliviensis (Nm = 0.413). This validates A. a. boliviensis as the most genetically disconnected of the Aotus taxa analyzed with microsatellites.

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Table 3. Genic differentiation at 12 nuclear DNA microsatellites taken simultaneously seven Aotus taxa by means of exact tests. * Significant probability with Bonferroni correction Locus Probability PEPC3 0.000001* PEPC8 0.000001* PEPC59 0.00316* AP68 0.000001* AP40 0.01890 D5S111 0.000001* D5S117 0.000001* D6S269 0.000001* D8S165 0.000001* D14S51 0.000001* D17S804 0.000001* 2 = Infinite Degree of freedom = 24 Overall probability with the Fisher method= Highly significant

Standard Error 0.00000 0.00000 0.00046 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000

Table 4. Genetic differentiation by means of the FST statistic (with correction by sample size) by Aotus taxa pairs (seven taxa) throughout the results of 12 nuclear DNA microsatellites. 1 = A. l. griseimembra; 2 = A. l. zonalis; 3 = A. brumbacki; 4 = A. trivirgatus; 5 = A. vociferans; 6 = A. nancymaae; 7 = A. azarae boliviensis. * P < 0.0001, significant heterogeneity Aotus taxa 1 2 3 4 5 6 7

1 0 0 0.151 0 0.026 0.467*

2

3

4

5

6

7

0 0 0 0 0.453*

0.007 0.101* 0.016 0.359*

0.173 0.097 0.133

0.023 0.377*

0.159*

-

Table 5. Gene flow estimates (Nm) by means of the FST statistic (with correction by sample size) by Aotus taxa pairs (seven taxa) throughout the results of 12 nuclear DNA microsatellites. 1 = A. l. griseimembra; 2 = A. l. zonalis; 3 = A. brumbacki; 4 = A. trivirgatus; 5 = A. vociferans; 6 = A. nancymaae; 7 = A. azarae boliviensis. Inf = Infinite Aotus taxa 1 2 3 4 5 6 7

1 Inf Inf 2.815 Inf 18.871 0.552

2

3

4

5

6

7

Inf Inf Inf Inf 0.603

62.823 4.414 31.572 0.891

2.390 4.640 3.246

21.012 0.826

2.644

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Table 6. Genetic diversity analysis for night monkeys (Aotus) by means of the Nei’s statistics (Ho = observed gene diversity in the total Aotus population; HS = average gene diversity within the Aotus taxa; HT = expected gene diversity in the total Aotus population; DST = absolute gene differentiation among Aotus taxa; GST = relative genetic differentiation among Aotus taxa with regard to the total gene diversity; HT,’ DST’ and GST’= the same as the previous statistics but corrected by sample size Locus PEPC3 PEPC8 PEPC59 AP68 AP40 AP74 D5S111 D5S117 D6S269 D8S165 D14S51 D17S804 All the loci taken together

Ho 0.296 0.239 0.639 0.237 0 0.308 0.403 0.283 0.326 0.423 0.431 0.376 0.330

HS 0.586 0.574 0.741 0.371 0.081 0.688 0.769 0.641 0.611 0.814 0.715 0.774 0.614

HT 0.809 0.884 0.871 0.820 0.083 0.946 0.910 0.880 0.913 0.898 0.915 0.859 0.816

DST 0.223 0.310 0.130 0.449 0.002 0.257 0.141 0.239 0.302 0.084 0.2 0.086 0.202

DST’ 0.278 0.387 0.173 0.539 0.003 0.3 0.169 0.318 0.362 0.098 0.240 0.1 0.252

H T’ 0.864 0.961 0.914 0.909 0.083 0.989 0.938 0.960 0.913 0.912 0.955 0.874 0.866

GST 0.276 0.351 0.149 0.548 0.028 0.272 0.155 0.271 0.330 0.093 0.219 0.1 0.247

GST’ 0.322 0.403 0.189 0.593 0.034 0.304 0.180 0.332 0.372 0.107 0.251 0.115 0.291

The Nei’s gene diversity analysis (Table 6) and the Wright F statistics analysis (Table 7) showed an overall significant genetic heterogeneity: GST = 0.247-0.291, FST = 0.238 ± 0.037 with jackknifing over loci, FST = 0.159-0.339 (99% confidence interval). However, some individual microsatellites did not show a significant genetic differentiation with 10,000 randomizations and the log-likelihood G test and the Bonferroni’s correction. This was true for PEPC59, AP40, D5S111, D5S117 and D6S269. The FIT and FIS statistics generally showed significant homozygote excess, with the exception of PEPC59 and AP40. These results agree well with the existence of different species of Aotus and with some genetic fragmentation within. This is true at least, in some of these species but with the differences among these species not being very high. If A. a. boliviensis is extracted from these analysis, the degree of genetic differentiation considerably decreases. The AMOVA with two groups (north and south of the Amazon River) for all the 12 microsatellites analyzed showed that 94.78% of the genetic variance was among the considered taxa (FST = 0.0522; P = 0.123 ± 0.008). Only 8.59% of the genetic variance was due to being north and south from the Amazon River (FCT = 0.085; P = 0.141 ± 0.009). The genetic variance among taxa within the groups was practically null (FSC = -0.03; P = 0.596 ± 0.017). If we only take the three most polymorphic loci (AP74, D6S269 and D17S804), the levels with significant genetic variance were within the considered taxa (75.72% of genetic variance; FST = 0.248; P = 0.000 ± 0.000) and among taxa within the groups (27.53% of genetic variance; FSC = 0.266; P = 0.000 ± 0.000). However, the genetic variance between those north and south of the Amazon River was practically nonexistent (FCT = -0.032; P = 0.569 ± 0.012).

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Table 7. Wright F statistics, estimated by jackknifing over loci, for the seven Aotus taxa studied at 12 nuclear DNA microsatellites. * P < 0.05 for FIT; + P < 0.05 for FIS; # P < 0.05 for FST and no assuming random mating within populations by means of the loglikelihood G test (Goudet et al., 1996) PEPC3 FIT 0.528* 0.122 PEPC8 FIT 0.619* 0.079 PEPC59 FIT 0.104* 0.109 AP68 FIT 0.732* 0.094 AP74 FIT 0.683* 0.070 D5S111 FIT 0.488* 0.130 D5S117 FIT 0.702* 0.068 D6S269 FIT 0.477* 0.109 D8S165 FIT 0.534* 0.110 D14S51 FIT 0.661* 0.147 D17S804 FIT 0.665* 0.095 All the loci FIT 0.586* 0.038

FST 0.215# 0.138

FIS 0.410+ 0.156

Average Standard Error

FST 0.224# 0.086

FIS 0.503+ 0.057

Average Standard Error

FST -0.027 0.185

FIS 0.120 0.055

Average Standard Error

FST 0.503# 0.175

FIS 0.480+ 0.145

Average Standard Error

FST 0.228# 0.090

FIS 0.592+ 0.089

Average Standard Error

FST 0.057# 0.065

FIS 0.460+ 0.148

Average Standard Error

FST 0.295 0.123

FIS 0.573+ 0.025

Average Standard Error

FST 0.144# 0.142

FIS 0.361+ 0.000

Average Standard Error

FST 0.099# 0.073

FIS 0.481+ 0.110

Average Standard Error

FST 0.303# 0.181

FIS 0.515+ 0.161

Average Standard Error

FST 0.185# 0.135

FIS 0.595+ 0.120

Average Standard Error

FST 0.238# 0.037

FIS 0.456+ 0.038

Average Standard Error

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Figure 11. Assignment of diverse Aotus individuals belonging to seven night monkey taxa by means of the Geneclass 2.0 Software by using the Bayesian method of Cornuet et al., (1999) and the “leave one out” procedure. Assignment of A. l. griseimembra (A); Assignment of A. l. zonalis (B); Assignment of A. brumbacki (C); Assignment of A. trivirgatus (D); Assignment of A. vociferans (E); Assignment of A. nancymaae (F); Assignment of A. a. boliviensis (G). Alg = A. l. griseimembra; Alz = A. l. zonalis; Ab = A. brumbacki; At = A. trivirgatus; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.

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Figure 12. Assignment of diverse Aotus individuals belonging to seven night monkey taxa by means of the Geneclass 2.0 Software by using the Nei’s (1972) standardized genetic distance and with the “as is” procedure. Assignment of A. l. griseimembra (A); Assignment of A. l. zonalis (B); Assignment of A. brumbacki (C); Assignment of A. trivirgatus (D); Assignment of A. vociferans (E); Assignment of A. nancymaae (F); Assignment of A. a. boliviensis (G). Alg = A. l. griseimembra; Alz = A. l. zonalis; Ab = A. brumbacki; At = A. trivirgatus; Av = A. vociferans; An = A. nancymaae; Aa = A. a. boliviensis.

The AMOVA with all the Aotus taxa in one group and A. a. boliviensis in another group with the 12 microsatellites analyzed showed that 76.71% of the genetic variance was among the considered taxa (FST = 0.233; P = 0.142 ± 0.009). Whereas the genetic variance between the groups (all Aotus taxa versus A. a. boliviensis) increased with reference to the previous AMOVA in 26.37% of the genetic variance (FCT = 0.264; P = 0.153 ± 0.012). The genetic variance among taxa within the groups was practically null (FSC = -0.041; P = 0.746 ± 0.011). If we only take the three most polymorphic loci (AP74, D6S269 and D17S804), the levels with significant genetic variance were among the considered taxa (66.28% of genetic

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variance; FST = 0.337; P = 0.000 ± 0.000), among taxa within the groups (17.86% of genetic variance; FSC = 0.212; P = 0.000 ± 0.000) and between all the Aotus taxa and A. a. boliviensis (15.86% of genetic variance FCT = 0.158; P = 0.005 ± 0.001). Thus, there is more genetic differentiation between A. a. boliviensis and the other Aotus taxa considered than the genetic differentiation caused by the Amazon River between the Northern and Southern Aotus populations. This disagrees with the Hershkovitz’s view of the Amazon River separating the two main Aotus groups.

Figure 13. Structure 2.3 Software analyses with specimens of seven Aotus taxa. Without origin and without admixture (A); without origin and with admixture (B); with origins and with admixture (C); with origins and without admixture (D).

Although, microsatellite data showed genetic heterogeneity among many pairs of Aotus taxa, this differentiation is not absolute. The Geneclass 2.0 Software showed this fact. The Bayesian method with the technique of Cornuet et al., (1999) and the “leave one out” procedure (58.86% of correct assignment, 83/141) showed that 18 individuals of other taxa

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were enclosed in A. l. griseimembra, nine individuals of other taxa in A. l. zonalis, four individuals of other taxa in A. brumbacki, one individual of other taxon in A. trivirgatus, 11 individuals of other taxa in A. vociferans, 14 individuals of other taxa in A. nancymaae and one individual of other taxon in A. a. boliviensis (Figure 11 a, b, c, d, e, f, g). The standardized genetic distance of Nei (1972) with the “as is” procedure (86.52% of correct assignment, 122/141) showed seven individuals of other taxa in A. l. griseimembra, three individuals of other taxon in A. l. zonalis, three individuals of other taxa in A. vociferans and five individuals of other taxa in A. nancymaae. In the cases of A. brumbacki, A. trivirgatus and A. a. boliviensis, none were mis-classified (Figure 12 a, b, c, d, e, f, g). Table 8. Number of possible different gene pools for seven Aotus taxa (A. l. griseimembra; A. l. zonalis; A. brumbacki; A. trivirgatus; A. vociferans; A. nancymaae; A. azarae boliviensis) analyzed using the Structure Program with 12 microsatellite loci. * = Most probable number of populations. K = number of populations. Without “a priori” origins and with admixture (A); Without “a priori” origins and without admixture (B); With “a priori” origins and with admixture (C); With “a priori” origins and without admixture (D) (A) Populations K=1 K=2 K=3 K = 4* K=5 K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -1,774.9 -1,604.6 -1.544,4 -1,536.1 -1,539.2 -1,558.3 -1,576.6 -1,594.1 -1,612.3 -1,629.5 -1,646.3 -1.663.3

(B) Populations K=1 K=2 K=3 K=4 K = 5* K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

Ln Likelihood -1,774.8 -1,591.9 -1.525.4 -1,505.4 -1,490.2 -1,499.7 -1,505.5 -1,513.1 -1,519.9 -1,524.5 -1,530.3 -1.535.8

Can Mitochondrial DNA, Nuclear Microsatellite DNA … (C) Populations K=1 K=2 K=3 K=4 K = 5* K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12 (D) Populations K=1 K=2 K=3 K=4 K = 5* K=6 K=7 K=8 K=9 K = 10 K = 11 K = 12

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Ln Likelihood -1,774.8 -1,609.0 -1.577.3 -1,525.4 -1,508.6 -1,515.9 -1,516.7 -1,523.5 -1,548.0 -1,534.7 -1,537.6 -1.542.7 Ln Likelihood -1,774.8 -1,611.5 -1.545.7 -1,531.1 -1,506.4 -1,512.2 -1,512.4 -1,528.1 -1,517.6 -1,521.3 -1,518.2 -1.517.7

For the Structure Software (Table 8 and Figure 13 a, b, c, d), four (without origin and with admixture) or five (without origin and without admixture; with origins with and without admixture) populations were detected as the more probable results. Without origins, it’s clearly difficult to classify one animal in a determined taxon. When we provide the origins of the individuals to the software, then it is easier to classify the exemplars to the corresponding taxa, especially when admixture is allowed. This last analysis distinguished (1) A. l. griseimembra and A. l. zonalis, (2) A. brumbacki, (3) A. vociferans, (4) A. nancymaae and (5) A. trivirgatus and A. a. boliviensis. Therefore, although we started this microsatellite analysis with seven “a priori” Aotus taxa, the Structure analyses detected only four or five different taxa. We applied Bottleneck Software to detect possible demographic changes for the three Aotus taxa with the greatest sample sizes (A. l. griseimembra, A. vociferans and A. nancymaae) (Table 9). For A. l. griseimembra, only the Wilcoxon test with the IAM (p = 0.027) supports a bottleneck. The remaining tests did not detect any evidence in favor of a bottleneck (Sign test: IAM [p = 0.282] and SMM [p = 0.209]; Standardized difference test: IAM [T2= 0.171, p = 0.189] and SMM [T2 = -0.883, p = 0.019]; Wilcoxon test: SMM [p = 0.902]). The significant negative value of the Standardized difference test is more related to a population expansion or to population fragmentation within this taxon. For A. vociferans, no tests detected any support in favor of a bottleneck (Sign test: IAM [p = 0.332] and SMM [p =

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0.273]; Standardized difference test: IAM [T2= 0.055, p = 0.478] and SMM [T2 = -2.301, p = 0.011]; Wilcoxon test: IAM [p = 0.289] and SMM [p = 0.927]). As in the previous case, the significant negative value of the Standardized difference test is more related with a population expansion or to population fragmentation within this taxon. For A. nancymaae, no tests detected any proof in favor of a bottleneck [Sign test: IAM (p = 0.087) and SMM (p = 0.151); Standardized difference test: IAM (T2= -1.154, p = 0.124) and SMM (T2 = -0.021, p = 0.221); Wilcoxon test: IAM (p = 0.527) and SMM (p = 0.215)]. The graphic descriptors for the three species (Figure 14 a, b, c) and an analysis by Garza and Williamson (2001) did not detect any sign of recent bottlenecks in these Aotus taxa (Table 10). Therefore, no evidence of bottlenecks were detected in the Aotus taxa analyzed for the microsatellites. Table 9. Application of the Bottleneck Software to detect possible recent bottleneck events in three Aotus taxa sampled. This Software employed the Cornuet and Luickart (1996)’s procedure. IAM = Infinite allele model; SMM = Stepwise mutation model Aotus lemurinus griseimembra Locus Expected heterozygosity in the sample PEPC3 0.742 PEPC8 0.561 AP40 Monomorphic AP74 0.718 D5S111 0.828 D5S117 0.563 D8S165 0.900 D145S51 0.812 D17S804 0.879 Aotus vociferans PEPC3 0.385 PEPC8 0.733 PEPC59 0.642 AP68 0.680 AP40 Monomorphic AP74 0.826 D5S111 0.867 D5S117 0.844 D6S269 0.908 D8S165 0.742 D14S51 0.233 D17S804 0.736 Aotus nancymaae PEPC3 0.735 PEPC8 0.871 PEPC59 0.541 AP68 0.440 AP40 Monomorphic AP74 0.714 D5S111 0.848 D6S269 0.933 D8S165 0.864 D15S51 0.821 D17S804 0.803

Expected heterozygosity under the IAM 0.759 0.607 0.683 0.760 0.551 0.864 0.716 0.847

Expected heterozygosity under the SMM 0.789 0.695 0.785 0.838 0.626 0.885 0.777 0.868

0.430 0.774 0.791 0.549 0.766 0.834 0.862 0.877 0.759 0.293 0.645

0.490 0.812 0.832 0.624 0.819 0.841 0.875 0.898 0.790 0.321 0.720

0.630 0.829 0.775 0.775

0.710 0.869 0.824 0.824

0.642 0.778 0.915 0.824 0.753 0.828

0.685 0.826 0.918 0.845 0.769 0.847

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Figure 14. Graphic descriptors to detect possible bottlenecks in three Aotus taxa. For A. l. griseimembra (A); for A. vociferans (B); for A. nancymaae (C).

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Table 10. Average M value in simulations with the Garza and Williamson (2001) procedure, Critical Mc value, average M value of analyzed data, and microsatellites showing an M value under Mc for six Aotus taxa

DISCUSSION Morphometrics, Molecular Population Genetics and Discrimination of Aotus Taxa It is important to take into account that the craniometric and the microsatellite analyses did not contain all the Aotus taxa recognized from a morphological or karyotype perspective. Also, the mt COII analyses contained all the described Aotus taxa to date. This is the first molecular analysis to enclose all of these taxa. Clearly, all the craniometrics analyses revealed that the skulls of A. l. zonalis, A. l. griseimembra, A. l. lemurinus, A. brumbacki, A. vociferans and A. nancymaae were intermixed. This was independent of the combined influence of size and shape or just shape. A. a. boliviensis was the unique taxon which showed some craniometric differentiation in MDS (the most external individuals), PCA (a uniquely different and significant group) and in UPGMA trees (they formed a nested group within all the other intermixed Aotus taxa skulls, although some A. a. boliviensis skulls were also intermixed with the other skulls). Only one skull of A. trivirgatus was analyzed and it was intermixed with the remaining skulls. Thus, craniometrics has a comparatively lower power to differentiate the Northern Aotus taxa (among and between taxa and A. nancymaae). Only the most Southern taxon (A. a. boliviensis) was positively discriminated. The microsatellite analyses with the same Aotus taxa showed a higher degree of differentiation among the Aotus taxa than did craniometrics. However, basically, the most differentiated taxon was again A. a. boliviensis, which was specially observed in the FST taxa pair comparisons, gene flow estimates, GeneClass 2.0 results and in the AMOVA analyses. The mitochondrial analyses revealed the highest discrimination power of all the analyses carried out. This procedure analyzed the most individuals including all the described Aotus taxa. However, if, in this last procedure we remove A. a. azarae, A. nigriceps and A. infulatus from the analyses, then A. a. boliviensis individuals appear as the most differentiated ones. Another commonality among the three procedures is that the differentiation among all the Northern Aotus taxa, excluding A. trivirgatus, is extremely limited. However, they do have

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different chromosome numbers (from 2n = 46, 47, 48 of A. vociferans to 2n = 58 of A. l. lemurinus) (Table 11). One fundamental difference between mitochondrial DNA and microsatellites is that the former could clearly differentiate A. vociferans from A. nancymaae, whereas the second had more limited power for this task. For instance, FST microsatellite comparision pairs and microsatellite gene flow estimates did not clearly differentiate both taxa. Similarly, the genetic assignment analysis with the worst assignation percentage (Bayesian with the Cornuet et al., 1999’s procedure) showed the nancymaae group including five A. vociferans individuals and the A. vociferans group including one specimen of A. nancymaae. The best percentage assignment analysis (with the Nei’s genetic distance) showed the A. nancymaae group including three individuals of A. vociferans. The Structure analysis could differentiate the multi-genotypes of A. vociferans and A. nancymaae when the different geographical origins were given to the software, but they were not differentiated when geographical origins were not specified. The differentiation of A. nancymaae and A. vociferans specimens is critical because of the intense illegal traffic of A. nancymaae from the Peruvian Amazon to Colombia. The illegal traffic is mainly due to malaria vaccine experiments in Colombia (note the Patarroyo case with its legal considerations; see Maldonado, 2011; Maldonado and Peck, 2014; Maldonado et al., 2009; Ruiz-García et al., 2012a). Thus, mitochondrial analyses seem to be more effective in differentiating individuals from both taxa. Also, mitochondrial analyses are completed more rapidly and are less expensive than those with microsatellites. Nevertheless, we revealed 22 Aotus sequences out of the 190 analyzed, which amplified numts in many taxa of the genus. Aotus is the first Neotropical primate genus where we detected numts. These sequences were characterized by missing and ambiguous data. The amino acid translations of the sequences did not have initial start and terminal stop codons but did have premature stop codons, typical of the numts (Chung and Steiper, 2008). Thus, caution must be taken with the use of mt COII gene sequences with Aotus for individual classifications and phylogeny. For instance, Menezes et al., (2010) analyzed the Aotus phylogeny, and concluded that Aotus diverged some 4.62 millions of years ago (MYA; with 95% HPD intervals of 3.07-6.43 MYA). Ashley and Vaughn (1995) and Plautz et al., (2009) professed similar dates (3.3-3.6 MYA and 4.7 MYA, respectively) for the initial divergence of Aotus. However, Ruiz-García et al., (2011) and Babb et al., (2011) estimated divergence times considerably older for the original diversification of Aotus (8.47 MYA, if the average temporal split between A. vociferans-A. nancymaae and A. a. boliviensis is estimated, and 8.95 MYA, respectively). These higher temporal splits could be an artifact caused by the presence of numts among the sequences studied both of these works. Nevertheless, paleontological evidence of A. didensis fossils (11.8–13.5 MYA) from La Venta, Colombia (Setoguchi and Rosenberger, 1987; Rosenberger et al., 2009; Takai et al., 2009) seems to be correlated with these last estimations. The first cited estimations are also not congruent with the temporal split of 22 MYA for an Aotusplatyrrhine divergence based on nuclear DNA data (Opazo et al., 2006), or 15 MYA for the origins of Aotus based on mitochondrial genomes (Hodgson et al., 2009). If the first estimates (around 4 MYA) for the diversification of Aotus were accurate, this could be correlated with more than 10 MYA of molecular stasis and nearly 14 MYA of morphological stasis (14 MYA to present). This could explain the very limited morphological and morphometric differences we found in Aotus. Maybe the fact that Aotus is nocturnal, and the consequence of little selection for phenotypic differences, could explain the small degree of morphological changes

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in the last 14 MYA (morphological stasis). The question of the temporal origin and the molecular diversification of Aotus needs to be deeply studied. Microsatellites did not detect any evidence of bottleneck affecting the three Aotus taxa with the highest sample sizes (A. l. griseimembra, A. vociferans and A. nancymaae). However, some tests detected possible population fragmentation within A. l. griseimembra and within A. vociferans. This agrees quite well with the Structure analysis without given origins. In other Aotus taxa, such as A. a. azarae, Babb et al., (2011) detected a high level of haplotypic diversity and a recent expansion of Azara’s owl monkeys into the Argentinean Chaco. Table 11. Aotus taxa, number of diploid chromosomes (2n), fundamental numbers (FN) estimated by Defler and Bueno (2007)/Ford (1994), Karyomorph nomenclature of Ma (1981a) and Ma et al., (1976a) (K1), of De Boer and Reumer (1978) and Reumer and De Boer (1980) (K2), and of Torres et al., (1998) (K3) and phenotypes defined by Ma et al., (1976a) (Ph). Nomenclature for Aotus taxa followed Hershkovitz (1983) and Groves (2001) Aotus taxa lemurinus lemurinus lemurinus zonalis lemurinus griseimembra jorgehernandezi brumbacki vociferans trivirgatus nancymaae miconax nigriceps infulatus azarae boliviensis azarae azarae

2n 58 (BS) 55,56 (BS) 52, 53, 54 (BS) 50 (BS) 50 (BS) 46, 47, 48 (BS) 50? 54? 51-52m/52f? 54 (BS) 51m/52f 49m/50f 49m/50f 49m/50f

FN 76/72/62 72/62 70/58 70/60 -/72 -/66 -/60-61 -/60-61 -/60-61

K1 VIII, IX II, III, IV V, X, XI I VII VI -

K2 1 2 6 7 3 4 5 -

K3 8 1 2 9 6 7 3 10 5 -

Ph B B B B B A C D D D

Phylogenetics and Systematics of Aotus There has been much disagreement among primatologists about the systematics of Aotus. However, when we consider the previous morphological, karyotypic and molecular studies along with the current results it is relatively easy to understand why disagreements exist. Primatologists often believe that the evolution and the speciation processes within different Neotropical primate genera can be similar and in parallel. However, this is not certain. Here are three examples that contest this. 1- The species of Alouatta (howler monkeys) are very homogeneous morphologically speaking (although they have a variety of remarkably different colors), but there are considerable karyotypic differences which are correlated with considerable molecular differences. Thus, there is a certain morphological stasis with a long evolution history reflected in parallel both in karyotypes and molecules (both revealed the same evolutionary history); 2- The taxa of Ateles (spider monkey), with the exception of A.

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paniscus (which is morphologically, karyotypically and molecularly different), are morphologically very similar (but with very different color pelages), with small molecular differences (which in turn reveals a short evolution) and some minor karyotype differences, which don’t correlate with the molecular evolution. Therefore, the Ateles evolution is relatively recent which is revealed by morphology (although color differences have been extremely emphasized by traditional primatologists) and molecular biology. The minor karyotypic differences within this genus have been of little value for reconstructing its phylogeny (a similar situation is found for Saimiri, the squirrel monkeys and in Lagothrix, the Humboldt woolly monkeys); 3- The taxa of Cebus (including Sapajus), capuchin monkeys, are characterized by considerable morphological differences, but very similar karyotypes and moderate molecular differences. Indeed, in the robust forms, the morphological differences are very outstanding but the molecular and karyotypic differences are relatively small. The temporal evolution of capuchin monkeys is between that observed in Alouatta and that observed in Ateles, but characterized by a rapid morphological evolution. Nevertheless, the Aotus case has a different history. The taxa of Aotus are extremely homogeneous from a morphological perspective (stasis), but with extreme karyotype differences. There are also very small molecular differences, at least, within the Northern and Southern groups. However the chromosomal differences within these groups are outstanding. This means that different speciation mechanisms are affecting these Neotropical primate genera. Thus we cannot apply the same rules to define species in these different Neotropical primate genera. The molecular genetic distances are very small especially in the Northern Aotus group, except for A. trivirgatus. For the mt COII gene in primates, Ascunce et al., (2003) and Collins and Dubach (2000b) reported an average genetic distance around 5.82% ± 1.64% among species within a genus, around 2-4% for subspecies within species and around 15.68% ± 1.73% among genera. The genetic distances for all the Northern Aotus taxa (A. vociferans, A. brumbacki, A. jorhehernandezi, A. l. griseimembra, A. l. lemurinus and A. l. zonalis) ranged from 1.8% to 4.3%, and are in the range of subspecies within a species, although each one if these named taxa have different 2n chromosomes (exception of A. brumbacki and A. jorgehernandezi). Conversely, the genetic distance percentages between A. trivirgatus and all the other Northern Aotus taxa ranged from 4.7% to 6.6%, typical of different species. The genetic distance percentages between the Northern group and A. trivirgatus in respect to A. nancymaae were 5.1-5.8% and 6.6%, respectively, which are in the range of different species. However, A. nancymaae and A. miconax only showed 2% of genetic differences, typical of subspecies. The genetic distance percentages among four taxa of the Southern Aotus group (A. a. azarae, A. a. boliviensis, A. nigriceps and A. infulatus) ranged from 1.7% to 3.8%, typical of subspecies. The genetic distance percentages between the Northern group, A. trivirgatus, A. nancymaae-A. miconax with reference to this Southern group were 3.9-6.1%, 4.3-6.2% and 4.7-7.3%, respectively, being in the range of different species. Our results agree extremely well with the results of Plautz et al., (2009). These authors found that A. l. griseimembra, A. vociferans, and A. brumbacki formed a very coherent group, with relatively, very low nucleotidic divergence. Their maximum parsimony and likelihood trees showed unresolved polytomy among these three taxa, while the NJ tree pointed to A. vociferans as the sister clade of the A. brumbacki–A. l. griseimembra cluster. The lack of resolution in this case does not necessarily reflect any specific shortcomings of the mt COII sequences, but may simply reflect relatively rapid dispersal and divergence in the Northern Aotus group.

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The small genetic distances (neutral markers) and mitochondrial haplotypes shared in the Northern group, although they have high karyotypic differences, as well as that found in the Southern group (although in this group the karyotypic differences are not so extreme) provide arguments in favor of recent chromosomal speciation in closely related Aotus populations. The extreme importance of chromosomal evolution in Aotus has been demonstrated by reciprocal chromosome painting between humans and A. nancymaae as well as between A. nancymaae and woolly monkey whole chromosome probes. Stanyon et al., (2004) showed that for the A. nancymaae karyotype, only three human syntenic groups were conserved. They coexisted with 17 derived human homologous associations. A minimum of 14 fissions and 13 fusions were required to derive the A. nancymaae karyotype from that of the ancestral karyotype of Neotropical primates. A. nancymaae is considered the ancestral karyotype by some authors because it is the least derived karyotype of all the current Aotus taxa. Meanwhile for Ateles, Lagothrix, Saimiri and Cebus we can invocate typical allopatric speciation following vicariant or peripatric events or even non-allopatric speciation with parapatric events, in the case of Aotus, the main process is chromosomal speciation following stasipatric, peripatric or parapatric models (Lewis, 1966; White, 1968, 1978; Reig, 1980; King, 1993). The Northern group case is very similar to the cases of some rodents such as Spalax ehrenbergii from the Middle East with 2n = 48, 52, 54, 56 and 58 chromosomes (Nevo, 1991) or the Venezuelan rodent Proechimys guairae with six karyomorphms forming a “rassenkreis” (2n = 42, 44, 46, 48, 50 and 62) (Reig, 1980). Of the three chromosomal speciation models, the stasipatric model seems to be globally the most improbable because within the geographical range of the Northern Aotus taxa, there isn’t a wide distributed karyotype with other karyotypes differentiated within the territory of the main distributed karyotype. Within the geographical range of A. vociferans, there are not specimens with karyotypes of A. brumbacki or A. l. griseimembra, for instance. However, the existence of several chromosome polymorphisms within several of these Aotus taxa should be cases of stasipatric events. For instance, consider A. vociferans with 2n = 46, 47, 48 and A. l. griseimembra with 2n = 52, 53, 54, and A. l. zonalis with 2n = 55, 56. Indeed, both microsatellites and mitochondrial DNA did not discriminate these two last forms, which means that they are, molecularly speaking, the same taxon. Nevertheless, the karyotypic differentiation for the Northern group taken as a whole is more related to parapatric or peripatric chromosomal differentiation. We suggest that there is a greater (major) influence of the chromosomal parapatric differences compared to peripatric ones, although both could have occurred during the evolution of these Aotus forms. Our reasoning for this suggestion is because each chromosome form is in different biomes or ecotones: A. vociferans in the Amazon rain forest, A. brumbacki in the Eastern Llanos, A. jorgehernandezi in the Andean areas of the Colombian Central Andean cordillera, A. l. griseimembra in the Magdalena River Basin, A. l. lemurinus in the high Andean areas of the Colombian Eastern Cordillera and A. l. zonalis in the Choco and Darien jungles of Western Colombia and Panama. The selective pressures of these different ecotones could have selected the different karyotypes of the Aotus of these areas. Additionally, in the three taxa of the Northern group where we analyzed relatively large samples (A. vociferans, A. brumbacki and A. l. griseimembra), the gene diversity for both microsatellite and mitochondrial genes was elevated and there was no evidence of bottlenecks. Both of these results support parapatric chromosomal differentiation. Ford’s (1994) study indicating morphometric variables, coat colors and karyotypes with clinal patterns provides more evidence in favor of parapatric chromosomal differentiation.

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However, if large samples of A. jorgehernandezi, A. l. lemurinus and A. l. zonalis will be studied for molecular markers and they reveal low gene diversity levels or bottlenecks, as we suspected, these cases could then be related to peripatric chromosomal differentiation. Therefore, although the parapatric chromosomal differentiation seems to be the most important process in the Northern Aotus group, both stasipatric and peripatric chromosomal differentiation could have simultaneously occurred. However, the molecular differences are extremely small for all these Aotus forms because their temporal splits are very recent. This could indicate a very rapid and successful colonization of many different biomes in the current Colombian territory by the night monkeys. The small molecular differences could also be due to the absence of total reproductive barriers although these forms have different chromosomal numbers. In fact, total reproductive barriers could be more important to the fundamental number (FN) than to the 2n (Martin, 1990). Defler and Bueno (2007) suggested that there would probably be no successful interbreeding among the putative subspecies of A. lemurinus, nor between any of them and A. brumbacki, nor between A. vociferans and populations of A. brumbacki or A. lemurinus. However, Ma et al., (1976a) and Pieczarka and Nagamachi (1988) claimed that these different chromosomal numbers in the Northern Aotus group do not constitute a reproductive barrier. In fact, there is no selection against heterokaryomorphms in A. l. griseimembra with diploid numbers of 52, 53 and 54 (Giraldo et al., 1986). A similar situation is true for A. vociferans with 2n = 46, 47 and 48 (Descailleaux et al., 1990) and perhaps for A. zonalis with 2n = 55 and 56 (in this taxon 2n = 54 which is theoretically possible but has not yet to be observed). If this last one is certain, there should be chromosomal number continuity in A. l. griseimembra and A. l. zonalis of 2n = 52, 53, 54, 55 and 56. Recall that from a molecular point of view, both mitochondrial DNA and microsatellites are extremely similar between both Aotus taxa. Defler and Bueno (2007) affirmed that A. l. zonalis is more closely related to A. l. lemurinus than it is to A. l. griseimembra, because A. l. zonalis and A. l. griseimembra differ in two distinct translocations of one chromosome (Ma et al., 1978). This put forward the extreme relationships among the most Northern Aotus taxa. It is clear that these three taxa (A. l. griseimembra, A. l zonalis and A. vociferans) appear to maintain their karyological identity with multiple chromosome differences. Only the study of possible hybridization or tension zones between some of these Aotus populations could help to distinguish between both hypotheses (Hewitt, 1989, 1993). Our chromosomal parapatric diversification model agrees well with the proposal by Ma (1981a). It suggests that geographic isolation in different geographic niches led to karyotypic diversity in the night monkeys. Related with this, Plautz et al., (2009) hypothesized that climatic and geological changes in the last 5 MYA (eustatic sea level changes, for instance) allowed the development of Aotus taxa in three refuge groups, one comprising A. vociferans, A. lemurinus and A. griseimembra in the Andean foothills. A second group contains A. trivirgatus in the northwestern Guyanan shield. The third refuge group consists of A. nigriceps, A. azarae, A. infulatus and A. nancymaae in the Brazilian shield refuge. Menezes et al., (2010) suggested that A. nancymaae should be included in the Andean foothill refuge rather than in the Brazilian shield refuge. We provide evidence more compelling than Menezes et al., (2010) that A. nancymaae was highly related with the Andean foothill refuge. Menezes et al., (2010) also claimed that A. nigriceps, A. azarae and A. infulatus must have diverged after the rise of sea level (5 MYA) while grey neck species and A. nancymaae could have diverged before this event. Babb et al., (2011) also detected older diversification

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processes in the Northern group, around 7.34 MYA, whereas that of the Southern group was 6.22 MYA. Similarly, the diversification process for A. a. azarae was 1.78 MYA (95% HPD: 0.24–3.99 MYA), whereas for A. nancymaae, it was 4.68 MYA (95% HPD: 1.93–8.10 MYA). The more recent diversification of the Southern Aotus group could be related with the more recent establishment of southern rivers and the draining of the South American Chaco (Rosenberger et al., 2009). Hershkovitz (1977, 1983), using the theory of metachromism, claimed that the red-necked Aotus species had to have derived from the gray-necked species in the North. Following this theory, these pigment changes are one-way and always proceed from the loss of eumelanin (although the molecular results contradicts this because the ancestor of A. nancymaae, a red-necked taxon, is in the origin of all the gray-necked Aotus taxa, except A. trivirgatus). However, our results don’t clearly show which of the two clades, A. nancymaae-miconax + Northern group or A. trivirgatus + Southern group, was the first to diverge. Two possible schemes could explain the order of appearance of the different Northern Aotus forms for the parapatric chromosomal differentiation: 1. The first hypothesis: A. nancymaae (2n = 54)  A. vociferans (2n = 46, 47, 48)  A. brumbacki (2n = 50) and/or A. jorgehernandezi (2n = 50) (and A. brumbacki  A. jorgehernandezi or A. jorgehernandezi  A. brumbacki)  A. l. griseimembra (2n = 52, 53, 54)  A. l. zonalis (2n = 55, 56) and A. l. lemurinus (2n = 58). This scheme shows that the parapatric chromosome differentiation is basically by fission events (with the exception of the first step) and not by fussion events as was suggested by Defler and Bueno (2007). These authors concluded that A. l. lemurinus with 2n = 58 was the original karyotype of these Aotus forms. However, this hypothesis is untenable because this form has a peripheric distribution and lives in a very specialized habitat (high Andes). It has the highest 2n chromosome number of all the Aotus and is more related to a derived form than to an original form. A higher number of chromosomes facilitates a higher degree of recombination which could be very important when adapting to new environmental conditions. Defler and Bueno (2007) and Defler et at., (2001) claimed that A. l. lemurinus has an acrocentric chromosome that is involved in two different rearrangements, in A. brumbacki and A. l. griseimembra. However, molecular and other karyotype characters show the reverse, A. l. lemurinus is the most derived of all the Northern Aotus group. 2. The second hypothesis should agree with the fact that some authors (Ma, 1981a and Galbreath, 1983) have claimed that the original Aotus had 54 chromosomes. Therefore: A. l. griseimembra (2n = 52, 53, 54)  A. l. zonalis (2n = 55, 56) and A. l. lemurinus (2n = 58) is on one side, and, A. l. griseimembra (2n = 52, 53, 54)  A. brumbacki (2n = 50) and/or A. jorgehernandezi (2n = 50) (and A. brumbacki  A. jorgehernandezi or A. jorgehernandezi  A. brumbacki)  A. vociferans (2n = 46, 47, 48) is on the other side. Ma et al., (1985) suggested that A. vociferans was derived from A. brumbacki by a single fusion event, and that the latter represents an intermediate form between A. vociferans and A. l. griseimembra. However, this second hypothesis is less credible than the first one because it means that in the area north of A. l. griseimembra’s distribution there were only fission events and in the area south of A. l. griseimembra’s distribution there were only fussion events, which

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is less probable. It’s the same fact detected for the Venezuelan rodent, Proechimys guairae (Reig, 1980). It’s remarkable that within the Northern Aotus taxa with a unique 2n, there is also considerable chromosomal heterogeneity. This is the case of A. brumbacki. None of the three previously published descriptions of chromosomal morphology for A. brumbacki (Brumback, 1974; Yunis et al., 1977; Torres et al., 1998) agreed completely in the chromosomal characteristics (all with 2n = 50) and showed considerable variation in the identification of numbers of metacentric, submetacentric and acrocentric chromosomes. Our mitochondrial analysis is the first including two individuals of A. miconax. This taxon was inside the large A. nancymaae cluster. Since the A. miconax’s karyotype is unknown, we cannot know which relationship exists with the A. nancymaae’s karyotype. But, the fact that both forms are distributed in contiguous but very different biomes (A. nancymaae in the lowland Amazon forest and A. miconax in the transition forests from high Andes to lowland Amazon), could be related with peripatric or parapatric chromosome differentiation. Thus, we believe that A. miconax should have a different karyotype from A. nancymaae. However, it must be investigated. The mitochondrial analysis revealed that the remaining four Southern taxa formed a monophyletic group with low genetic distances and relatively minor karotypic differences with regard to the Northern group (all the Southern taxa possess 2n = 49(male)/50(female) chromosomes, with the exception of A. nigriceps with 2n =51m/52f). A. nigriceps, A. a. boliviensis, and A. a. azarae show a difference in chromosome numbers between males and females as a consequence of a fusion of the Y chromosome with an autosome. Ma et al., (1980) and Ma (1981a) compared the karyotypes of A. nigriceps and A. a. boliviensis. They found that the fusion of the Y chromosome occurs with the short arm of a medium-size subtelocentric autosome. Ma et al., (1980) and Ma (1981a, 1984), by using G-banding and gene mapping, showed evidence that this autosome is the same on both karyotypes. In addition, in A. a. boliviensis the fusion of the Y with the short arm of the autosome is followed by a pericentric inversion, whereby the short arm bearing the Y chromosome becomes a portion of the long arm of the resulting submetacentric. In A. a. azarae, the Y fusion is to a small acrocentric autosome, but all other chromosomes are identical on the Gand C-banding level to A. a. boliviensis chromosomes (Mudry et al., 1984). Galbreath (1983) assumed on the basis of geographic distribution and similarities in fur pattern that the karyotype of A. infulatus should not be very different from the karyotypes of A. nigriceps and A. azarae. Our mitochondrial result agrees quite well with Galbreath’s (1983) assumption, as well as the chromosomal study of Pieczarka and Nagamachi (1988). These authors showed that in the A. infulatus karyotype, except for pair B12, all the autosomic chromosomes and the X chromosome are identical on G- and C-banding levels to the corresponding chromosomes of A. a. boliviensis described by Ma et al., (1976a). The only differences between A. infulatus and A. a. boliviensis are on the G-banding pattern of B12 and on the G- and C-banding patterns of the Y/autosome. Pieczarka and Nagamachi (1988) tentatively explained that the origin of the Y/autosome of A. infulatus and A. a. boliviensis could be related with an ancestral population with a karyotype similar to that of A. nigriceps (2n = 51m/52f), which gave rise to a group of animals that fixed three autosomal rearrangements, originating the 2n=49m/50f karyotype and maintaining the Y/autosome chromosome of the ancestral population. This newly derived population was further divided, with a pericentric inversion of

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the Y/autosome becoming fixed in one group (A. infulatus), and another peri centric inversion of the Y/autosome, becoming fixed on the other group (A. a. boliviensis). They concluded that there are insufficient differences between A. infulatus and A. a. boliviensis to support two distinct species. Also, our results agree quite well with Babb et al., (2011), who suggested that the Southern expansion of Aotus was gradual (only one chromosomal fission event and the maintenance of the Y-autosomal fusion event in Southern males), with taxa diversifying steadily at different points in time, not through multiple splits or population bottlenecks (Pieczarka et al., 1993, 1998; Torres et al., 1998). However, we obtained two samples in Rondonia (Brazil) from two specimens with some morphological resemblance to specimens of A. a. boliviensis. Pieczarka et al., (1993) established that these two morphologically similar A. a. boliviensis individuals were karyotypically intermediate between A. a. boliviensis and A. infulatus. These authors assumed that the Rondonia individuals, A. a. boliviensis and A. infulatus formed only one species. Our mitochondrial results placed the two Rondonia specimens we sequenced within the A. infulatus cluster, thus agreeing with the karyotype study of Pieczarka et al., (1993). Regrettably, we don’t know if the relationship among the three animals we sampled in Bolivia and amplified for numts is similar to the morphotypes from Rondonia. This too must be investigated. Plautz et al., (2009) stated that A. nigriceps was clearly divergent from all other species suggesting that it is closest to the ancestral form, contrary to the hypothesis of Ruiz-Herrera et al., (2005). They proposed that the most divergent species is a karyotypically distinct northern form. Clearly this statement of Plautz et al., (2009) is incorrect because their alleged A. nigriceps sequence is from an A. trivirgatus. Menezes et al., (2010) used a maximum likelihood tree from SRY data (nuclear gen) to show three collapsed lineages, one leading to A. vociferans, a second one leading to A. l. griseimembra and A. l. lemurinus, and a third one leading to A. trivirgatus, A. nigriceps, A. azarae boliviensis and A. infulatus. Indeed, some SRY haplotypes differed by only one nucleotide such as the case of A. infulatus when compared to A. azarae boliviensis and A. nigriceps. The karyotypic similarity between A. infulatus and A. azarae suggested a close proximity and recent common ancestry, a finding coincident with their low genetic distance estimates and by the recent time of their evolutionary divergence (0.53 MYA) following Menezes et al., (2010). Thus, this Southern lineage agrees quite well with what we detected with the mt COII gene. Another relevant question is if the Northern and Southern Aotus groups (sensu Hershkovitz, 1983) are real. Three of our analyses, AMOVA and Structure with microsatellites and the mitochondrial trees, clearly revealed that one Northern form, A. trivirgatus, is more related to the Southern group than to the remaining Northern Aotus forms. For instance, the Structure analysis with microsatellites revealed a strong relationship between A. trivirgatus and A. a. boliviensis. Similarly, the Southern forms, A. nancymaae and A. miconax, are not placed together with the rest of the Southern taxa group. In our mitochondrial analysis, we detected A. nancymaae-A. miconax as the sister clade of all the Northern Aotus forms, excluding A. trivirgatus. However, Menezes et al., (2010), studying three mitochondrial genes (COI, COII and Cyt-b) but only with 18 individuals (versus 190 individuals herein reported), detected that the ancestor of A. nancymaae should be in the origin of all the other Aotus species. Their maximum likelihood and Bayesian reconstructions used these three mitochondrial genes and showed two sister lineages, one leading to the most basal offshoot represented by A. nancymaae and another to a clade grouping seven other Aotus taxa. This clade split into two sister clades, one leading to A. vociferans and the other

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one further splitting in A. l. griseimembra and A. l. lemurinus and to a more derived clade (A. trivirgatus (A. nigriceps (A. infulatus, A. azarae))). Indeed, A. nancymaae has 2n = 54, which was suggested as the original karyotype in Aotus, such as we previously commented (Ma, 1981a and Galbreath, 1983). However, in contrast to this view, Ruiz-Herrera et al., (2005), combined G-banding comparisons and molecular cytogenetic techniques, and described the most likely pattern of chromosome evolution and phylogenetic position of two Aotus karyomorphs from Venezuela. They indicated that the homologies between these two Aotus karyomorphs and human chromosomes agree well with a karyotype of 2n = 50 (they classified this karyomorph as A. sp.; this specimen could be A. trivirgatus or A. brumbacki). They are closer to the ancestral Platyrrhini karyotype, whereas a specimen of A. nancymaae (2n = 54) presented a more derived karyotype with respect to the ancestral Platyrrhini karyotype. It’s intriguing that this last exemplar was present as part of the Venezuelan fauna because this Aotus taxon is not reported for this country. A clear result is that, although A. nancymaae could be in the origin of the Northern group, there is a complete reproductive barrier between this taxon and A. vociferans (2n = 46). Pieczarka et al., (1992) studied the karyotypes of 11 A. vociferans and 11 A. nancymaae using G- and C-banding and NORstaining. Five A. nancymaae and five A. vociferans were sympatric in the Aramaza Island in Brazil (not in Colombia as cited the authors), three A. nancymaae and one A. vociferans were sympatric in Yahuma in Peru and three A. nancymaae and one A. vociferans were sympatric in Terezinha in Brazil. In no case, were signs of hybridization detected showing clear chromosomal reproductive barriers between both taxa. The real position of A. nancymaae in the Aotus phylogeny must be carefully studied. Therefore, with the results obtained here and taking into consideration the previous karyotype studies, we propose the existence of four superspecies within the Aotus genus: 1. A. vociferans (Spix 1823). We agree with the viewpoint of Ford (1994) and the taxa named vociferans, brumbacki, lemurinus, jorgehernandezi, zonalis and griseimembra should be treated as a superspecies (very noteworthy chromosomal differences but with very limited molecular and morphological differences). This superspecies should be named A. vociferans because it is the oldest name (brumbacki, Hershkovitz 1983; lemurinus, Geoffroy 1843; jorgehernandezi, Defler and Bueno 2007; zonalis, Goldman 1914; griseimembra, Elliot 1912). This superspecies, A. vociferans, could contain six subspecies: A. v. vociferans, A. v. brumbacki, A. v. lemurinus, A. v. jorgehernandezi, A. v. zonalis and A. v. griseimembra or better yet, this species contains six karyomorphms (1, 2, 6, 7, 8 and 9) following the nomenclature of De Boer and Reumer (1978), Reumer and De Boer (1980) and Torres et al., (1998). Menezes et al., (2010) claimed that A. lemurinus, A. griseimembra and A. vociferans were valid species, arguing that genetic distance estimates between A. lemurinus and A. griseimembra were higher than many other interspecific estimates and even higher when comparing A. vociferans with A. lemurinus and A. griseimembra (Table 4). These findings argue against the proposition that A. lemurinus and A. griseimembra are junior synonyms of A. vociferans (Ford, 1994) and are in agreement with Defler and Bueno (2007) indicating that these taxa are valid species. However, we disagree with this claim for three reasons. First, within the genetic distances they published, the values between A. lemurinus and A. griseimembra are among the lowest values between taxa considered “a priori” different

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species (around 2% of differences which are values typical of subspecies). Second, they used many sequences obtained in the GenBank and many of the Aotus sequences in GenBank are erroneously classified. Third, the number of sequences they used for the Northern Aotus group is very limited (five individuals). As we demonstrated in Ruiz-García et al., (2016a, this volume, for Cebus), at the moment that we considerably augment the sample size of the Northern group (we herein employed 87 Northern Aotus specimens, excluding A. trivirgatus and the numt sequences), many haplotypes were intermixed and thus reducing the reciprocal monophylia among the Northern Aotus taxa. 2. A. trivirgatus (Humboldt 1812). We again agree with Ford’s (1994) perspective only distinguishing two northern species of Aotus, A. vociferans and A. trivirgatus. One unclear question with this species (if it is a single unique species) is its karyotype make-up. Menezes et al., (2010) analyzed an A. trivirgatus female (from Barcelos, left side of the Negro River in the Amazonas State in Brazil) with 2n = 50. Its chromosome complement contained 12 pairs of biarmed chromosomes varying in size from large to small and 13 pairs of acrocentric chromosomes varying in size from medium to small. A Maipures Aotus specimen (frontier between Colombia and Venezuela) karyotyped by Monsalve (unpublished; see Defler and Bueno, 2007) also presented 2n = 50 (13 pairs of biarmed chromosomes and 11 pairs of acrocentric chromosomes). Hershkovitz examined color slides of Maipures specimens and stated that he believed they were A. trivirgatus and that, therefore, A. trivirgatus had 2n = 50 (see Defler and Bueno, 2007). However, a renowned Colombian primatologist, Jorge I. Hernández Camacho did not agree with this interpretation, believing that the Maipures specimen was A. brumbacki or a new species. Defler and Bueno (2007) were unable to distinguish if these Maipures specimens were A. trivirgatus or A. brumbacki. Other authors showed other possible karyotypes for A. trivirgatus. Santos-Mello and Thiago de Mello (1985, 1986) described a karyomorph 2n = 51 for one male, 2n = 52 for one female and 2n = 51 or 52 for another male, for some Aotus exemplars collected around Manaus (right side of the Negro River). According to the authors, this is the true karyotype for A. trivirgatus. However, as the distribution of the Aotus taxa is not clearly delimited, their identification is questionable. Indeed, Manaus is located at the confluence of the distribution of A. vociferans, A. nigriceps and A. trivirgatus. The karyotypes of the work of Santos-Mello and Thiago de Mello (1986) coincided with that of A. nigriceps. Also, the karyotypes of Menezes et al., (2010) and Monsalve (unpublished) coincided with the karyotypes of A. brumbacki (10-11 pairs of biarmed chromosomes and 13-14 pairs of acrocentric chromosomes following Torres et al., 1998). Indeed, the question could be more complex. Torres et al., (1998), citing Chu and Bender (1961) and Descailleux et al., (1990), showed 2n = 50-54 for A. trivirgatus. As we previously commented, Ruiz-Herrera et al., (2005) reported a Venezuelan specimen with 2n = 54 and classified it as A. nancymaae, but this taxon is not reported for Venezuela (Bodini and Pérez-Hernández, 1989). Should this specimen with 2n = 54 represent the original A. trivirgatus, whereas the animals with 2n = 50 are A. brumbacki and those with 2n = 51m/52f are A. nigriceps? We don’t know. Thus, determining the sex chromosome system and the real karyotype of A. trivirgatus might be essential in determining the status of this species. However, at the molecular (this work) and morphometrics (Ford, 1994) levels, A. trivirgatus

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seems to be differentiated. Ford (1994) showed that whorls, crests or tufts and the head stripes do not unite posteriorly. The dorsum is usually grayish, sometimes with buffy agouti and with a narrow and strongly contrasting orange middorsal band. Morphometrically this species is also easily distinguishable from the rest of the Northern Aotus with a canonical variate from cranial measurements separating A. trivirgatus completely from the other Northern group. 3. A. miconax (Thomas 1927). This superspecies should be named A. miconax, because this name is older than A. nancymaae (Hershkovitz 1983). Both Hershkovitz (1983) and Ford (1994) also suspected that both forms could be conspecific. As we don’t know the karyotype of miconax, we could define two different taxa as A. miconax miconax and A. miconax nancymaee. 4. A. azarae (Humboldt 1812). Within this superspecies we could include azarae, boliviensis, infulatus and nigriceps. This superspecies should be named A. azarae because it is the oldest name (boliviensis, Elliot 1907; infulatus, Kuhl 1820; nigriceps, Dollman 1909). The superspecies, A. azarae, could contain four subspecies: A. a. azarae, A. a. boliviensis, A. a. infulatus and A. a. nigriceps. Or, even better, this species could contain two or three karyomorphms (4, 5 and possibly 10) following the nomenclature of De Boer and Reumer (1978), Reumer and De Boer (1980) and Torres et al., (1998). The monophyly of this superspecies was in agreement with karyologic data showing that they shared the same X1X1X2X2/X1X2Y sex chromosome system, contrary to other Aotus taxa with an XX/XY sex chromosome system (Torres et al., 1998). Menezes et al., (2010) claimed that A. a. boliviensis is a full species because the insertion of one cytosine in position 59 of MT-TS1. This is exclusive to this taxon. This difference as well as the presence of different SRY haplotypes was claimed by these authors as a fundamental fact to consider A. infulatus as a different species from A. azarae. However, these very limited differences don’t necessarily mean that they are different species. They probably don’t translate into any reproductive isolation mechanism. Thus, we believe that these four Southern Aotus taxa are a valid superspecies. A variety of molecular markers should be applied to the Aotus taxa to validate the phylogenetic inferences we described.

ACKNOWLEDGMENTS Thanks go to the SDA (Secretaria Distrital Ambiental of Bogota DC, Colombia) for the project entitled “Fortalecimiento del control y prevención del tráfico ilegal de fauna silvestre, especialmente de Primates, a través de la determinación de zonas sometidas a extracción ilegal utilizando pruebas de genética molecular de poblaciones,” and to Corpoamazonía (Leticia-Colombia), which allowed us to obtain the necessary financial resources to carry out the current study. Additional thanks to P. Escobar-Armel, L. Castellanos-Mora and N. Lichilín for their respective help in obtaining Aotus samples during the last 18 years. Many thanks go to the Bolivian and Peruvian Ministry of Environment, to the Dirección General de Biodiversidad, to the Wildlife Conservation Society and CITES from Bolivia, to the

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PRODUCE, Dirección Nacional de Extracción and Procesamiento Pesquero from Peru, the Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales (INRENA) and to the Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador) for their role in facilitating the obtainment of the collection permits. Special thanks goes to the Colección Boliviana de Fauna (Dr. Julieta Vargas) in La Paz (Bolivia). We also thank the following Indian communities for helping to obtain monkey samples: Ticuna, Yucuna, Yaguas, Witoto and Cocama in the Colombian Amazon, Bora, Ocaina, Shipibo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana and Alama in the Peruvian Amazon, the Movima, Moxeño, Sirionó, Canichana, Cayubaba and Chacobo in Bolivia and to the Kichwa, Huaorani, Shuar and Achuar in Ecuador.

REFERENCES Adkins, R. M., Honeycutt, R. L. (1991). Molecular phylogeny of the superorder archonta. Proceedings of the National Academy of Sciences USA 88:10317–10321. Adkins, R. M., Honeycutt, R. L. (1994). Evolution of the primate cytochrome c oxidase subunit II gene. Journal of Molecular Evolution 38: 215–231. Akaike, H. (1974). A new look at the statistical model identification. IEEE Transations on Automatic Control, AC-19: 716–723. Ascunce, M. S., Hasson, E., Mudry, M. D. (2003). COII: a useful tool for inferring phylogenetic relationships among New World monkeys (Primates, Platyrrhini). Zoologica Scripta 32: 397-406. Ashley, M. V., Vaughn, T. A. (1995). Owl monkeys (Aotus) are highly divergent in mitochondrial cytochrome c oxidase (COII) sequences. International Journal of Primatology 5: 793–807. Babb, P. L., Fernandez-Duque, E., Baiduc, C. A., Gagneux, P., Evans, S., Schurr, T. G. (2011). mtDNA diversity in Azara’s owl monkeys (Aotus azarai azarai) of the Argentinean Chaco. Am. J. Phys. Anthropol. 146:209–224. Barton, N. H., Slatkin, M. (1986). A quasi-equilibrium theory of the distribution of rare alleles in a subdivided population. Heredity 56: 409-416. Beumont, M., Nichols R. (1996). Evaluating loci for use in the genetics analysis of population structure. Proceedings of the Royal Society of London, Series B 263: 1619-1626. Bodini, R., Pérez-Hernández, R. (1987). Distribution of the species and subspecies of Cebids in Venezuela. Fieldiana Zoology 39: 231-244. Bruford, M. W., Wayne, R. K. (1993). Microsatellites and their application to population genetics studies. Current Opinion in Genetics and Development 3: 939-943. Brumback, R. A. (1973). Two distinctive types of owl monkeys (Aotus). Journal of Medical Primatology 2: 284-289. Brumback, R. A. (1974). A third species of the owl monkey (Aotus). Journal of Heredity 65: 321-323. Brumback, R. A. (1976). Taxonomy of the owl monkey (Aotus). Laboratory Primate Newsletter 15: 1-2. Brumback, R. A., Willenborg, D. O. (1973) Serotaxonomy of Aotus. A preliminary study. Folia Primatologica 83: 100-125.

Can Mitochondrial DNA, Nuclear Microsatellite DNA …

337

Brumback, R. A., Staton, R. D., Benjamin, S. A., Land, C. M. (1971). The chromosomes of Aotus trivirgatus Humboldt, 1812. Folia Primatoogica 15: 264 – 273. Burnaby, T. P. (1966). Growth-invariant discriminant functions and generalized distances. Biometrics 22: 96-110. Chu, E. H. Y, Bender, M. A. (1961). Chromosome cytology and evolution in primates. Science 133: 1399-1405. Chung, W. K., Steiper, M. E. (2008). Mitochondrial COII introgression into the nuclear genome of Gorilla gorilla. International Journal of Primatology 29: 1341–1353. Collins, A. C., Dubach, J. M. (2000a). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology 21: 381– 420. Collins, A. C., Dubach, J. M. (2000b) Biogeographic and ecological forces responsible for speciation in Ateles. International Journal of Primatology 21: 421-444. Cornuet, J. M., Luikart, G. (1996). Description of power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144:2001-2014. Cornuet, J. M., Piry, S, Luikart, G, Estoup, A, Solignac, M. (1999). New methods employing multilocus genotypes to select or exclude populations as origins of individuals. Genetics 153:1989-2000. Cuadras, C. M. (1991). Métodos de Análisis multivariantes. Promociones y Publicaciones Universitarias, Barcelona. De Boer, L. E. M., Reumer, J. W. F. (1979). Reinvestigation of the chromosomes of a male owl monkey (Aotus trivirgatus) and its hybrid son. Journal of Human Evolution 8: 479489. Defler, T. R., Bueno, M. L. (2007). Aotus diversity and the species problem. Primate Conservation 22: 55–70. Defler, T. R., Bueno, M. L., Hernández-Camacho, J. I. (2001). Taxonomic status of Aotus hershkovitzi: its relationship to Aotus lemurinus lemurinus. Neotropical Primates 9: 37– 52. Descailleaux, J., Fujita, R., Rodríguez, L. A., Aquino, R., Encarnación, F. (1990). Rearreglos cromosómicos y variabilidad cariotípica del género Aotus (Cebidae: Platyrrhini). In La Primatología en el Perú. Invetigaciones Primatológicas (1973 – 1985), Proyecto Peruano de Primatologia “Manuel Moro Sommo.” (pp 572 – 577). Lima, Perú. Disotell, T. R., Honeycutt, R. L., Ruvolo, M. (1992) Mitochondrial DNA phylogeny of the Old-World monkey tribe Papionini. Molculare Biology and Evolution 9: 1-13. Ellesworth, J. A., Hoelzer, G. A. (1998). Characterization of microsatellite loci in a new world primate, the mantled howler monkey (Alouatta palliata). Molecular Ecology 7: 657–658. Excoffier, L., Smouse, P. E., Quattro, J. M. (1992). Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479-491. Excoffier, L., Laval, G., Schneider, S. (2005). Arlequin (version 3.0): An integrated software Packaged for population genetics data analysis. Evolutionary Bioinformatics 1: 47 –50. Falush, D., Stephens, M., Pritchard, J. K. (2007). Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 7:574–578.

338

Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al.

Figueiredo, W. B., Carvalho-Filho, N. M., Schneider, H., Sampaio, I. (1998). Mitochondrial DNA sequences and the taxonomic status of Alouatta seniculus populations in Northeastern Amazonia. Neotropical Primates 6: 73-77. Ford, S. M. (1994). Taxonomy and distribution of the owl monkey. In J. F. Baer, R. E. Seller, and I. Kakoma (Eds.), Aotus: the owl monkey (pp. 1–57). Maryland Heights: Academic Press. Galbreath, G. J. (1983). Karyotypic evolution in Aotus. American Journal of Primatology 4: 245–251. Garza, J., Williamson, E. (2001). Detection of reduction in population size using data from Microsatellite loci. Molecular Ecology 10:305-318. Giraldo, A., Bueno, M. L., Silva, E., Ramírez, J., Umaña, J., Espinal, C. (1986). Estudio citogenético de 288 Aotus colombianos. Biomédica 61: 5 – 13. Goudet, J. 1995. Fstat (ver1.2): a programm to calculate F-statistics. Journal of Heredity 86:485-486. Goudet J., Raymond M., Demeeus T., Rousset F. (1996). Testing differentiation in diploid populations. Genetics 144: 1933–1940. Gower, J. C., Ross G. J. S. (1969). Minimum spanning trees and single cluster analysis. Applicative Statistics 18: 54-64. Groves, C. P. (2001). Primate taxonomy. Washington, DC: Smithsonian Institution Press. Hershkovitz, P. (1949). Mammals of northern Colombia. Preliminary report no. 4: monkeys (primates), with taxonomic revisions of some forms. Proceedings of the United States National Museum 98: 323–427. Hershkovitz, P. (1977). Living new world monkeys (Platyrrhini). Chicago: University of Chicago Press. Hershkovitz, P. (1983). Two new species of night monkeys, genus Aotus (Cebidae, Platyrrhini): a preliminary report on Aotus taxonomy. American Journal of Primatology 4: 209–243. Hewitt, G. W. (1989). The subdivision of subspecies by hybrid zones. In Otte D, Endler JA (Eds.): Speciation and its consequences, Pp. 85-115. Sinauer, Sunderland, US. Hewitt, G. W. (1993). After the ice: Parallelus meets Erythropus in the Pyrenees. In Harrison RG (Ed.): Hybrid zones and the evolutionary process. (pp. 140-164). Oxford University Press, New York and Oxford, US. Hodgson, J. A., Sterner, K. N., Matthews, L. J., Burrell, A. S., Jani, R. A., Raaum, R. L., Stewart, C. B., Disotell, T. R. (2009). Successive radiations, not stasis, in the South American primate fauna. Proceedings of the National Academy of Sciences USA 106:5534–5539. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120. Kimura, M. (1983). The neutral theory of molecular evolution. Cambridge University Press, Great Britain. King, M. (1993). Species Evolution. Cambridge University Press, Cambridge. Kruskal, J. B. Jr. (1964a). Multidimensional scaling by optimizing goodness of fit to a nonmetric hypothesis. Psychometrika 29: 1-27. Kruskal, J. B. Jr. (1964b). Nonmetric multidimensional scaling: a numerical method. Psychometrika 29: 28-42.

Can Mitochondrial DNA, Nuclear Microsatellite DNA …

339

Lewis, H. (1966). Speciation in flowering plants. Science 152: 167-172. Luikart, G., Allendorf, F., Sherwin, B., Cornuet, J. M. (1998). Distortion of allele frequency distribution provides a test for recent population bottleneck. The Journal of Heredity 86: 319-322. Ma, N. S. F. (1981a). Chromosome evolution in the owl monkey, Aotus. American Journal of Physical Anthropology 54: 293–303. Ma, N. S. F. (1981b). Errata. Chromosome evolution in the owl monkey Aotus. American Journal of Physical Anthropology 54: 326. Ma, N. S. F. (1983). Gene map of the new world Bolivian owl monkey, Aotus. Journal of Heredity 74:27–33. Ma, N. S. F. (1984). Linkage and syntenic relationship in chromosomes of owl monkeys (with karyotypes I, III, V, VI, VII homologous to man. In: Genetic Maps, Vol. 3. O’Brien, S. J (Ed). New York, Cold Spring HarbourPress. Pp. 410-413. Ma, N. S., Jones, T. C., Miller, A. C., Morgan, L. M., Adams, E. A. (1976a) Chromosome polymorphism and banding patterns in the owl monkey (Aotus). Laboratory Animal Science 26: 1022-1036. Ma, N. S. F., Elliott, M. W., Morgan, L. M., Miller, A. C., Jones, T. C. (1976b). Translocation of Y chromosome to an autosome in the Bolivian owl monkey, Aotus. American Journal of Physical Anthropology 45: 191-202. Ma, N. S. F., Jones, T. C., Bedard, M. T., Miller, A. C., Morgan, L. M., Adams, E. A. (1977). The chromosome complement of an Aotus hybrid. Journal of Heredity 68: 409-412. Ma, N. S. F., Rossan, R. N., Kelley, S. T., Harper, J. S., Bedard, M. T., Jones, T. C. (1978). Banding patterns of the chromosomes of two new karyotypes of the owl monkey, Aotus, captured in Panama. Journal of Medical Primatology 7: 146 – 155. Ma, N. S. F., Renquist, D. M., Hall, R., Sehgal, P. K., Simeone, T., Jones, T. C. (1980). XX/XO sex determination systems in a population of Peruvian owl monkeys, Aotus. Journal of Heredity 71: 336–342. Ma, N. S. F., Simeone, T., McLean, J., Parham, P. (1982). Erythrocyte glyoxalase I polymorphism in the owl monkey, Aotus. Immunogenetics 15: 1–16. Ma, N. S. F., Aquino, R., Collins, W. E. (1985). Two new karyotypes in the Peruvian owl monkey (Aotus trivirgatus). American Journal of Primatology 9: 333–341. Mahalanobis, P. C. (1936). On the generalized distance in statistics. Proceeding of National Institute of Science India 2: 9-55. Maldonado, A. M. (2011). Tráfico de monos nocturnos Aotus spp. en la frontera entre Colombia, Perú y Brasil: Efectos sobre sus poblaciones silvestres y violación de las regulaciones internacionales de comercio de fauna estipuladas por CITES. La Revista de la Academia Colombiana de Ciencias 35: 225-242. Maldonado, A. M., Peck, M. R. (2014). Research and in situ conservation of owl monkeys enhances environmental law enforcement at the Colombian-Peruvian border. American Journal of Primatology 76: 658-669. Maldonado, A. M., Nijman, V., Bearder, S. K. (2009). Trade in night monkeys Aotus spp. In the Brazil-Colombia-Peru tri-border area: international wildlife trade regulations are ineffectively enforced. Endangered Species Research 9: 143–149. Marcus, L. F. (1990). Traditional Morphometrics. In Proceedings of the Michigan Morphometrics Workshop. F. J. Rohlf and F. L. Bookstein (Eds.). Special Publication Number 2. (pp. 77-122). The University of Michigan Museum of Zoology.

340

Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al.

Martin, R. D. (1990). Primate origins and evolution. Princeton: Princeton University Press. Menezes, A. N., Bonvicino, C. R., Seuánez, H. N. (2010). Identification, classification and evolution of Owl Monkeys (Aotus, Illiger 1811). BMC Evolutionary Biology 10: 248. Michalakis, Y., Excoffier L. (1996). A generic estimation of population subdivision using distances between alleles with special reference to microsatellite loci. Genetics 142: 1061–1064. Mudry, M. P., Colillas, O. J., Brieux, S. S. (1984). The Aotus of northern Argentina. Primates 25:530–537. Nei, M. (1972). Genetic distance between populations. American Naturalist 106: 283-292. Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the USA 70: 3321–3323. Nevo, E. (1991) Evolutionary Theory and processes of active speciation and adaptative radiation in subterranean mole rats, Spalax ehrenbergi superspecies, in Israel. Evolutionary Biology 25: 1-125. Nielsen, R. (1997). A likelihood approach to population samples of microsatellite alleles. Genetics 146: 711-716. Opazo, J. C., Wildman, D. E., Prychitko, T., Johnson, R. M., Goodman, M. (2006). Phylogenetic relationships and divergence times among New World monkeys (Platyrrhini, Primates). Molecular Phylogenetics and Evolution 40: 274-280. Pieczarka, J. C., Nagamachi, C. Y. (1988). Cytogenetic studies of Aotus from eastern Amazonia Y autosome rearrangement. American Journal of Primatology 14: 255–264. Pieczarka, J. C., Barros-R., M. D. S., Nagamachi, C., Rodríguez, R., Espinel, A. (1992). Aotus vociferans × Aotus nancymai sympatry without chromosomal hybridization. Primates 33: 239- 245. Pieczarka, J. C., Barros, R. M., Faria, J. R., Nagamachi, C. Y. (1993). Aotus from the southwestern Amazon region is geographically and chromosomally intermediate between A. azarae boliviensis and A. infulatus. Primates 34: 197–204. Pieczarka, J. C., Nagamachi, C. Y., Muniz, J. A., Barros, R. M., Mattevi, M. S. (1998). Analysis of constitutive heterochromatin of Aotus (Cebidae. Primates) by restriction enzyme and fluorochrome bands. Chromosome Research 6:77–83. Piry, S., Luikart, G., Cornuet, J. M. (1999). BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. Journal of Heredity 90: 502–503. Piry S., Alapetite, A., Cornuet, J. M., Paetkau, D., Baudouin, B and Estoup, A. (2004). GENECLASS2: a software for genetic assignment and first-generation migrant detection. Journal of Heredity 95: 536-539. Plautz, H. L., Goncalves, E. C., Ferrari, S. F., Schneider, M. P. C., Silva, A. (2009). Evolutionary inferences on the diversity of the genus Aotus (Platyrrhini, Cebidae) from mitochondrial cytochrome c oxidase subunit II gene sequences. Molecular Phylogenetics and Evolution 51: 382–387. Posada, D., Buckley, T. R. (2004). Model selection and model averaging in phylogenetics: advantages of akaike information criterion and Bayesian approaches over likelihood ratio tests. Systematic Biology 53: 793–808. Ramírez-Cerqueira., J. (1983). Reporte de una nueva especie de primate del género Aotus de Colombia. Resúmenes de las Comunicaciones Científicas del IX Congreso Latinoamericano de Zoología, Arequipa, Perú, October 9-15, 1983. Pp. 146.

Can Mitochondrial DNA, Nuclear Microsatellite DNA …

341

Rao, C. R. (1951). Advanced statistical methods in biometric research. Hafner Publishing Company, Darien. Raymond, M., Rousset, F. (1995). GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity 86: 248–249. Reig, O. (1980). Modelos de especiación cromosómica en las casiraguas (Género Proechimys) de Venezuela. In Reig, O (Ed.). Ecología y Genética de la especiación animal. (pp. 149-190). Equinoccio, Editorial de la Universidad Simón Bolívar, Caracas, Venezuela. Reumer, J. W. F., de Boer, L. E. M. (1980). Standardization of Aotus chromosome nomenclature with description of the 2n = 49-50 karyotype and that of a new hybrid. Journal of Human Evolution 9: 461-482. Rohlf, F. J. (1970). Adaptive hierarchical clustering schemes. Systematic Zoology 19: 145153. Rohlf, F. J. (2000). NTSYS-pc, Numerical Taxonomy and Multivariate Analysis System, Version 2.1. Exeter Publications, New York, USA. Rosenberger, A. L., Tejedor, M. F., Cooke, S. B., Pekar, S. (2009). Platyrrhine ecophylogenetics in space and time. In Garber PA, Estrada A, Bicca-Marques JC, Heymann EW, and Strier KB, (Eds.). South American primates: comparative perspectives in the study of behavior, ecology and conservation. (pp 69-116). New York: Springer Science. Ruiz-García, M. (2005). The use of several microsatellite loci applied to 13 Neotropical primates revealed a strong recent bottleneck event in the woolly monkey (Lagothrix lagotricha) in Colombia. Primate Report 71: 27–55. Ruiz-García, M., Pinedo-Castro, M. (2010). Molecular systematics and phylogeography of the genus Lagothrix (Atelidae, Primates) by means of mitochondrial COII gene. Folia Primatologica 81: 109–128. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P., Shostell, J. M. (2006). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report 73: 3–47. Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., GutierrezEspeleta, G., Shostell, J. M. (2007). Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: Genetic structure and recent bottlenecks. Folia Primatologica 78: 73–87. Ruiz-García, M., Castillo, M. I., Vásquez, C., Rodriguez, K., Pinedo-Castro, M., et al., (2010). Molecular phylogenetics and phylogeography of the white-fronted capuchin (Cebus albifrons; Cebidae, Primates) by means of mtCOII gene sequences. Molecular Phylogenetetics and Evolution 57: 1049-1061. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L. F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). Internation Journal of Primatology 32: 1218–1241. Ruiz-García, M., Castillo, M. I., Lichilin, N., Pinedo-Castro, M. (2012a). Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, C. xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene sequences. Folia Primatologica 83: 100-125.

342

Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al.

Ruiz-Garcia, M., Castillo, M. I., Ledezma, A., Leguizamon, N., Sánchez, R., et al., (2012b). Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of Primatology 74: 366-380. Ruiz-García, M., Vásquez, C., Camargo, E., Castellanos-Mora, L. F., Gálvez, H., et al., (2013). Molecular genetics analysis of mtDNA COII gene sequences shows illegal traffic of night monkeys (Aotus, Platyrrhini, Primates) in Colombia. Journal of Primatology 2: 107. doi:10.4172/2167-6801.1000107. Ruiz-García, M., Pinedo-Castro, M., Shostell, J. M. (2014a). How many genera and species of wolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular analysis of Lagothrix flavicauda, an endemic Peruvian primate species. Molecular Phylogenetics and Evolution 79: 179–198. Ruiz-García, M., Escobar-Armel, P., Leguizamón, N., Manzur, P., Pinedo-Castro, M., Shostell, J. M. (2014b). Genetic characterization and structure of the endemic Colombian silvery brown bare-face tamarin, Saguinus leocopus (Callitrichinae, Cebidae, Primates). Primates 55: 415-435. Ruiz-García, M., Luengas, K., Leguizamón, N., Thoisy, B., Gálvez, H. (2015). Molecular Phylogenetics and Phylogeography of all the Saimiri species (Cebidae, Primates) inferred from mt COI and COII gene sequences. Primates 56: 145-161. Ruiz-García, M., Lichilín, N., Escobar-Armel, P., Rodríguez, G. E., Gutierrez-Espeleta, E. (2016a). Historical Genetic demography and some insights into the systematics of Ateles (Atelidae, Primates) by means of diverse mitochondrial genes. In Ruiz-García, M., and Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I. (2016b). Genetic structure, spatial patterns and historical demographic evolution of white-throated capuchin (Cebus capucinus, Cebidae, Primates) populations of Colombia and Central America by means of DNA microsatellites. In RuizGarcía, M., and Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I., Luengas, K., Leguizamon, N. (2016c). Invalidation of three robust capuchin species (Cebus libidinosus pallidus, C. macrocephalus and C. fatuellus; Cebidae, Primates) in the Western Amazon and Orinoco by analyzing DNA microsatellites. In Ruiz-García, M., and Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I., Luengas, K. (2016d). It is misleading to use Sapajus (robust capuchins) as a genus? A review of the evolution of the capuchins and suggestions on their systematics. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-Herrera, A., Garcia, F., Aguilera, M., Garcia, M., Fontanals, M.P. (2005). Comparative chromosome painting in Aotus reveals a highly derived evolution. American Journal of Primatology 65: 73–85.

Can Mitochondrial DNA, Nuclear Microsatellite DNA …

343

Ruvolo, M., Disotell, T. R., Allard, M. W., Brown, W. M., Honeycutt, R. L. (1991). Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence. Proceedings of the National Academy of Sciences of the USA 88: 1571–1574. Santos-Mello, R., de Mello, M. T. (1986). Cariótipo de Aotus trivirgatus (macaco-da-noite) das proximidades de Manaus, Amazonas. Nota preliminar. In Mello, M. T. (Ed.) A Primatologia no Brasil. Volumen 2, de Sociedade Brasileira de Primatología, Belo Horizonte: Imprensa Universitária (pp. 139-1460). Universidade Federal de Minas Gerais. Schmidt, L. H. (1973) Infections with Plasmodium falciparum and Plasmodium vivax in the owl monkey-model systems for basic biological and chemotherapeutic studies. Transactions of the Royal Society of Tropical Medicine and Medicine 67: 446-474. Schwarz, G. E. (1978). Estimating the dimension of a model. Annals of Statistics 6: 461–464. Sena, L., Vallinoto, M., Sampaio, I., Schneider, H., Ferrari, S. F., et al., (2002). Mitochondrial COII gene sequences provide new insights into the phylogeny of marmoset species groups (Callitrichidae, Primates). Folia Primatologica 83: 100- 125. Setoguchi, R., Rosenberger, A. L. (1987). A fossil owl monkey from La Venta, Colombia. Nature 326: 692-694. Slatkin, M (1985). Rare alleles as indicators of gene flow. Evolution 39: 53-65. Sneath, P. H., and R. R. Sokal. (1973). Numerical Taxonomy. Freeman. San Francisco. Spence, I. (1972). A Monte Carlo evaluation of three nonmetric multidimensional scaling algorithms. Psychometrika 37: 461-486. Spence, I., Ogilvie, S. C. (1973). A table of expected stress values for random rankings in nonmetric multidimensional scaling. Mult. Behavior Research 8: 511-517. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688-2690. Stanyon, R., Bigoni, F., Slaby, T., Muller, S., Stone, G., Bonvicino, C. R., Neusser, M., Seuánez, H. N. (2004). Multi-directional chromosome painting maps homologies between species belonging to three genera of New World monkeys and humans. Chromosoma 113:305-315. Takai, M., Nishimura, T., Shigehara, N., Setoguchi, T. (2009). Meaning of the canine sexual dimorphism in fossil owl monkey, Aotus dindensis from the middle Miocene of La Venta, Colombia. Frontiers of Oral Biology 13:55–59. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 27312739. Torres, O. M., Enciso, S., Ruiz, F., Silva, E., Yunis, I. (1998). Chromosome diversity of the genus Aotus from Colombia. American Journal of Primatology 44: 255–275. Thorington, R. W., Jr., Vorek, R. E. (1976). Observations on the geographic variation and skeletal development of Aotus. Laboratory Animal Science 26: 1006-1021. Walsh, P. S., Metzger, D. A., Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506513. Weber, J. L., May, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. The American Journal of Human Genetics 44: 388–396.

344

Manuel Ruiz-García, Adriana Vallejo, Emily Camargo et al.

White, M. J. D. (1968). Models of speciation. New concepts suggest that the classical sympatric and allopatric models are not the only alternatives. Science 159: 1065-1070. White, M. J. D. (1978). Modes of speciation. W. H. Freeman, San Francisco. Wright, S. (1951). The genetical structure of populations. Annals of Eugenics 15: 323–354. Yunis, E., Torres de Caballero, O. M., Ramírez, C. (1977). Genus Aotus Q-and G-band karyotypes and natural hybrids. Folia Primatologica 27: 165-177.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 10

PHYLOGENETIC RELATIONSHIPS OF PITHECIDAE AND TEMPORAL SPLITS IN REFERENCE TO CEBIDAE AND ATELIDAE BY MEANS OF MITOGENOMICS Manuel Ruiz-García1,, Geven Rodríguez1, Myreya Pinedo-Castro1 and Joseph Mark Shostell2 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética. Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC., Colombia 2 Department of Math Science and Technology, University of Minnesota Crookston, Crookston, MN, US

ABSTRACT We carried out a mitogenomic analysis of 41 Neotropical primate individuals belonging to 10 different taxa (Pithecia monachus monachus, P. m. milleri, Callicebus torquatus lugens, C. cupreus ornatus, Ateles hybridus, A. fusciceps, A. belzebuth, Aotus vociferans, A. lemurinus griseimembra, and A. nancymaae). Three main results were obtained. 1- Pitheciidae was the most basal family of the Platyrrhini monkeys, with Cebidae + Atelidae forming a more recent clade. 2- We estimated temporal splits throughout this mitogenomics analysis such as the diversification of the current Platyrrhini which began around 24.3 millions of years ago (MYA) with the split of the Pitheciidae. Also, there was a split between Cebidae and Atelidae around 22.01 MYA. The diversification within Pitheciidae occurred around 17.4 MYA, whereas the mitochondrial diversification within Callicebus and within Pithecia happened approximately 7.8 and 6.8 MYA, respectively. The split between Aotus vociferans and A. nancymaae began around 4.3 MYA and the mitochondrial diversification within Ateles was around 3.23 MYA; 3- The current results allowed us to make some inferences on the systematics of Pithecia, Aotus and Ateles.



Correspondence: [email protected], [email protected].

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Manuel Ruiz-García, Geven Rodríguez, Myreya Pinedo-Castro et al.

Keywords: Ateles, Aotus, Callicebus, Pithecia, mitogenomics, molecular phylogenies, temporal splits

INTRODUCTION There is little consensus among primatologists as to the number of Neotropical primate families and the relationships among them. One, two or three Platyrrhini families have been proposed from a morphological perspective (Simpson, 1945; Hershkovitz, 1977). These traditional classifications of the Platyrrhini divided them into Cebidae and the Callitrichidae, with Callimico being allocated to either the first (Simpson, 1945; Simons, 1972) or the second family (Pocock, 1925; Napier and Napier, 1967; Szalay, 1979). Hershkovitz (1972, 1977) proposed a third family (Callimiconidae, with a genus Callimico). According to Hershkovitz (1977), then, the Platyrrhini should be included in three families: Callitrichidae, which comprised the marmosets (Callithrix and Cebuella), tamarins (Saguinus) and lion tamarins (Leontopithecus), Callimiconidae (Callimico) and the Cebidae (Cebus, Saimiri, Aotus, Alouatta, Ateles, Lagothrix, Brachyteles, Callicebus, Pithecia, Chiropotes, and Cacajao). Two or three families have also been claimed from a molecular point of view. Schneider et al., (1993, 1996) proposed the existence of two families by analyzing the nuclear sequences of the long intron 1 of the interstitial retinol-binding protein (IRBP) and -globin gene sequences. They defended the existence of the Cebidae family, including the subfamilies Callitrichinae (subtribe Callitrichina, genera Callithrix and Cebuella; subtribe Callimiconina, genus Callimico; subtribe Leontopithecina, genus Leontopithecus; subtribe Saguina, genus Saguinus), Aotinae (Aotus) and Cebinae (Cebus and Saimiri) and the Atelidae family, including two subfamilies, Pitheciinae (tribe Callicebini, genus Callicebus; tribe Pitheciini, subtribe Chiropotina, genera Chiropotes and Cacajao and subtribe Pitheciina, genus Pithecia) and Atelinae (tribe Alouattini, genus Alouatta, and tribe Atelini with subtribes Brachytelina, genera Brachyteles and Lagothrix and Atelina, genus Ateles). This arrangement differed from that offered by Rosenberger (1984). His analysis of morphology led him to also consider two families, Cebidae and Atelidae, because in the last arrangement, Aotus was enclosed within Atelidae. However, Harada et al., (1995) and Barroso et al., (1997) considered the subfamily Pitheciinae as a full family, Pitheciidae. Since then, the major part of the molecular works considered the existence of three families (Atelidae, Cebidae and Pitheciidae). Von Dornum and Ruvolo (1999), used a 1.3 kb sequence from two introns of the glucose-6-phosphate dehydrogenase (G6PD) gene in 24 platyrrhine species and presented a phylogeny, where Pitheciidae was differentiated from the Atelidae. They also determined Pitheciidae to be the most basal branch in the phylogeny of the Platyrrhini. Steiper and Ruvolo (2003) subsequently added two G6PD introns, with a total sequence of 2.1 kb, and reconfirmed the results of Von Dornum and Ruvolo (1999). Later, Opazo et al., (2006) sequenced six nuclear genes (B2M, -globin, G6PD4, G6PD5, IRBP, vWF and TOM) and one mitochondrial gene (16S rRNA) (9,137 base pairs, bp) for 15 genera of Neotropical primates. They showed the existence of three clades (families Pitheciidae, Cebidae and Atelidae). Ray et al., (2005) studied 183 Alu markers that were specific to nine platyrrhines and detected three families with Pitheciidae as the most basal one. Osterholz et al. (2009) analyzed the presence/absence

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pattern of 128 SINEs in15 platyrrhine genera and they detected forty Alu insertions, which were informative for phylogenetics tasks among genera. The most important results were the confirmation of the monophyly of Cebidae, Atelidae, and Pitheciidae, as well as the evidence for a sister grouping of the Cebidae with the Atelidae (they shared five Alu insertions), with the Pitheciidae positioned externally. Osterholz et al., (2009) showed that the monophyly of the Pitheciidae, Atelidae and Cebidae, were supported by seven, 11, and 10 Alu insertions respectively. The most recent works on molecular primate phylogenetics ratified the existence of three differentiated families. These are the cases of Wildman et al., (2009), Perelman et al., (2011), Springer et al., (2012), Jameson Kiesling et al., (2015) and Schneider and Sampaio (2015). However, the phylogenetics relationships among these three families is controversial and may be a function of the characters or molecular markers used. Mitochondrial genes are interesting markers for phylogenetic tasks because they include a rapid accumulation of mutations, rapid coalescence time, lack introns, have a high number of copies per cell (which makes mitogenomic data easy to obtain especially in low quality samples, such as hairs, teeth of hunted animals or museum specimens; Mason et al., 2011; Guschansli et al., 2013), a negligible recombination rate, and haploid inheritance (Avise et al., 1987). Mitochondrial gene trees are more precise in reconstructing the divergence history among species than other molecular markers (Moore, 1995). Cummings et al., (1995) showed that mitochondrial genomes have higher information content per base than nuclear DNA. When mitochondrial genes were used for the reconstruction of the phylogeny of the Platyrrhini, no clear results were obtained. Horovitz and Meyer (1995) used maximum parsimony and successive weighting to analyze 16S rRNA sequence data of 13 genera (Figure 1a). They showed Atelidae as the most basal family, with Pithecia clustered together with Cebus and Callicebus together with Saimiri (therefore, no monophyletic clade exists for the Pitheciidae) within the Cebidae. Later, Horovitz et al., (1998) expanded the analysis to 15 genera for the mitochondrial genes, 16S rRNA and 12 rRNA (Figure 1b,c). For the first tree, Callicebus was clustered with Cebus + Saimiri within the Cebidae. Pithecia and Chiropotes formed the most external branch of the Platyrrhini. The second showed Pithecia and Chiropotes as the most external clade followed by Callicebus, although the tree did not form a monophyletic clade. Cebidae and then Atelidae followed. The Atelidae was determined to be a monophyletic clade but not the Cebidae. Recently, many mitogenomic analyses have shown to be extremely useful in determining phylogenetic relationships and in estimating divergence times in many different groups of organisms, such as fish (Miya et al., 2003; Inoue et al., 2010), amphibians (Zhang et al., 2005; Zhang and Wake, 2009), birds (Pereira and Baker, 2006; Slack et al., 2007) and mammals (Arnasson et al., 2002, 2004, 2008; Hodgson et al., 2009; Matsui et al., 2009; Chiou et al., 2011; Finstermeier et al., 2013; Pozzi et al., 2014). The study of Pozzi et al., (2014) was the first to use the complete mitochondrial genome to reconstruct the phylogeny of primates. That study only included six Platyrrhini species (one individual per species) (Aotus lemurinus, Saguinus oedipus, Cebus albifrons, Saimiri sciureus, Ateles belzebuth and Callicebus donacophilus). Aotus, Saguinus, Cebus and Saimiri formed a clade (Cebidae). Ateles formed another sister branch of the Cebidae (Atelidae) and the most divergent and basal branch consisted of Callicebus (Pitheciidae). However, the number of individuals in that study was very limited.

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Figure 1. Maximum parsimony tree of Horovitz and Meyer (1995) analyzing 13 genera of Platyrrhini with the mitochondrial 16S rRNA gene and successive weighting (A); Maximum parsimony tree of Horovitz et al., (1998) analyzing 15 genera of Platyrrhini with the mitochondrial 16S rRNA gene (B); Maximum parsimony tree of Horovitz et al., (1998) analyzing 15 genera of Platyrrhini with the mitochondrial 12S rRNA gene (C).

Herein, we also sequenced the complete mitochondrial genomes of 41 Neotropical primates, including Pitheciidae (Callicebus with two taxa, C. torquatus lugens, following Groves, 2001, or C. lugens following Van Roosmalen et al., 2002, and C. cupreus ornatus, following Goves, 2001, or C. ornatus, following Van Roosmalen et al., 2002; Pithecia with two taxa, P. monachus milleri, following Groves, 2001, or P. milleri, following Marsh, 2014, and P. monachus monachus, following Groves, 2001, or P. monachus or P. hirsuta, following

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Marsh, 2014). We also included Atelidae (with three Ateles species following Collins and Dubach, 2000ab, 2001, although we discuss the systematics of the spider monkeys in another chapter of this book: A. belzebuth, A. fusciceps rufiventris and A. hybridus). Cebidae was also included (represented by Aotus and three taxa, A. vociferans, A. lemurinus griseimembra and A. nancymaae, following Groves, 2001, although we discuss the systematics of the night monkeys in another chapter of this book). Taking all of this into consideration, we have three main aims in the current work: 1- to determine the phylogenetics relationships among the three Platyrrhini families; 2- to estimate temporal splits among the three families and also among the genera and species of each family and 3- to determine systematics of taxa under the genus level and to compare them with other systematics studies presented in the current book and other works.

MATERIAL AND METHODS A total of 41 individuals were sequenced for their entire mitochondrial genomes. We followed Groves’s (2001) nomenclature. There were: one individual of Callicebus torquatus lugens (Meta Department, Colombia), three individuals of Callicebus cupreus ornatus (Meta Department, Colombia), two individuals of Pithecia monachus milleri (Putumayo Department, Colombia), two individuals of Pithecia monachus (Amazon River, Peru), one individual of Aotus nancymaae (Quebrada Yanayacu, Peru), 15 individuals of Aotus vociferans (from Leticia to San Juan de Atacuarí in the Amazon River, Colombia), one individual of Aotus lemurinus griseimembra (Cordoba Department, Colombia), three individuals of Ateles belzebuth (Pastaza province, Ecuador), eight individuals of Ateles fusciceps rufiventris (Choco and Antioquia Departments, Colombia) and five individuals of Ateles hybridus (Antioquia Department, Colombia). Sampling methods complied with all the protocols approved by the Ethical Committee of the Pontificia Universidad Javeriana (No. 45677) and the laws of the Ministerio de Ambiente, Vivienda y Desarrollo Territorial (R. 1252) from Colombia. This research also adhered to the American Society of Primatologists’ Principles for the Ethical Treatment of Primates.

Molecular Procedures DNA was extracted and isolated from either blood or muscle samples using the QIAamp DNA Micro Kit (Qiagen, Inc.) following the protocol provided by the manufacturer. Blood extractions followed the protocol “DNA Purification from Blood or Body Fluids (Spin Protocol),” while muscle extractions followed the protocol “DNA Purification from Tissues.” Mitochondrial genomes were sequenced by long-template PCR, which minimizes the chance of amplifying mitochondrial pseudogenes from the nuclear genome (numts) (Thalmann et al., 2004; Raaum et al., 2005). PCR amplification of mitochondrial DNA was carried out using a LongRange PCR Kit (Qiagen, Inc.), with a reaction volume of 25 l and a reaction mix consisting of 2.5 l of 10x LongRange PCR Buffer, 500 M of each dNTP, 0.6 M of each primer, 1 unit of Long-Range PCR Enzyme, and 70–250 ng of template DNA. Cycling conditions were as follows: 93°C for 5 min, followed by 45 cycles denaturing at 93°C for 30

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s, primer annealing at 51–59°C (depending on primer set) for 30 s, and extension at 72°C for 10 min, followed by 30 cycles of denaturing at 93°C for 30 s, annealing at 46–51°C (depending on primer set) for 30 s, and extension at 72°C for 5 min, with a final extension at 72°C for 10 min. To minimize the possibility of amplifying nuclear mitochondrial pseudogenes (numts), four sets of primers were used to generate overlapping amplicons from 2,500 to 6,000 bp in length, thereby enabling a quality test for genome circularity (Thalmann et al., 2004). Both mtDNA strands were sequenced directly using BigDye Terminator v3.1 (Applied Biosystems, Inc.) and a suite of sequencing primers including primers previously designed for different platyrrhine species (Hodgson et al., 2009). Sequencing products were analyzed on an ABI 3730 DNA Analyzer system (Applied Biosystems, Inc.). Sequences were then assembled and edited using Sequencher 4.7 (Gene Codes, Corp., Ann Arbor, MI). Overlapping regions were examined for irregularities such as frameshift mutations and premature stop codons. A lack of such irregularities indicates an absence of contaminating numt sequences. We used 12 heavy-strand protein-coding genes (ND1, 962 bp; ND2, 1,050 bp; COI, 1,569 bp; COII, 696 bp; ATP8, 225 bp; ATP6, 681 bp; COIII, 804 bp; ND3, 355 bp; ND4L, 296 bp; ND4, 1,386 bp; ND5, 1846 bp and Cyt-b, 1,144 bp) and RNA sequences (2 rRNAs genes, 1,708 bp and 22 tRNAs, 1,313 bp) summing up a total of 14,035 bp (around 83% of the total mitochondrial genome). The control region was excluded because of alignment difficulties due to its high variability among different genera. The ND6 gene was also deleted because it is encoded on the mitochondrial L-strand which has a different nucleotide composition from the H-strand, and has been shown to have poor phylogenetic signal (Gissi et al., 2000). Protein-coding genes were then aligned based on their corresponding amino acid translations using the TranslatorX Software (Abascal et al., 2010). Since homology is best identified at the amino acid level, we used this software because it translates the DNA sequences into amino acids, aligns the amino acid sequences, and then back-translates the alignment to the nucleotide sequences. The alignments with all the genes employed (14,035 bp) were concatenated after removing problematic regions using Gblocks 0.91 (Talavera and Castresana, 2007) under a relaxed approach. This software removes all poorly aligned regions and it has been shown to be particularly effective in phylogenetic studies including very divergent sequences (Castresana, 2000; Talavera and Castresana, 2007). The individual alignments were then concatenated by means of the SequenceMatrix v1.7.6 Software (Vaidya et al., 2011) to create a master alignment.

Phylogenetics Procedures The MrModeltest v2.3 Software (Nylander, 2004) and the Mega 6.05 Software (Tamura et al., 2013) were applied to determine the best evolutionary mutation model for the sequences analyzed for each individual gene, for different partitions and for all the concatenated sequences. Akaike information criterion (AIC; Akaike, 1974; Posada and Buckley, 2004) and the Bayesian information criterion (BIC; Schwarz, 1978) were used to determine the best evolutionary nucleotide model. Additionally, we obtained maximum likelihood estimates of transition/transversion bias as well as maximum likelihood estimates of the gamma parameter for site rates for the best evolutionary nucleotide model obtained (Tamura et al., 2013).

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Phylogenetic trees were constructed by using two procedures: Maximum Likelihood tree and Bayesian analysis (BI). A Maximum Likelihood tree (MLT) was obtained using RAxML v.7.2.6 Software (Stamatakis, 2006). To select the best fitting model, 50 independent iterations were run using three data partitions (codon 1, codon 2, codon 3). Additionally, 50 iterations were run using two data partitions (codons 1+2 combined, codon 3). For each analysis, the HKY + G model (Hasegawa-Kishino-Yano, 1985) was used to search for the MLT and topologic support was estimated with 500 bootstrap replicates using the HKYGAMMA (Stamatakis, 2006). Maximum-likelihood bootstrap proportions (MLBS) higher than 70% were considered strong support (Hillis and Bull, 1993; Wilcox et al., 2002). 2- A Bayesian analysis was performed using a HYK + G model with the gamma distributed rate varying among sites, because it was determined to be the better model using the MrModeltest v2.3 Software. This Bayesian analysis was completed with the BEAST v. 1.8.1 program (Drummond et al., 2012). Four independent iterations were run using three data partitions (codon 1, codon 2, codon 3) with six MCMC chains sampled every 10,000 generations for 40 million generations after a burn-in period of 4 million generations. We checked for convergence using Tracer v1.6 (Rambaut et al., 2013). We plotted the likelihood versus generation and estimated the effective sample size (ESS > 200) of all parameters across the four independent analyses. Additionally, AWTY (Are We There Yet?) Software (Wilgenbusch et al., 2004; Nylander et al., 2008) was used to plot pairwise split frequencies for the four independent MCMC runs and to check the posterior probabilities of clades for non-overlapping trees in the sample using the compare and slide commands. The results from different runs were combined using the LogCombiner v1.8.0 Software (Rambaut and Drummond, 2013a) and the TreeAnnotator v1.8.0 Software (Rambaut and Drummond, 2013b). A Yule speciation model and a relaxed molecular clock with an uncorrelated lognormal rate of distribution (Drummond et al., 2006) was used. Posterior probability values provide an assessment of the degree of support of each node on the tree. Values higher than 0.95 were considered strong support for monophyletic clades (Erixon et al., 2003; Huelsenbeck and Rannala, 2004). We estimated the lower and upper 95% highest posterior densities (HPD), the means, geometric means, medians, marginal densities and traces with the Tracer v1.6 Software. Trees were visualized in the FigTree v. 1.4 Software (Rambaut, 2012). This program was run to estimate the time to most recent common ancestor (TMRCA) for different nodes of the BI. We employed two priors. One was around 24.17 ± 1.0 Millions of years ago (MYA) for the initial diversification of the current Platyrrhini. The second was about 18.3 ± 0.5 MYA for the diversification within Pitheciidae. These average temporal priors were obtained from the data offered by Harada et al., (1995), Kay et al., (1998, 2005), Jameson Kiesling et al., (2015), Opazo et al., (2006), Osterholz et al., (2009), Perelman et al., (2011), Schneider and Sampaio (2015), Schneider et al., (1993), Springer et al., (2012), Steiper and Ruvolo (2003), Von Dornum and Ruvolo (1999) and Wildman et al., (2009). To estimate possible divergence times among the haplotypes found in the genera studied, we constructed a Median Joining Network (MJ) (Bandelt et al., 1999) using Network 4.2.0.1 Software (Fluxus Technology Ltd). Additionally, the  statistic (Morral et al., 1994) and its standard deviation (Saillard et al., 2004) were estimated and transformed into years. The  statistic is unbiased and highly independent of past demographic events. We used the rate of one mutation each 80,000 years (based on an average of the 12 heavy-strand protein-coding genes).

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Figure 2. Maximum likelihood tree analyzing the phylogenetic relationships of 41 individuals of Callicebus torquatus lugens, Callicebus cupreus ornatus, Pithecia monachus milleri, Pithecia monachus monachus (= hirsuta), Aotus nancymaae, Aotus vociferans, Aotus lemurinus griseimembra, Ateles belzebuth, Ateles hybridus and Ateles fusciceps rufiventris, by means of mitogenomics (14, 035 base pairs). The number in the nodes are bootstrap percentages.

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RESULTS Based on the BIC and the Akaike information criterion, the HKY + G nucleotide substitution model was the better fit with the mitochondrial genome of the Neotropical primates (235,996.23 and 228,529.51, respectively). The maximum likelihood estimate of transition/transversion bias was 4.67 (maximum log likelihood was -111,802.96). The MLT (Figure 2) showed Pithecia as the first clade to diverge (100% bootstrap) which in turn contained two sub-clades (100%, respectively), one containing two P. m. milleri and another containing two P. m. monachus. Thus, both taxa of Pithecia are highly differentiable from a molecular point of view. The next genus to diverge was Callicebus (90%) with a clear differentiation between C. t. lugens and C. c. ornatus (100%). Thus, Pithecia and Callicebus, although they did not form a monophyletic clade in this tree, their ancestors diverged before those of Ateles (representing Atelidae) and Aotus (representing Cebidae). These two last genera formed a monophyletic clade (Atelidae + Cebidae; 100%). Each genus also formed a monophyletic clade. Ateles (100%) showed two sub-clades (92 and 100%, respectively). One sub-clade contained a cluster of three A. belzebuth (100%), one A. hybridus and three A. f. rufiventris. The other sub-clade had five A. f. rufiventris and four A. hybridus. Aotus (100%) showed two clear sub-clades, one containing the unique A. nancymaae and another contained all of the A. vociferans individuals plus one A. l. griseimembra. Thus, this tree showed a more intense phylogenetic relationship between Ateles (Atelidae) and Aotus (Cebidae) than either with Pitheciidae. However, Pithecia and Callicebus did not form a monophyletic clade. However, both genera showed that their ancestors were more related with the hypothetical and original Platyrrhini ancestor. The BI (Figure 3) basically showed the same topology to that of the previous tree with the exception that Callicebus and Pithecia formed a monophyletic clade (Pithecidae). The majority of a posteriori probabilities were one or very close to one. Thus, all the clades we found were very significant. The temporal split between Pitheciidae and Cebidae + Atelidae (diversification of the current Platyrrhini) was around 24.33 MYA (95% HPD: 22.97-24.91 MYA). The mitochondrial diversification within Pitheciidae began around 17.43 MYA (95% HPD: 17.27-19.22 MYA) and the mitochondrial diversification within Cebidae + Atelidae was around 22.01 MYA (95% HPD: 21.11-23.05). The mitochondrial differentiation between C. t. lugens and C. c. ornatus began around 7.82 MYA (95% HPD: 6.4-8.34 MYA), whereas the haplotype diversification detected in the three C. c. ornatus individuals analyzed was around 0.31 MYA (95% HPD: 0.04-1.55 MYA). The mitochondrial differentiation between the two Pithecia taxa occurred around 6.76 MYA (95% HPD: 1.88-13.14 MYA), with the mitochondrial diversification in P. m. milleri occurring around 0.31 MYA (95% HPD: 0.011.25 MYA) and around 1.65 MYA (95% HPD: 0.69-5.87 MYA) for P. m. monachus. In the case of Aotus, the split between A. nancymaae and A. vociferans + A. l. griseimembra happened around 4.32 MYA (95% HPD: 4.19-5.88 MYA) and the mitochondrial diversification within A. vociferans + A. l. griseimembra took place about 3.51 MYA (95% HPD: 3.19-5.23 MYA). The mitochondrial diversification within Ateles was estimated to have occurred around 3.23 MYA (95% HPD: 3.23-3.99 MYA). Thus, of all the genera studied, the youngest mitochondrial diversification seems to be that of Ateles.

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Figure 3. Bayesian tree analyzing the phylogenetic relationships of 41 individuals of Callicebus torquatus lugens, Callicebus cupreus ornatus, Pithecia monachus milleri, Pithecia monachus monachus (= hirsuta), Aotus nancymaae, Aotus vociferans, Aotus lemurinus griseimembra, Ateles belzebuth, Ateles hybridus and Ateles fusciceps rufiventris, by means of mitogenomics (14, 035 base pairs). The first number in the nodes are posterior probabilities higher than 0.5 (green); the second number in the nodes are mean temporal splits (red); the third number in the nodes are 95% HPD of temporal splits (black). These temporal splits are in millions of years.

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Figure 4. Median Joining Network (MJN) with haplotypes found by mitogenomics for the 41 individuals of Callicebus torquatus lugens, Callicebus cupreus ornatus, Pithecia monachus milleri, Pithecia monachus monachus (= hirsuta), Aotus nancymaae, Aotus vociferans, Aotus lemurinus griseimembra, Ateles belzebuth, Ateles hybridus and Ateles fusciceps rufiventris analyzed. Light blue circles = Pithecia monachus milleri; brown circles = Pithecia monachus monachus (= hirsuta); lilac circle = Callicebus torquatus lugens; grey circles = Callicebus cupreus ornatus; green circles = Ateles belzebuth; black circles = Ateles hybridus and Ateles fusciceps rufiventris; yellow circles = Aotus vociferans; dark blue circles = Aotus nancymaae and pink circle = Aotus lemurinus griseimembra. Red circles indicate missing intermediate haplotypes.

The MJN procedure (Figure 4) ratified many of the aspects observed in both of the described trees. The oldest diversification is on the left and the newest is on the right. The two Pithecia taxa diversified first. Both are clearly differentiated. Later, Callicebus’ mitochondrial diversification began. This was followed by Ateles’s mitochondrial diversification (with mitochondria of A. hybridus and A. fusciceps intermixed) and finally by the Aotus’s mitochondrial diversification, where A. l. griseimembra presented mitochondria not differentiated from those of A. vociferans. The temporal splits are as follows: Pithecia-Ateles and Pithecia-Aotus showed temporal splits of 13.02 ± 0.11 MYA and 19.57 ± 0.13 MYA, with an average of around 16.3 MYA. The split between Pithecia and Callicebus was around 11.31 MYA (Pithecia-C. c. ornatus: 13.35 ± 0.10 MYA and Pithecia-C. torquatus: 9.28 ± 0.31 MYA, respectively). The split between Ateles and Aotus was around 10.96 ± 0.13 MYA. All of these temporal splits were somewhat lower than those obtained with the BI. However, the split estimations among more related taxa were similar to those obtained for the BI. The split between the two Callicebus

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taxa was estimated to have occurred around 4.15 ± 0.20 MYA and between both Pithecia taxa around 5.74 ± 0.05 MYA. The temporal split between the haplotypes of P. m. milleri occurred around 0.14 ± 0.05 MYA. A little older, the haplotypes of P. m. monachus are estimated to have split approximately 0.42 ± 0.03 MYA. The temporal split for the different C. c. ornatus’s haplotypes took place around 0.14 ± 0.02 MYA. The temporal divergence between A. nancymaae and A. vociferans was estimated to have occurred 3.96 ± 0.09 MYA, while that of A. belzebuth and A. hybridus was a little more recent, at around 3.57 ± 0.21 MYA.

Figure 5. Molecular phylogeny from two introns studied of the G6PD gene (1,286 bp; Von Dornum and Ruvolo, 1999), which coincides with our phylogenetic trees with mitogenomics.

DISCUSSION Our mitogenomic data confirmed the existence of three main and different clades (especially the BI). The data support the existence of three families (Pitheciidae, Cebidae and Atelidae) with Pitheciidae as the most basal family in the current Platyrrhini. Therefore, the overall mitochondrial information helped to reconstruct the phylogenetic relationships among these families as did other works with other kind of markers. Barroso et al., (1997) analyzed the long intron 1 of the IRBP gene (1,800 bp). Von Dornum and Ruvolo (1999) studied two introns of the G6PD gene (1,286 bp) (Figure 5) and later Steiper and Ruvolo (2003) added two G6PD introns (2,100 bp). Kay et al., (2005) studied 183 Alu markers, while Osterholz et al., (2009) analyzed 128 SINEs elements. Prychitko et al., (2005) analyzed -globin gene sequences. Opazo et al., (2006) sequenced six nuclear genes (B2M, -globin, G6PD4, G6PD5, IRBP, vWF and TOM) and one mitochondrial gene (16S rRNA) (a total of 9,137 bp) with a maximum parsimony tree. Wildman et al., (2009) sequenced 11 non-coding genomic

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markers from a shotgun library (7,665 bp). Perelman et al., (2011) sequenced 34,927 bp from 54 nuclear genes (27,427 bp from autosome genes, 4,870 bp from X chromosome and 2,630 bp from Y chromosome). Springer et al., (2012) analyzed 61,199 bp and 69 nuclear gene segments as well as 10 mitochondrial genes. Jameson Kiesling et al., (2015) analyzed 40,986 bp with a multiple sequence alignment of 64 non-genic markers. Schneider and Sampaio (2015) reanalyzed with Bayesian procedures the sequences of 25 autosome introns (16,656 bp) obtained by Perelman et al., (2011). All these studies support Pitheciidae as the first to diverge, followed by the more closely related Cebidae and Atelidae. Our mitogenomics data complement these earlier findings. Thus, findings of molecular studies are similar as long as there are sufficient data and taxa.

Figure 6. Morphological and parasitological phylogenies of the Platyrrhini: Rosenberger (1981, 1984) (A); Ford (1986) (B); Kay (1990) (C); Sorci et al., (1997) (D).

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However, other morphological and parasitological studies as well as those with insufficient molecular data do not provide phylogenetic results of the three families of Platyrrhini with Pitheciidae being the most basal one. The morphological phylogenies (Figure 6a, b, c) don’t completely agree with our results. Rosenberger (1981, 1984) studied a variety of morphological characters and showed a clade with Pitheciidae + Atelidae, plus a close relationship between Callicebus and Aotus within Pitheciidae. Ford (1986), used cladistics procedures to analyze dental, cranial and postcranial characters. She showed a clade of Pitheciidae + Atelidae, but with Callicebus outside of Pitheciidae and clustered with Aotus and maybe with Saimiri. She also stated that Cebidae was paraphyletic with Callitrichinae and more related to Pitheciidae + Atelidae. Kay (1990), also used cladistics procedures and applied them to dental characters. Kay determined the branch of Callicebus to be the most basal of all the current Platyrrhini genera with the Atelidae more related to the Cebidae genera with the exception of Cebus. Cebus was outside of the Cebidae and the second basal branch after Callicebus. Sorci et al., (1997) studied the morphology of parasites of Neotropical primates (Figure 6d) and found Pitheciidae + Atelidae to exclude Callicebus from the Pitheciidae. Pitheciidae + Atelidae were closely related to Aotus and were the most basal branch of Platyrrhini. The first molecular phylogenies also yielded controversial results (Figure 7a, b, c, d, e, f, g). Schneider et al., (1993) analyzed introns 1, 2 and 3’ of the noncoding region of -globin gene sequences and detected the clade Pitheciidae + Atelidae. It included Callicebus. They also detected Cebidae as a monophyletic clade. Harada et al., (1995), also used -globin gene sequences, but increased the number of taxa. Their findings were the same as that of Schneider et al., (1993). Based on intron 1 of the IRBP gene sequences, Schneider et al., (1996), showed Pitheciidae to be more related to Cebidae. Furthermore, they showed the Atelidae to be the most basal family of the Platyrrhini. The same authors showed the relationship of Pitheciidae + Atelidae when a maximum parsimony tree was obtained with both IRBP + -globin gene sequences. Porter et al., (1997), used the-globin gene and 5´flanking region (2,000 bp), to determine Pitheciidae + Atelidae in one side, and the Cebidae on the other side. The super tree of Hugot (1998), including these molecular data, plus the morphological and parasitological data, showed Pitheciidae + Atelidae, but with Callicebus + Aotus conforming an external clade not clearly related with the other Platyrrhini clades. Canavez et al., (1999), based on the 2-microglobuline gene sequences, also showed Pitheciidae + Atelidae versus Cebidae. Finally, Opazo et al., (2006), with the quoted six nuclear genes and one mitochondrial gene sequences, determined Pitheciidae versus Atelidae + Cebidae with a maximum parsimony tree. They also showed Pitheciidae + Atelidae versus Cebidae when maximum likelihood and Bayesian trees were obtained. This last work was the unique it had a large molecular data and it did not show Pitheciidae as the most basal family in the Platyrrhini with Cebidae + Atelidae. It seems clear that the morphological and parasitology characters used to reconstruct the Platyrrhini phylogeny are not neutrally derived homologies because these kind of characters are extremely affected by natural selection. This creates homoplasies by convergent selection or extreme divergent characters by anagenesis in closely related taxa. The molecular data (including mitogenomics) clearly supports the view of Schneider (2000), with three defined families in the Platyrrhini (Cebidae with three subfamilies, Callitrichinae, Cebinae and Aotinae; Pitheciidae with two subfamilies, Pitheciinae and

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Callicebinae; Atelidae with two subfamilies, Alouattinae and Atelinae). In contrast, other classifications from morphological perspectives considered a different number of families. For instance, Groves (2001) considered the existence of four families, the three previous ones plus Aotidae (and other subfamily within Cebidae, Samiriinae). Rylands and Mittermeier (2009) considered five families (Callitrichidae, Cebidae, Aotidae, Pitheciidae and Atelidae). However, we prefer those classifications which are more related to the phylogeny and evolutionary history of the organisms and, indisputably, many molecular markers show fewer levels of homoplasies and ancestral homologies, as well as less heterogenic evolutionary rates motivated by different and rapidly changeable natural selection pressures, than the morphological characters.

Figure 7. (Continued).

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Figure 7. Molecular phylogenies of the Platyrrhini: Schneider et al., (1993) with introns 1, 2 and 3’ non coding region of -globin gene sequences (A); Harada et al., (1995) with the same -globin gene sequences as Schneider et al., (1993) but with more taxa (B); Schneider et al., (1996) with a maximum parsimony tree and the first intron of IRBP gene sequences (C); Porter et al., (1997) with the -globin and 5´flanking region gene sequences (D); Hugot (1998) with a super tree with molecular + morphological + parasitology data (E); Canavez et al., (1999) with 2-microglobuline gene sequences (F); Opazo et al., (2006) with maximum likelihood and Bayesian trees and six nuclear gene sequences and one mitochondrial gene sequence (G).

Our temporal split estimations for the divergence of these three families as well as the diversification within them and the split among genera and among species within genera were also very similar to that obtained by other works. Here we provide some examples. For the crown diversification of the current Platyrrhini, our Bayesian estimation was around 24.3 MYA, which agrees extremely well with estimates obtained by many other authors

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(MacFadden, 1990 [25.7 MYA]; Opazo et al., 2006 [26 MYA]; Chatterjee et al., 2009 [26.6 MYA, interval: 23.5-30.0 MYA]; Perelman et al., 2011 [24.8 MYA, interval: 20.6-29.3 MYA]; Wilkison et al., 2011 [25.1 MYA, interval: 20.1-31.0 MYA]; Springer et al., 2012 [23.3 MYA, interval: 19.2-27.5 MYA]; Finstermeier et al., 2013 [22.0 MYA, interval: 19.224.4 MYA]; Pozzi et al., 2014 [21 MYA, interval: 17.9-24.4 MYA]; Schneider and Sampaio, 2015 [25 MYA]; and Jameson Kiesling et al., 2015 [25.5 MYA, interval: 25.1-26.4 MYA]). Other estimations were lower than ours (Schneider et al., 1993 [20.1 MYA]; Steiper and Young, 2006 [20.8 MYA]; Fabre et al., 2009 [15.9 MYA, interval: 9.7-19.9 MYA]; and Meredith et al., 2011 [14.6 MYA]). Our Bayesian estimate for the diversification within Pitheciidae was around 17.4 MYA. This was relatively similar to the temporal estimations obtained by Schneider et al., (1993) (14.5 MYA), Kay et al., (1998) (15.7 MYA), Opazo et al., (2006) (21.3 MYA for a maximum parsimony tree; 19.3 MYA for a maximum likelihood and Bayesian tree), Perelman et al., (2011) (20.2 MYA, interval: 15.3-25.2 MYA), Springer et al., (2012) (20.9 MYA, interval: 15.9-23.8 MYA), Schneider and Sampaio (2015) (20 MYA) and Jameson Kiesling et al., (2015) (18.1 MYA, interval: 15.8-21.0 MYA). Our Bayesian estimation for the split of Cebidae and Atelidae (22.0 MYA) is also supported by different studies as those from Schneider et al., (1993) (20 MYA), Opazo et al., (2006) (23.0 MYA for a maximum parsimony tree), Perelman et al., (2011) (22.8 MYA, interval: 18.127.1 MYA), Springer et al., (2012) (23 MYA), Schneider and Sampaio (2015) (23.0 MYA) and Jameson Kiesling et al., (2015) (24.0 MYA, interval: 22.0-25.3 MYA). We determined a Bayesian estimation of 7.8 MYA for the diversification within Callicebus. This value is also relatively similar to estimations made by other authors such as Schneider et al., (1993) (5.5 MYA), Perelman et al., (2011) (9.8 MYA, interval: 6.2-14.0 MYA), Springer et al., (2012) (7.6 MYA, interval: 5.2-10.4 MYA) and Jameson Kiesling et al., (2015) (6.6 MYA, interval: 4.3-9.5 MYA). Our Bayesian split estimation within Pithecia was around 6.8 MYA, which is somewhat higher than other molecular estimations on the diversification within this genus (Perelman et al., 2011 [4.0 MYA, interval: 1.6-7.1 MYA] and Springer et al., 2012 [3.5 MYA, interval: 2.0-6.0 MYA]). Our Bayesian split estimation between some species of Aotus, 4.3 MYA, was relatively similar to those obtained by Perelman et al., (2011) (5.5 MYA, interval: 3.2-7.8 MYA), Springer et al., (2012) (3.3 MYA, interval: 2.0-4.4 MYA) and Jameson Kiesling et al., (2015) (4.4 MYA, interval: 3.1-5.7 MYA). Finally, our Bayesian estimate for divergence within Ateles was 3.2 MYA and extremely similar to other estimations such as from Collins and Dubach (2000a,b) (3.3-3.6 MYA), Perelman et al., (2011) (5.1 MYA, interval: 2.9-7.5 MYA) and Springer et al., (2012) (2.8 MYA, interval: 1.6-4.4 MYA). Therefore, mitogenomics provide temporal splits similar to that provided by using other molecular markers. Our mitogenomics results also contribute systematics information to be considered within the genera analyzed. Following Hershkovitz (1987), Groves (2001) and Defler (2003, 2010), in Colombia, two Pithecia taxa are present: P. m. milleri and P. m. monachus. Two of the saki monkeys we analyzed are, without any doubt, milleri. If we follow these authors, the other two saki monkeys are monachus. However, if we take into consideration the recent monography of Marsh (2014), these two animals should be Pithecia hirsuta or Pithecia monachus. Whatever is the correct classification of those two individuals, it seems clear that they belong to a different species from milleri because the mitochondrial differentiation was considerable. In regards to the individuals of Aotus analyzed, the overall mitochondrial DNA data clearly differentiated both A. vociferans and A. nancymaae, similar to what we

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previously demonstrated with individual mitochondrial genes such as the COII gene (RuizGarcía et al., 2011, 2012). Additionally, and as we also demonstrated only with the mitochondrial COII gene, A. vociferans and A. l. griseimembra are intermixed and they did not form monophyletic clades. Thus, mitochondrial DNA could support our interpretation that many of the Aotus taxa north of the Amazon River constitute a super-species, A. vociferans. This super species includes different populations, probably reproductively isolated by stasipatric or parapatric (chromosomal) mechanisms, but not molecularly differentiated because they split in fairly recent times. This correlates well with the systematics proposed by Ford (1994) which considered only two Aotus species north of the Amazon River. In the case of Ateles, our mitogenomic results also agree quite well with some of our previous systematics interpretations of this genus. Ruiz-García et al., (2016) (in this book) considered only two or three Ateles species (A. paniscus and A. belzebuth or A. paniscus, A. belzebuth and A. geoffroyi). The present mitogenomics results should be even more related with the existence of two species because belzebuth was in a clade with some hybridus and fusciceps individuals. The intermixing of hybridus and fusciceps individuals doesn’t agree with the view of Collins and Dubach (2000a,b, 2001) and Nieves et al., (2005) who state that hybridus is a full and separated species from other spider monkey taxa. Mitogenomics could be an indispensable tool in helping to clarify the systematics of the Platyrrhini below to the genus level.

ACKNOWLEDGMENTS Thanks to Hugo Gálvez, Luz Mercedes Botero, Marcela Ramírez, Jorge Gardeazabal and Luis Carrillo for their respective help in obtaining samples. Thanks to Corpoamazonas and Piscilago in Colombia, to the Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de Extracción y Procesamiento Pesquero), Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales from Peru. We also thank the many people of diverse Indian tribes in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana and Alamas), Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna) and Ecuador (Kichwa, Huaorani, Shuar and Achuar) for their help in obtaining monkey samples.

REFERENCES Abascal, F., Zardoya, R., Telford, M. J. (2010). TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Research 38: W7–W13. Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on Automatic Control, AC-19: 716–723. Arnason, U., Adegoke, J. A., Bodin, K., Born, E. W., Esa, Y. B., Gullberg, A., Nilsson, M., Short, R. V., Xu, X., Janke, A. (2002). Mammalian mitogenomic relationships and the root of the eutherian tree. Proceedings of the National Academy of Sciences 99: 8151– 8156.

Phylogenetic Relationships of Pithecidae and Temporal Splits …

363

Arnason, U., Gullberg, A., Janke, A. (2004). Mitogenomic analyses provide new insights into cetacean origin and evolution. Gene 333: 27–34. Arnason, U., Adegoke, J. A., Gullberg, A., Harley, E. H., Janke, A., Kullberg, M. (2008). Mitogenomic relationships of placental mammals and molecular estimates of their divergences. Gene 421: 37–51. Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, J. E., Reeb, C. A., Saunders, N. C. (1987). Intraspecific phylogeographic: the mitochondrial DNA, bridge between population genetics and systematics. Annual Review of Ecology, Evolution, and Systematics 18: 489-522. Bandelt, H-J., Forster, P., Rohl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16: 37-48. Barroso, C. M. L., Schneider, H., Schneider, M. P. C., Sampaio, I., Harada, M. L., Czelusniak, J., Goodman, M. (1997). Update on the phylogenetic systematics of New World monkeys: further DNA evidence for placing the pygmy marmoset (Cebuella) within the genus Callithrix. International Journal of Primatology 18: 651–674. Canavez, F., Moreira, M., Ladasky, J., Pissinatti, A., Parham, P., Seuanez, H. (1999). -microglobulin DNA sequences. Molecular Phylogenetics and Evolution 12: 74–82. Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17 (4): 540–552. Chatterjee, H. J., Ho, S. Y. W., Barnes, I., Groves, C. (2009). Estimating the phylogeny and divergence times of primates using a supermatrix approach. BMC Evolutionary Biology 9: 259. Chiou, K. L., Pozzi, L., Lynch Alfaro, J. W., Di Fiore, A. (2011). Pleistocene diversification of living squirrel monkeys (Saimiri spp.) inferred from complete mitochondrial genome sequences. Molecular Phylogenetics and Evolution 59: 736–745. Collins, A. C., Dubach, J. M. (2000a). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology 21: 381– 420. Collins, A. C., Dubach, J. M. (2000b) Biogeographic and ecological forces responsible for speciation in Ateles. International Journal of Primatology 21: 421-444. Collins, A. C., Dubach, J. M. (2001). Nuclear DNA variation in spider monkeys (Ateles). Molecular and Phylogenetics Evolution 19: 67–75. Cummings, M. P., Otto, S. P., Wakeley, J. (1995). Sampling properties of DNA sequence data in phylogenetic analysis. Molecular Biology and Evolution 12: 814–822. Defler, T. R. (2003). Primates de Colombia. Colombia, Bogotá: Conservación Internacional. Defler, T. R. (2010). Historia natural de los primates colombianos. Editorial Universidad Nacional de Colombia. Pp. 1-609. Drummond. A. J., Ho, S. Y. W., Phillips, M. J, Rambaut, A. (2006). Relaxed phylogenetics and dating with confidence. PLOS Biology 4: e88. Drummond, A. J., Suchard, M. A., Xie, D., Rambaut, A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1. 7. Molecular Biology and Evolution 29: 1969–1973. Erixon, P., Svennblad, B., Britton, T., Oxelman, B. (2003). Reliability of Bayesian posterior probabilities and bootstrap frequencies in phylogenetics. Systematic Biology 52: 665– 673.

364

Manuel Ruiz-García, Geven Rodríguez, Myreya Pinedo-Castro et al.

Fabre, P. H., Rodrigues, A., Douzery, E. J. P. (2009). Patterns of macroevolution among Primates inferred from a supermatrix of mitochondrial and nuclear DNA. Molecular Phylogenetics and Evolution 53: 808–825. Finstermeier, K., Zinner, D., Brameier, M., Meyer, M., Kreuz, E., Hofreiter, M., Roos, C. (2013(. A mitogenomic phylogeny of living primates. PLoS One 8: e69504. Ford, S. M. (1986). Systematics of the New World monkeys. In Swindler, D. R., Erwin, J. (Eds.). Comparative Primate Biology, Systematics, Evolution and Anatomy, vol. I. Alan R. Liss, New York, Pp. 73–135. Gissi, C., Reyes, A., Pesole, G., Saccone, C. (2000). Lineage-specific evolutionary rate in mammalian mtDNA. Molecular Biology and Evolution 17: 1022–1031. Groves, C. P. (2001). Primate Taxonomy. Smithsonian Institution Press, Washington, DC. Guschanski, K., Krause, J., Sawyer, S., Valente, L. M., Bailey, S., Finstermeier, K., Sabin, R., Gilissen, E., Sonet, G., Nagy, Z. T., Lenglet, G., Mayer, F., Savolainen, V. (2013). Nextgeneration museomics disentangles one of the largest primate radiations. Systematic Biology 62: 539–554. Harada, M. L., Schneider, H., Schneider, M. P. C., Sampaio, I., Czelusniak, J., Goodman, M. (1995). DNA evidence on the phylogenetic systematics of New World monkeys: support for the sister-grouping of Cebus and Saimiri from two unlinked nuclear genes. Molecular Phylogenetetics and Evolution 4: 331–349. Hasegawa, M., Kishino, H., Yano, T. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22: 160-174. Hershkovitz, P. (1972). Notes on New World monkeys. International Zoo Yearbook 12: 3–12. Hershkovitz, P. (1977). Living New World Monkeys (Platyrrhini), With an Introduction to Primates. University of Chicago Press, Chicago. Hershkovitz, P. (1987). The taxonomy of South American sakis, genus Pithecia (Cebidae, Platyrrhini): a preliminary report and critical review with the description of a new species and a new subspecies. American Journal of Primatology 12: 387-468. Hillis, D., Bull, J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42: 182–192. Hodgson, J. A., Sterner, K. N., Matthews, L. J., Burrell, A. S., Jani, R. A., Raaum, R. L., Stewart, C. B., Disotell, T. R. (2009). Successive radiations, not stasis, in the South American primate fauna. Proceedings of the National Academy of Sciences USA 106:5534–5539. Horovitz, I., Meyer, A. (1995). Systematics of New World monkeys (Platyrrhini, Primates) based on 16S mitochondrial DNA sequences: a comparative analysis of different weighting methods in cladistic analysis. Molecular Phylogenetics and Evolution 4: 448– 456. Horovitz, I., Zardoya, R., Meyer, A. (1998). Platyrrhine systematics: a simultaneous analysis of molecular and morphological data. American Journal of Physical Anthropology 106: 261–281. Huelsenbeck, J. P., Rannala, B. (2004). Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology 53: 904–913. Hugot, J-P. (1998). Phylogeny of Neotropical monkeys: The interplay of morphological, molecular, and parasitological data. Molecular Phylogenetics and Evolution 9: 408-413.

Phylogenetic Relationships of Pithecidae and Temporal Splits …

365

Inoue, J. G., Miya, M., Lam, K., Tay, B.-H., Danks, J. A., Bell, J., Walker, T. I., Venkatesh, B. (2010). Evolutionary origin and phylogeny of the modern holocephalans (Chondrichthyes: Chimaeriformes): a mitogenomic perspective. Molecular Biology and Evolution 27: 2576–2586. Jameson Kiesling, N. M., Yi, S. V., Xu, K., Sperone, F. G., Wildman, D. E. (2015). The tempo and mode of New World monkey evolution and biogeography in the context of phylogenomic analysis. Molecular Phylogenetics and Evolution 82: 386-399. Kay, R. F. (1990). The phyletic relationships of extant and fossil Pitheciinae (Platyrrhini, Anthropoidea). Journal of Human Evolution 19: 175–208. Kay, R. F., Johnson, D., Meldrum, D. J. (1998). A new pitheciin primate from the middle Miocene of Argentina. American Journal of Primatology 45: 317–336. MacFadden, B. J. (1990). Chronology of Cenozoic primate localities in South America. Journal of Human Evolution 19: 151–156. Marsh, L. K. (2014). A taxonomic revision of the saki monkeys, Pithecia Desmarest, 1804. Neotropical Primates 21: 1-163. Mason, V. C., Li, G., Helgen, K. M., Murphy, W. J. (2011). Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens. Genome Research 21: 1695–1704. Matsui, A., Rakotondraparany, F., Hasegawa, M., Horai, S. (2007). Determination of a complete lemur mitochondrial genome from feces. Mammal Study 32: 7–16. Meredith, R. W., Janecka, J. E., Gatesy, J., Ryder, O., Fisher, C., Teeling, E. C., Goodbla, A., Eizirik, E., Simao, T. L. L., Stadler, T., Rabosky, D. L., Honeycutt, R. L., Flynn, J.J., Ingram, C. M., Steiner, C., Williams, T. L., Robinson, T. J., Burk-Herrick, A., Westerman, M., Ayoub, N., Springer, M. S., Murphy, W. J. (2011). Impacts of the cretaceous terrestrial revolution and KPg extinction on mammal diversification. Science 334: 521–524. Miya, M., Takeshima, H., Endo, H., Ishiguro, N. B., Inoue, J. G., Mukai, T., Satoh, T. P., Yamaguchi, M., Kawaguchi, A., Mabuchi, K., Shirai, S. M., Nishida, M. (2003). Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 26: 121–138. Moore, W. (1995). Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution 49: 718–726. Morral, N., Bertrantpetit, J., Estivill, X. et al. (1994). The origin of the major cystic fibrosis mutation (delta F508) in European populations. Nature Genetics 7: 169-175. Napier, J. R., Napier, P. H. (1967). A handbook of living primates. London: Academic Press. Nieves, M., Ascunce, M. S., Rahn, M. I., Mudry, M. D. (2005). Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters. Primates 46: 155–164. Nylander, J.A. (2004). MrModeltest v2. Evolutionary Biology Center, Uppsala University. Nylander, J., Wilgenbusch, J. C., Warren, D. L., Swofford, D. L. (2008). AWTY (are we there yet?): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics 24: 581–583. Opazo, J. C., Wildman, D. E., Prychitko, T., Johnson, R. M., Goodman, M. (2006). Phylogenetic relationships and divergence times among New World monkeys (Platyrrhini, Primates). Molecular Phylogenetics and Evolution 40: 274-280.

366

Manuel Ruiz-García, Geven Rodríguez, Myreya Pinedo-Castro et al.

Osterholz, M., Walter, L., Roos, C. (2009). Retropositional events consolidate the branching order among New World monkey genera. Molecular Phylogenetics and Evolution 50: 507–513. Pereira, S. L., Baker, A. J. (2006). A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock. Molecular Biology and Evolution 23: 1731–1740. Perelman, P., Johnson, W. E., Roos, C., Seuanez, H. N., Horvath, J. E., Moreira, M. A. M., Kessing, B., Pontius, J., Roelke, M., Rumpler, Y., Schneider, M. P. C., Silva, A., O’Brien, S. J., Pecon-Slattery, J., (2011). A molecular phylogeny of living primates. PLoS Genetics 7: e1001342. Pocock, R. I. (1925). The external characters of the Catarrhine monkeys and Apes. Proceedings of the Zoological Society of London 95: 1479-1579. Porter, C. A., Czelusniak, J., Schneider, H., Schneider, M. P. C., Sampaio, I., Goodman, M. (1997). Sequences of the primate e-globin gene: implications for systematics of the marmosets and other New World primates. Gene 205: 59–71. Posada, D., Buckley, T. R. (2004). Model selection and model averaging in phylogenetics: advantages of akaike information criterion and Bayesian approaches over likelihood ratio tests. Systematic Biology 53: 793–808. Pozzi, L., Hodgson, J. A., Burrell, A. S., Sterner, K. N, Raaum, R. L., Disotell, T. R. (2014). Primate phylogenetic relationships and divergence dates inferred from complete mitochondrial genomes. Molecular Phylogenetics and Evolution 75: 165-183. Prychitko, T., Johnson, R. M., Wildman, D. E., Gumucio, D., Goodman, M. (2005). The phylogenetic history conversion in the atelid ancestry. Molecular Phylogenetics and Evolution 35: 225–234. Rambaut, A. (2012). FigTree v1.4. http://tree.bio.ed.ac.uk/software/figtree/. Rambaut, A., Drummond, A.J. (2013a). LogCombiner v1.8.0. http://beast.bio.ed.ac.uk/. Rambaut, A., Drummond, A.J. (2013b). TreeAnnotator v1.8.0. http://beast.bio.ed.ac.uk/. Rambaut, A., Suchard, M.A., Xie, W., Drummond, A.J. (2013). Tracer v1.6. http://tree.bio.ed.ac.uk/software/tracer/. Ray, D. A., Xing, J., Hedges, D. J., Hall, M. A., Laborde, M. E., Anders, B. A., White, B. R., Stoilova, N., Fowlkes, J. D., Landry, K. E., Chemnick, L. G., Ryder, O. A., Batzer, M. A. (2005). Alu insertion loci and platyrrhine primate phylogeny. Molecular Phylogenetics and Evolution 35: 117–126. Raaum, R. L., Sterner, K. N., Noviello, C. M., Stewart, C.-B., Disotell, T. R. (2005). Catarrhine primate divergence dates estimated from complete mitochondrial genomes: concordance with fossil and nuclear DNA evidence. Journal of Human Evolution 48: 237–257. Rosenberger, A. L. (1981). Systematics: the higher taxa. In: Coimbra-Filho, A. F., Mittermeier, A. R. (Eds.). Ecology and Behavior of Neotropical Primates. Academia Brasileira de Ciências, Rio de Janeiro. Pp. 9–27. Rosenberger, A. L. (1984). Fossil New World monkeys dispute the molecular clock. Journal of Hum. Evolution 13: 737–742. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L. F., Vallejo, A., Gálvez, H., Shostell, J., Alvarez, D. (2011). The molecular phylogeny of the Aotus genus (Cebidae, Primates). Internation of Journal of Primatology 32:1218–1241.

Phylogenetic Relationships of Pithecidae and Temporal Splits …

367

Ruiz-García, M., Vásquez, C., Camargo, E., Castellanos-Mora, L. F., Gálvez, H., et al. (2013). Molecular genetics analysis of mtDNA COII gene sequences shows illegal traffic of night monkeys (Aotus, Platyrrhini, Primates) in Colombia. Journal of Primatology 2: 107. doi:10.4172/2167-6801.1000107. Ruiz-García, M., Lichilín, N., Escobar-Armel, P., Rodríguez, G. E., Gutierrez-Espeleta, E. (2016). Historical Genetic demography and some insights into the systematics of Ateles (Atelidae, Primates) by means of diverse mitochondrial genes. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Rylands, A. B., Mittermeier, R. A. (2009). The diversity of the New World primates (Platyrrhini): An annotated taxonomy. In South American Primates. Comparative perspectives in the study of behavior, ecology and conservation. Garber, P. A., Estrada, A., Bicca-Marques, J. C., Heymann, E. W., Strier, K. B. (Eds.). Springer Science+Business Media, New York, USA. Pp. 23-54. Saillard, J., Forster, P., Lynnerup, N., Bandelt, H-J., Norby, S. (2000). mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion. American Journal of Human Genetics 67: 718-726. Schneider, H. (2000). The current status of the New World Monkey Phylogeny. Annals de Academia Brasileira de Ciencias 72: 165-172. Schneider, H., Sampaio, I. (2015). The systematics and evolution of New World primates – A review. Molecular Phylogenetics and Evolution 82: 348–357. Schneider, H., Schneider, M. P., Sampaio, I., Harada, M. L., Stanhope, M., Czelusniak, J., Goodman, M. (1993). Molecular phylogeny of the New World monkeys (Platyrrhini, primates). Molecular Phylogenetics and Evolution 2: 225–242. Schneider, H., Sampaio, M. I. C., Harada, M. L., Barroso, C. M. L., Schneider, M. P. C., Czelusniak, J., Goodman, M. (1996). Molecular phylogeny of the New World monkeys (Platyrrhini, Primates) based on two unlinked nuclear genes: IRBP intron -globin sequences. American Journal of Physical Anthropology 100: 153–179. Schneider, H., Canavez, F. C., Sampaio, I., Moreira, M. A. M., Tagliaro, C. H., Seuánez, H. N. (2001). Can molecular data place each Neotropical monkey in its own branch? Chromosoma 109: 515–523. Schwarz, G. E. (1978). Estimating the dimension of a model. Annals of Statistics 6: 461-464. Simons, E. L. (1972). Primate Evolution: An Introduction to Man’s Place in Nature. Macmillan, New York. Simpson, G. G. (1945). The principles of classification and a classification of mammals. Bulletin of the American Museum of Natural History 85: 1–350. Slack, K. E., Delsuc, F., McLenachan, P., Arnason, U., Penny, D. (2007). Resolving the root of the avian mitogenomic tree by breaking up long branches. Molecular Phylogenetics and Evolution 42: 1–13. Sorci, G., Morand, S., Hugot, J-P. (1997). Host parasite coevolution: comparative evidence for covariation of life history traits in Primates and oxyurid parasites. Proceedings of the Royal Society of London B 264: 285-289. Springer, M. S., Meredith, R. W., Gatesy, J., Emerling, C., Park, J., Rabosky, D. L., Stadler, T., Steiner, C., Ryder, O., Janecka, J. E., Fisher, C., Murphy, W. J. (2012).

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Macroevolutionary dynamics and historical biogeography of primate diversification inferred from a species supermatrix. PLoS One 7: e49521. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688– 1243. Steiper, M. E., Young, N. M. (2008). Timing primate evolution: Lessons from the discordance between molecular and paleontological estimates. Evolutionary Anthropology Issues, News, Rev. 17: 179–188. Szalay, F. S., Delson, E. (1979). Evolutionary history of the primates. Academic Press, New York. Talavera, G., Castresana, J. (2007). Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology 56: 564–577. Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 27252729. Thalmann, O., Hebler, J., Poinar, H. N., Paabo, S., Vigilant, L. (2004). Unreliable mtDNA data due to nuclear insertions: A cautionary tale from analysis of humans and other apes. Molecular Ecology 13: 321–335. Vaidya, G., Lohman, D. J., Meier, R. (2011). SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27: 171–180. Van Roosmalen, M. G. M., Van Roosmalen, T. and Mittermeier, R. A. (2002). A taxonomic review of the titi monkeys, genus Callicebus Thomas, 1903, with the description of two new species, Callicebus bernhardi and Callicebus stephennashi, from Brazilian Amazonia. Neotropical Primates 10: 1–52. Von Dornum, M., Ruvolo, M. (1999). Phylogenetic relationships of the New World monkeys (Primates, Platyrrhini) based on nuclear G6PD DNA sequences. Molecular Phylogenetics and Evolution 11: 459–476. Wildman, D. E., Jameson, N. M., Opazo, J. C., Yi, S. V. (2009). A fully resolved genus level phylogeny of Neotropical primates (Platyrrhini). Molecular Phylogenetics and Evolution 53: 694–702. Wilcox, T. P., Zwickl, D.J., Heath, T. A., Hillis, D. M. (2002). Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support. Molecular Phylogenetics Evolution 25: 361–371. Wilgenbusch, J. C., Warren, D. L., Swofford, D. L. (2004). AWTY: A System for Graphical Exploration of MCMC Convergence in Bayesian Phylogenetic Inference. . Wilkinson, R. D., Steiper, M. E., Soligo, C., Martin, R. D., Yang, Z., Tavare, S. (2011). Dating primate divergences through an integrated analysis of palaeontological and molecular data. Systematic Biology 60: 16–31. Zhang, P., Wake, D. B. (2009). Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes. Molecular Phylogenetics Evolution 53: 492–508. Zhang, P., Zhou, H., Liang, D., Liu, Y.-F., Chen, Y.-Q., Qu, L.-H. (2005). The complete mitochondrial genome of a tree frog, Polypedates megacephalus (Amphibia: Anura: Rhacophoridae), and a novel gene organization in living amphibians. Gene 346: 133–143.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 11

MICROSATELLITE DNA ANALYSES OF FOUR ALOUATTA SPECIES (ATELIDAE, PRIMATES): EVOLUTIONARY MICROSATELLITE DYNAMICS Manuel Ruiz-García1,*, Pablo Escobar-Armel1, Marta Mudry2, Marina Ascunce2, Gustavo Gutierrez-Espeleta3 and Joseph Mark Shostell4 1

Unidad de Genética (Grupo de Genética de Poblaciones-Biología Evolutiva) Departamento de Biología. Facultad de Ciencias Pontificia Universidad Javeriana Bogotá DC., Colombia 2 GIBE. Departamento de Biología. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Buenos Aires, Argentina 3 Escuela de Biología. Universidad de Costa Rica. San José, Costa Rica 4 Department of Math Science and Technology, University of Minnesota Crookston, Crookston, MN, US

ABSTRACT We discuss the evolution of nine microsatellite DNA markers in four Alouatta species (howler monkeys; A. palliata, A. seniculus, A. macconnelli, and A. caraya) as well as evolutionary population parameters. There are five main findings from this study: 1.

2.

*

Four microsatellite central moments (mean, variance, skewness, and kurtosis) exhibited different distributions among species, with A. palliata being the most differentiated. This may suggest that central moments provide important phylogenetic signals. The “As is” bayesian method had the best percentage of classification (86.15%) of the assignment analyses. A. seniculus was the most incorrectly classified of all Alouatta species, which may suggest that some of their multigenotypes are original and that this species may be in the origin of other Alouatta species.

Corresponding author: [email protected], [email protected].

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5.

A. seniculus and A. caraya showed the highest and lowest effective numbers respectively. Only 9% of the detected microsatellite mutations were multi-step and, therefore, the majority of the mutations were of uni-step origin (91%). D5S117 was the unique microsatellite that clearly showed a multi-step mutation model. Different microsatellites frequently had different mutation rates per generation. An average estimate value of 7 x 10-5 did not support Quaternary refuges as sufficiently important for the molecular evolution of Alouatta. All but two of the microsatellites (D14S51, diversifying selection and D8S165, constrictive selection) behaved neutrally.

Keywords: Alouatta, central and south America, DNA microsatellites, molecular evolution, mutation models, natural selection

INTRODUCTION The Alouatta genus (howler monkeys) has the widest distribution of all Neotropical primates with a distribution that extends from Southern Veracruz (Mexico) to Northern Argentina. This genus, along with Cebus and Saimiri (Ayres 1986) consists of generalist species that can occupy poor quality forest habitats and thus permits them to inhabit a wide range of environments (Eisenberg, 1979). Alouatta is the first genus to colonize new islands in the Amazon Basin followed by other genera such as Cebus and Saimiri. In addition, they can live at sea level to elevations greater than 3,300 meters above sea level in the Andes. Together with the genera Ateles, Lagothrix and Brachyteles, they are the largest Neotropical primates (Milton, 1982). We herein studied four Alouatta species (A. palliata, A. seniculus, A. macconnelli and A. caraya). A. palliata ( = villosa), the mantled howler, has a distribution that extends from southern and eastern Mexico, southern Guatemala and south through the remainder of Central America to the Pacific area of Colombia, Ecuador and Northern Perú. We studied animals from México, Costa Rica and Colombia. A. seniculus, the red howler, has a geographic distribution that extends from Northern Colombia crossing the majority of Northern South America including north of the Amazon River in the east and extending south into Northern Bolivia in the west. Although many different subspecies of A. seniculus have been defined (among others, A. s. insulanus, A. s. stramineus, A. s. amazonica, A. s. juara, A. s. puruensis, A. s. seniculus, see Rylands et al., 1997), all the samples we analyzed came from Colombia, where “a priori” only the A. s. seniculus subspecies lives. A. macconnelli is distributed in Surinam, Guyana, French Guiana and the northern-eastern Amazonian area of Brazil. Although traditionally, A. macconnelli has been considered a subspecies of A. seniculus, the microsatellite differences we observed (shown elsewhere), together with the nucleotide sequence differences reported by Cortés-Ortiz et al., (2003) and the karyotypic differences previously found by Lima and Seuánez (1991) and Vassart et al. (1996), in our opinion, support the existence of a real and differentiated species. For example, while A. seniculus has a diploid chromosome number of 44 (males) and 45 (females), including 4 microchromosomes, A. macconnelli presents a diploid chromosome number of 47, 48 and 49. In fact, de Oliveira et al. (2002) revealed that howlers represent the genus with the most

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extensive karyotype diversity within Platyrrhini so far analyzed with high levels of intraspecific chromosomal variability. We also studied A. caraya (black howler) which has a habitat that includes Southern Brazil through Eastern Bolivia, Paraguay and Northern Argentina. The samples we analyzed were from Bolivia and Northern Argentina. All four of the species we analyzed were found in essentially allopatric ranges, with the exception of a small area in Northwestern Colombia (Sinú Valley), where both A. palliata and A. seniculus are found in sympatry (Hernández-Camacho and Cooper, 1976). The analyses of DNA G6PD gene sequences by Von Dornum and Ruvolo (1999) supported that the split between Alouatta and the Atelini (Ateles, Lagothrix, Brachyteles) occurred about 15.1 million of years ago (MYA). The development over the last few years of molecular procedures based upon the advent of the polymerase chain reaction (PCR), has enabled population geneticists to analyze and determine the genetic structure and the levels of genetic variability of many wild species from small tissue fragments (blood, bones, epithelial). Among the most remarkable molecular markers for these tasks are the STRPs (Short Tandem Repeat Polymorphisms, microsatellites) (Weber and May, 1989). These kinds of markers are composed of short sequences of nucleotides (one to six nucleotide base pairs) repeated in tandem arrays. These markers are frequently inside the eukaryotic genomes, are randomly distributed, and are highly polymorphic. Additionally, one determinant property of these markers is that the DNA amount needed to carry out these molecular analyses is very small, which permit the investigator to use non-invasive procedures to sample wild animals and to successfully examine population biology dynamics on a molecular genetic level (Bruford and Wayne, 1993). The main aims of the present work are as follows: 1- To determine the central moments of nine STRPs studied in four Alouatta species and to investigate their degree of similarity among the species analyzed. Diverse central moments could have some phylogenetic signal information. 2- To investigate the degree of microsatellite divergence among the four species of Alouatta studied and to determine possible original multigenotypes for these species. 3- To determine effective and total numbers of these species by means of maximum likelihood estimates using the  (= 4Ne) parameter. 4- To determine the percentages of uni-step and multi-step mutations affecting the evolution of the microsatellites analyzed. 5- To determine an approximate global value of  (mutation rate per generation) for the set of microsatellites employed and to provide insights that support the existence of different mutation rates for each of the microsatellites studied. 6- To determine if there is evidence of natural selection that affects some of the markers using the Beaumont and Nichols (1996) method.

MATERIALS AND METHODS Samples and Molecular Procedures Twenty (20) Alouatta caraya individuals were caught in Isla Brasilera at the Argentinean Chaco and in Southeastern Bolivia. Eighty four (84) A. seniculus individuals were sampled from Colombia in the Departments of La Guajira, Meta, Antioquia, Chocó, Amazonas,

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Vichada, Putumayo, Magdalena and Atlántico. A total of forty eight (48) A. palliata individuals were sampled. Two samples were from México (A. palliata mexicana; States of Costuxtla and Tabasco), three samples were from the Colombian Chocó (A. palliata aequatorialis) and the remaining samples were from Costa Rica (A. palliata palliata). Seven (7) A. macconnelli samples were obtained in French Guiana at the Carnopi River. In total, 159 Alouatta samples were collected and analyzed (Table 1). Table 1. Sample sizes, morphological subspecies, countries, specific geographic origins, type of samples and sources of the 159 Alouatta samples from four different species (A. seniculus, A. palliata, A. caraya and A. macconnellii) studied herein Species

N

Alouatta seniculus

84

Alouatta palliata

48

Subspecies and Origin A. s. seniculus COLOMBIA

A. p. palliata COSTA RICA

A. p. mexicana MEXICO

Alouatta caraya

20

Alouatta macconnelli

7

A. p. aequatorialis COLOMBIA A. caraya ARGENTINA A. caraya BOLIVIA

A. macconnellii FRENCH GUIANA

Geographic origin, type of samples and sources La Guajira (20 samples, all blood drop), Meta (7 samples, blood, hair and teeth), Antioquia (16 samples, all blood) Chocó (7 samples, blood and teeth), Amazonas (17 samples, blood, hair, teeth and bones), Vichada (3 samples, teeth), Putumayo (3 samples, teeth and bones), Magdalena (2 samples, teeth), Atlántico (2 samples, teeth), Caquetá (2 samples, hair and teeth), Arauca (1 sample, teeth), Bolivar (4 samples teeth and bones). M. Ruiz-García, P. Escobar-Armel, D. Alvarez, Indian tribes across all Colombia, F. Nassar, J. Gardeazábal, L. M. Borrero, D. M. Ramírez, Instituto Von Humboldt (4 skin samples). Across all Costa Rica (43 samples, blood and hairs). G. Gutierrez-Espeleta, M. Ruiz-García D. Alvarez, P. Escobar-Armel Costuxtla (1 sample, DNA), Tabasco (1 sample, DNA). L. Cortés-Ortiz Chocó (3 samples, teeth and bones). M. Ruiz-García Isla Brasilera and other three geographical points, Argentinan Chaco (14 samples, blood drops). M. Mudry, M. Ascunce Santa Cruz Department, and diverse localities at the Mamoré River (6 samples, hairs). M. Ruiz-García, D. Alvarez Carnopi River (4 samples of muscle tissue and 3 samples of hairs from three localities). F. Catzeflis, M. Ruiz-García.

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Table 2. Microsatellites, length size in base pairs and forward and reverse primer sequences of the nine DNA microsatellite markers applied to four Alouatta species Microsatelite AP40

Length size 168

AP68

190

AP74

154

D5S111

165

D5S117

157

D6S260

170

D8S165

142

D14S51

175

D17S804

181

Forward Primer 5'-CCACGGTGGCA GAGGAGATTT-3' 5'-TGTTGGTATAAT CTTTCCTA-3' 5'-TGCACCTCATC TCTTTCTCTG-3' 5’-GGCATCATTTT AGAAGGAAAT-3’ 5’-TGTCTCCTGAGA ATAG-3’ 5’-TTTTCACTATCA ATGGCAGC-3’ 5’-ACAAGAGCACA TTTAGTCAG-3’ 5’-GATTCTGCACCC CTAAATCC-3” 5’-GCCTGTGCTGC TGATAACC-3’

Reverse Primer 5'-AGAGGCACGAA GACAAGGACA-3' 5'-ACATACACCTT TGAGTTTCT-3' 5'-CATCTTTGTTT TCCTCATAGC-3' 5’-ACATTTGTTC AGGACCAAAG-3’ 5’-TAATATCCAAA CCACAAAGGT-3’ 5’-TTCATTTTCAGC AGCAATTT-3’ 5’-AGCTTCATTTT TCCCTCTAG-3’ 5’-ATGCTCAATGA ACAGCCTGA-3’ 5’-CACTGTGATG AGATGTCATTCC-3’

DNA, from blood, hair (with roots), skin, bones and teeth samples, was extracted using the phenol-chloroform procedure (Sambrock et al., 1989). The Chelex 10% method (Walsh et al., 1991) was also used to extract DNA from blood droplets and hairs. Nine microsatellites molecular markers (AP40, AP68, AP74, D5S111, D5S117, D6S260, D8S165, D14S51 and D17S804) were used in the current study (Table 2). AP68 did not amplify in A. caraya probably due to the poor quality of the DNA used. The final PCR volume of the STRPs for DNA extracted from blood, skin, bone, and teeth was 25 l (3 l of MgCl2 3 mM, 2.5 l of Buffer 10x, 1 l of dNTPs 1 mM, 10 pmol of forward and reverse primers, 13.5 l of H2O, 2 l of DNA (50-100 ng per l), and one Taq Polymerase unit. For the DNA extracted from hair and blood (droplets), the overall volume of the PCR reactions was 50 l, with 20 l of DNA and twofold amounts of MgCl2, Buffer, dNTPs, primers and Taq Polymerase. The PCR reactions were carried out in a Geneamp PCR System 9600 Perkin Elmer thermocycler. The temperatures used were as follows: 95°C for 5 minutes, 30 cycles of 1 minute at 95°C, 1 minute at the most accurate annealing temperature (57°C for AP40, 50°C for AP68 and 52°C for the remaining markers), one minute at 72°C, and 5 minutes at 72°C. The amplification products were kept at 4°C until used. The PCR amplification products were run in denaturant 6% polyacrilamide gels within a Hoefer SQ3 sequencer vertical chamber. Gels migrated for 2-3 hours depending on marker sizes, and were then stained with AgNO3 (silver nitrate). Every sixth line in the gel contained molecular markers (174 cut with Hind III and Hinf I). The PCR reactions were repeated three times for DNA extracted from hairs, teeth and bones in order to confirm the genotypes obtained from these tissues. Therefore, allelic dropout was highly improbable. The existence of null alleles cannot be totally excluded, which could

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increase the number of false homozygous genotypes. Nevertheless, it is improbable that all loci were affected in the same way.

Population Genetics Analyses Microsatellite Central Moments The central moments of the microsatellite distributions were calculated in order to analyze interspecific and intraspecific distribution dynamics. We used the analytical equations for moments up to the fourth order within a locus and the between-locus variance at mutation-drift equilibrium (Zhivotovsky and Feldman, 1995). The main power of these statistics is to test the effectiveness of the one-step mutation model as well as to detect between-locus variation in the mutation rate per generation. A description of the analytical expressions used to accomplish this follows. The total mutation rate for the one and two-step stepwise models is w = c vc c2 = v 2m, (v = c different to 0 vc; c = j – i, the change in repeat number due to mutation of the allele carrying j repeats to the allele with i repeats; vc is the probability of a mutational change by c with the repeat number not depended on the mutated allele; 2m is the variance of changes in repeats among the new mutations). Similarly, the first four central sample moments of the allele frequency distributions were calculated as r = i i pi (mean of repeat tandems), V = i pi (i – r)2 (variance of the repeat tandems), S = i pi (i – r)3 (skewness of repeat tandems), K = i pi (i – r)4 (kurtosis of repeat tandems), where pi is the frequency of allele Ai which carries i repeats. Those microsatellites affected by negative selection, or mutation constriction, will show statistical values that are clearly differentiated compared to microsatellites with more neutral dynamics. The GENECLASS program (Cornuet et al., 1999) was used to determine the capacity of the microsatellite markers to differentiate Alouatta species. This method of analysis could also be indirectly useful to determine the degree of molecular evolution (rates of evolution and selective constrictions) of microsatellites. The frequency (Paetkau et al., 1995), Bayesian (Rannala and Mountain, 1997) and genetic distance-based methods were applied. The distance-based methods assign individuals to the “closest” species and requires the defining of a distance between the individuals and the species considered. For this, the following genetic distances were employed: Nei (1978), Cavalli-Sforza and Edwards (1967), DAS (shared allele distance; Chakraborty and Jin, 1993) and the 2 genetic distance (Goldstein et al., 1995). Distance methods have an advantage over other methods because they work without meeting the Hardy-Weinberg equilibrium and linkage equilibrium assumptions. The “leave one out” and “as is” statistical methods were applied to the above assigning analysis. Exclusion methods without any prior information were also employed (Cornuet et al., 1999). For each one of the Alouatta species studied, the historical effective number was calculated by using a maximum likelihood procedure with a Markov chain recursion method (Nielsen, 1997), to estimate the probable  (= 4Ne) values, where Ne is the historical effective number of the species studied and  is the mutation rate per generation. Once the  value is known, Ne can be obtained which indicates the historical reproductive population sizes of the four Alouatta species. Estimations of mutation values in humans, pigs, and rats are 5.6 x 10-4 (Weber and Wong, 1993), 7 x 10-5 (Ellegren, 1995) and 1.5 x 10-4 (Serikawa, 1992) respectively. Therefore, to obtain a wide range of feasibly effective numbers in the

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Alouatta species analyzed, the mutation rates employed in this work ranged from 5.6 x 10-4 to 7 x 10-5. The Nielsen (1997)’s model is based on the likelihood function of , calculated as L() = P(), where  is a vector with the observed data in the obtained samples. A onestep mutation model typical of the microsatellites was adopted. Nielsen’s model is also based on the recursivity of the coalescence theory to obtain the likelihood functions of  for samples of a determined size. The coalescence time between two alleles is exponentially distributed with a mean equal to 1 and the conditional number of mutations in each lineage follows a Poisson distribution with a mean of t/2. It is feasible to calculate the probabilities to observe an allele sample determined in the previous generations by means of recursion considering the allele genealogies of the sample and summing up all the possible previous states in the time. This result is obtained by conditioning the last event that occurs prior to the present, by mutation or coalescence and following a symmetric random walk with k-allele states reflecting some types of barriers. The probability q () of the sample is determined by the addition of all the possible states that are previous to the probability of being in a determined state, multiplied by the transition probability of these states to the current state. With the chosen mutation model (uni-step), this probability is q () = (n +  - 1))  (ni + 1/(n)) 1/2 q ( + i – j) + (n – 1)/(n +  -1)  (nj – 1)/(n – 1) q ( - j), where i is a unity vector which adds values equal to 1 to the entry of i in . This recursive procedure is easy to obtain if we have the capacity to obtain the values of ( /(n +  - 1)), which is the probability that the last event before the present moment is a mutation and that a mutational or a coalescence event of (n-1)/(n +  -1) had previously occurred. This event is the probability of a coalescence event when considering that a previous mutation or coalescence of (ni – 1)/(n –1) occurred. The equation ((ni – 1)/(n –1)) represents the probability that a mutation occurs in an i allele, given that a mutation of (nj – 1)/(n –1) occurred. This is the probability that two alleles belonging to the j state will be coalescent, given that a coalescence event occurred. There is a 0.50 probability that a mutation occurs from an i state to a j state given that a mutation in the i state occurred. We calculated q() with the Monte Carlo method after an evaluation of the likelihood functions (Griffiths and Tavaré, 1994, based on the recursion of the expression) were evaluated. The likelihood surfaces for  were estimated with the MISAT program (Nielsen, 1997) and the 5% confidence interval was calculated by multiplying the log likelihood of the maximum likelihood value by 2. We used a grid size of 40, a previously calculated  (calculated with the method of the moments, 0) and a mutation one-step model with a 1,000,000 Markov chains. The estimate of the  maximum likelihood (the  value with the least negative log likelihood) was used to estimate Ne for each Alouatta species studied. In addition, the greatest possible multi-step mutation percentages (ranging from 0 to 0.5) were calculated through the maximum likelihood of  by means of 3,000,000 Markov chains. We analyzed the different possible mutation rates affecting each one of the microsatellites for each Alouatta species studied. For this, we started from the hypothesis 1 = 2 =  (the values of  for two different microsatellites) and we tested it by a likelihood ratio test with the expression -2 log [L1(L2()]/[L(,2)], which follows a 2 with one degree of freedom. A

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probability lower than  = 0.05 indicates that both microsatellites have different mutation rates. Likewise, we measured if the estimated multi-step mutation models were significantly better than the uni-step mutation models within each Alouatta species. We applied the likelihood ratio of -2 log [L(, p = 0)/L(, p)], which for large samples is similar to 2 with one degree of freedom under the null hypothesis that p = 0, to the obtained maximum likelihood multi-step p percentage. A probability lower than  = 0.05 indicates that the multistep mutation percentage is significantly different from the uni-step mutation model and this last model is rejected. Lastly, the coalescence theory generated by Beaumont and Nichols (1996) was used to detect whether DNA-microsatellites were effected by constrictive or diversifying natural selection within the Alouatta genus. We used the fdist program and we obtained the observed and expected FST statistical values for each marker used throughout the samples. Both the infinite allele and the step-wise mutation models were considered. A total of 5,000 iterations were completed to calculate the values that represented the relationship between the FST statistic and the expected heterozygosity of the markers. The iterations were grouped into batches of 200 from which the medians and the 2.5% and the 97.5% quartiles were calculated. The observed FST and the heterozygosity values were superimposed under this distribution and conformed by the median and the quartiles. Values that are outside of this theoretical distribution obtained indicate that the microsatellites in question are being affected by natural selection.

RESULTS The central moments of the microsatellite distributions for the four Alouatta species depicted dissimilar intraspecific trends among different microsatellites as well as interspecific differences among the same markers (Table 3). For example, within A. caraya, we can observe that all microsatellites had a moment of order 1 (mean, r) and were relatively similar with the exception of AP40, which had an r-value lower than the remaining markers. The variances (moment of order 2, V) were relatively similar for AP40, AP74 and D5S111, considerably higher for D8S165 (11 times higher) and considerably lower for D14S51 (6 times lower). The asymmetry coefficient (moment of order 3, S) was small (therefore there is symmetry) for all microsatellites studied with the exception of D8S165 which yielded clear evidence of positive asymmetry in its distribution. The kurtosis coefficient (moment of order 4, K) showed a platykurtic distribution for this species for AP40, AP74, D5S111 and D14S51, while D5S117 and, especially, D8S165 showed a well developed leptokurtic trend. Therefore, it seems probable that the microsatellites studied in A. caraya have different mutation rates and different selective constrictions. No microsatellite studied in this species showed a perfectly normal distribution, which would be expected if no mutation or selective constrictions were present. The comparative analysis of A. seniculus revealed that the dynamics of some microsatellites were different to that determined in the first species. For example, in A. caraya, AP40 showed the lowest value of r (also present in A. macconelli), lower than in A. seniculus. All the other microsatellites in A. seniculus yielded similar r values. The r value of DS5111 was seven times higher in A. caraya compared to A. seniculus. The markers AP40, AP68, AP74 and D5S111 showed the lowest variance (V) values for A.

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seniculus, whereas D6S260 and especially D5S117 yielded more pronounced variances. D5S117’s variance in A. seniculus was 24 times greater than in A. caraya, showing a clear difference in mutation rate or selective constriction for both species. A conspicuous difference in the variance of allele repeats between these two species was recorded for D14S51, which denotes the possibility of a different selective constraint in both species. The allele distribution for AP40, AP68, AP74 and D5S111 in A. seniculus was nearly symmetric whereas D14S51 and mainly D5S117 had a negative asymmetric trend. In contrast, D8S165 and D17S804 manifested a clear positive asymmetric trend. Only two microsatellites (AP40 and AP68) showed platykurtic distributions in A. seniculus. All other markers in this species showed noteworthy leptokurtic levels, especially D5S117, D6S260, D17S804 and D14S51. These levels of leptokurtic distributions are noticeably greater in this species than in A. caraya. The r values for A. macconnelli were more heterogeneous than for the other two species commented probably as a consequence of the smaller sample size used for this taxon. Nonetheless, as it was previously said, AP40 showed the same dynamics in this species as in the two previous ones and was the marker with the lowest r value. D5S117 showed a clear lower r value in A. macconnelli than in A. caraya and in A. seniculus (a reduction of five and four times respectively). Similar to a trend observed in A. seniculus (but not in A. caraya) the V value for AP40 was exceedingly low relative to the V values reported for the other microsatellites. Nonetheless, the V values for several markers such as AP74 and D5S111 (variances clearly higher in A. macconnelli than in A. caraya and in A. seniculus although the sample size of the first species was smaller compared to the others), D5S117 and D6S260 revealed marked differences between the three Alouatta species. No symmetrical distributions were determined in A. macconnelli (with the exception of AP40) and this species yielded relevant differences from the other Alouatta species studied. AP68, AP74 and D5S111 showed negative asymmetric distributions, while D8S165, D17S804 and, especially, D5S117 and D6S260 presented conspicuously positive asymmetries. For example, D6S260 showed astonishing asymmetric differences between A. macconnelli and A. seniculus. In A. macconnelli, only AP40 showed a platykurtic distribution whereas all the other microsatellites were clearly leptokurtic. Some markers, such as AP74, D5S111, D5S117 and D6S260 (especially the two last), were extremely leptokurtic, which denotes possible selective or mutation constrictions in this species as well. No other Alouatta species presented such extreme leptokurtic values, with the exception of A. seniculus for D17S804 and especially D5S117. A. palliata yielded values of r relatively similar to those observed in the other three Alouatta species. The two loci which showed the greatest heterogeneity of r among the four Alouatta species studied were D5S111 and D5S117. AP68 showed the highest V value in A. palliata while D5S111, D6S260 and D17S804 yielded the smallest variances of all species studied. D14S51 and D17S804 practically presented symmetric distributions, which is in contrast to that found in the other species with the exception of D14S51 in A. caraya. AP68, AP74 and D5S117 showed negative asymmetry, whereas D5S111 and D6S260 presented positive asymmetries. D5S117 and D6S260 markers yielded the greatest heterogeneity for S among the four Alouatta species studied. D5S111 was the only marker that had a normal distribution for A. palliata. AP68 showed the highest leptokurtic distribution in A. palliata among the four species analyzed. The only two microsatellites presenting platykurtic distributions were D14S51 and D17S804 in contrast with the findings determined for the other Alouatta species with the exception of D14S51 in A. caraya. Therefore, this provides

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more evidence that supports different microsatellite dynamics for the same markers in different Alouatta species. Table 3. Four central moments of DNA microsatellites studied in four Alouatta species A.seniculus r v s k AP40 2.000 0.560 0.240 2.000 AP68 20.706 0.706 0.106 1.042 AP74 24.064 1.336 0.217 5.341 D5S111 23.293 1.678 1.613 7.463 D5S117 12.206 40.320 -276.001 4212.200 D6S260 30.100 17.090 -9.828 550.406 D8S165 18.960 2.746 4.125 28.940 D14S51 18.946 5.681 -17.938 112.621 D17S804 18.812 5.277 24.162 177.570 A.caraya AP40 1.750 0.437 -0.656 1.176 AP74 23.750 0.437 -0.656 1.176 D5S111 24.334 0.888 -0.583 1.186 D5S117 15.830 1.693 -0.965 8.102 D8S165 20.600 4.940 4.692 51.879 D14S51 20.070 0.066 0.056 0.053 A.palliata AP68 15.613 6.698 -3.174 127.903 AP74 18.812 2.277 -3.025 16.812 D5S111 21.364 0.698 1.352 3.483 D5S117 16.094 8.460 -3.036 566.411 D6S260 29.660 6.053 11.867 72.065 D14S51 23.750 0.312 -0.375 0.629 D17S804 17.315 0.554 0.768 1.346 A.macconnelli AP40 1.5 0.25 0 0.062 AP68 17.7 4.61 -5.124 52.453 AP74 16.75 8.187 -3.093 86.082 D5S111 28.3 8.01 -1.536 153.210 D5S117 3.1 20.09 172.872 2078.5957 D6S260 35.3228 124.913 3355.5088 91199.854 D8S165 23.100 1.999 2.000 6.000 D17S804 17.75 4.437 6.093 2078.596 r = Mean of repeat tandems; v = Variance of the repeat tandems; S = skewness of repeat tandems; K = kurtosis of repeat tandems

Table 4 shows the assignment analyses for all the markers within the 65 Alouatta individuals with complete multi-genotypes (nearly 41% of the animals studied). The Bayesian procedure with the “As is” technique (86.15%) yielded the best assignment percentages. Other methods which offered elevated percentages of correct assignment included the DA “as

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is” (84.62%) and Cavalli “as is” (84.62%) (Cavalli-Sforza and Edwards, 1967). The worst results (lowest percentages) were obtained by means of the standard and minimum Nei’s genetic distance with the “leave-one-out” technique (73.85%) and the Cavalli-Sforza and Edwards’s genetic distance with the “leave one out” method (72.31%). We provide comments about the results of two methods. The Bayesian procedure with “as is” incorrectly classified 9 out of 65 animals: one A. caraya was classified as A. seniculus, four A. seniculus were assigned as A. caraya, two as A. macconnelli and two as A. palliata. The DA genetic distance with “as is” incorrectly assigned 10 individuals out of 65 studied: one A. caraya like A. seniculus, three A. seniculus as A. caraya, four as A. palliata and two as A. macconnelli. Therefore, A. seniculus had many characteristics that were also dispersed in the other species. Neither A. macconnelli nor A. palliata were incorrectly classified, which complements the fact that both species have peculiar gene pools well differentiated from the other Alouatta species. Conversely, some A. seniculus and A. caraya were similar enough to be misidentified in all analyses. This means that both species are relatively similar from a genetic standpoint at least in consideration of the microsatellites used in this study. Table 4. Population assignment results for the four Alouatta species studied (A. palliata, A. seniculus, A. macconnelli and A.caraya) using nine DNA Microsatellite markers Method procedure Bayesian – “leave one out” Bayesian – “as is” Nei – “leave one out” Nei – “as is” Nei minimun – “leave one out” Nei minimun – “as is” DA – “leave one out” DA – “as is” Cavalli – “leave one out” Cavalli – “as is”

% of correct assignment 75.38 86.15 73.85 81.54 73.85 81.54 80.00 84.62 72.31 84.62

Table 5. Effective number estimates by using uni-step and a multi-step mutation models by means of two extreme mutation rates per generation (5.6 x 10-4 and 7 x 10-5) throughout maximum likelihood estimates of (= 4Ne). Historical total population sizes were calculated by means of the Ne/N ratio = 0.4. XA =Average means. Xh = harmonic means Effective numbers Uni-step model Mutation rates 5.6  10-4 7  10-5 Alouatta palliata = 8.811 ± 5.772 Alouatta seniculus = 19.803 ± 12.662

Total numbers Ne/N = 0.4 Mutation rates 5.6  10-4 7  10-5

XA = Xh =

3,934 1,060

31,470 8,482

9,835 2,650

78,675 21,205

XA = Xh =

8,841 2,342

70,726 18,740

22,103 5,855

176,815 46,850

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Manuel Ruiz-García, Pablo Escobar-Armel, Marta Mudry et al. Table 5. (Continued) Effective numbers Uni-step model Mutation rates 5.6  10-4 7  10-5

Alouatta macconnelli = 16.121 ± 6.983 Alouatta caraya = 5.268 ± 4.631

Total numbers Ne/N = 0.4 Mutation rates 5.6  10-4 7  10-5

XA = Xh =

7,197 2,114

57,574 16,907

17,993 5,285

143,935 42,267

XA = Xh =

2,352 395

18,818 3,163

5,880 988

47,045 7,908

Multi-step model 5.6  10-4 Alouatta palliata = 7.063 ± 3.556 Alouatta seniculus = 15.872 ± 11.334 Alouatta macconnelli = 16.218 ± 7.537 Alouatta caraya = 4.551 ± 3.882

7  10-5

5.6  10-4

7  10-5

XA = Xh =

3,153 954

25,227 7,640

7,883 2,385

63,068 19,100

XA = Xh =

7,086 2,118

56,687 16,941

17,715 5,295

141,717 42,352

XA = Xh =

7,241 1,537

57,925 16,941

18,102 3,843

144,812 30,730

XA = Xh =

2,032 285

16,254 2,280

5,080 712

40,635 5,700

Table 5 shows effective number estimates by using uni-step and a multi-step mutation models by means of two extreme mutation rates per generation (5.6 x 10-4 and 7 x 10-5) throughout maximum likelihood estimates of (= 4Ne). Historical total population sizes were calculated by means of the Ne/N ratio = 0.4. A. seniculus presented the highest  and Ne values (8,841 and 70,726, for the one-step mutation model, arithmetic mean and depending on the selected mutation rate). A. macconnelli showed the second highest effective numbers (7,197 and 57,574). Considerably smaller effective numbers occurred in A. palliata and A. caraya. The first species showed a mean Ne range of 3,934-31,470. The historical effective sizes of A. seniculus and A. macconnelli were approximately between 1.8 and 2.2 times higher than that of A. palliata. Indisputably, A. caraya had the lowest historically effective size and total numbers (2,352 and 18,818, respectively). The Ne for A. caraya was 3.76 times lower than for A. seniculus and 3.01 times lower than for A. macconnelli. Therefore, A. caraya and A. seniculus seem to have the (historically) smallest and largest of the Alouatta populations studied respectively. Although the average multi-step mutation percentages were different for each Alouatta species studied, we detected no significant difference among the averages. A. caraya (p =

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0.158) presented the highest multi-step mutation percentage and A. palliata presented the lowest (p = 0.039). Both, A. seniculus and A. macconnelli, yielded very similar multi-step mutation percentages (0.087 and 0.066, respectively). This mean percentage oscillated for the genus Alouatta from p = 0.0879 to p = 0.0841 depending as the average was calculated. Therefore, on average, 91-92% of the microsatellite mutations studied were uni-step and only 8-9% were multi-step. The Ne values were extremely similar when they were calculated with either the  multi-step or uni-step mutation models. For example, when only the 5.6 x 10-4 mutation rate is considered, the values for the four Alouatta species were 3,153 vs. 3,934 (A. palliata), 7,086 vs 8,841 (A. seniculus), 7,242 vs. 7,197 (A. macconnelli) and 2,032 vs. 2,352 (A. caraya). Therefore, there is no obvious bias when effective numbers are estimated with a uni-step mutation model instead of the more accurate multi-step mutation model, at least not in the Alouatta of this study. The historical total number of individuals in each species generated with the uni-step model oscillated from 4,711 to 44,255 (A. palliata), 10,409 to 99,459 (A. seniculus), 9,395 to 80,964 (A. macconnelli) and 1,757 to 26,462 (A. caraya). These values were only slightly different from those generated with the multi-step model. As expected, the harmonic averages calculated from all the obtained estimates for each species showed a trend in favor of the smallest values. Table 6 displays the significant multi-step mutation percentages in regard to the uni-step model. Nonetheless, very few cases of multistep percentages were significant, with one in A. palliata (D5S117: 7.5%, 2 = 3.427, P < 0.05), two in A. seniculus, (D5S117: 10%, 2= 4.208, P < 0.05 and D14S51: 32.5%, 2 = 7.802, P < 0.01), one in A. macconnelli (D5S117: 20%, 2 = 6.91, P < 0.01) and none in A. caraya. Even though, A. seniculus showed the highest significant multi-step percentage (2/9 = 22.22%, 2 = 1.135, 1 df, NS), no percentage was significantly greater than the type I error of 5%. Consequently, the overall trends do not favor a generalized multi-step mutation model over a uni-step mutation model in the Alouatta species studied. Only D5S117 presented a noteworthy trend in support of a multi-step mutation model inside of this genus. However, there is incontrovertible evidence in favor of generalized differences in the mutation rates at the microsatellites analyzed within each one of the four species studied. The percentages of significantly different mutation rate pairs were 86.66% (A. caraya, for both uni-step and multi-step models), 80.85% (A. palliata for both models), 82.14-78.57% (A. macconnelli for both models, respectively) and 77.77-75% (A. seniculus, for both models, respectively). There was no significant difference in percentage pairs between A. caraya and A. seniculus (2 = 2.257, 1 df, NS). Additionally, all of these percentages were significantly greater than the 5% type I error. This means that the four species have similar percentage of mutation rate differences among the microsatellite pairs studied and that these different mutation rates are systematic and not due to chance. As the majority of comparison pairs were significant, we have focused our comments on non-significant pairs. For A. caraya, only the D5S111D14S51 and the AP40-AP74 pairs did not have significantly different mutation rates (considering both uni-step and multi-step models). For A. palliata, the pairs AP74-D6S260, D5S111-AP74, D6S260-D5S111, and D14S51-D17S804 showed similar mutation rates. In A. macconnelli, the microsatellite pairs with non significant mutation rates were D5S111-AP68, D5S111-D17S804 (uni-step model only), AP68-D17S804, AP74-D6S260, AP74-D8S165 and D8S165-D6S260; while in A. seniculus, these pairs were D5S111-D17S804, D5S111-AP40,

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D5S111-AP74, D14S51-D8S165 (uni-step model only), D17S804-D8S165, D17S804-AP74, D8S165-D6S260, AP40-AP74, and AP74-D6S260. Only a few pairs were repeated among several species combinations: AP74-D6S260 (A. palliata, A. macconnelli and A. seniculus), D5S111-AP74 (A. palliata and A. seniculus) and D5S111-D17S804 and D8S165-D6S260 (A. macconnelli and A. seniculus). This could mean that these microsatellites have similar mutation rates for these groups of species and that A. macconnelli and A. seniculus were the species with the greatest number of microsatellites suffering similar mutation rates. These analyses could also provide an interesting phylogenetic signal. Nevertheless, in general the majority of the microsatellites studied inside each species have significantly different mutation rates. Furthermore, with the exceptions aforementioned, the same microsatellites have different mutation rates in each species analyzed. For instance, for A. palliata the order of mutation rates was (from higher to lower): D5S117 > AP68 > (AP74 = D5S111 = D6S260) > (D14S51 = D17S804), meanwhile in A. seniculus this order was D5S117 > (D6S260 = D17S804 = D14S51 = D8S165) > (AP74 = D5S111 = AP40) > AP68. D5S117 provided the only similarity of mutation rates among the four Alouatta species studied. This was always the microsatellite with the highest mutation rate per generation in all four species. The last analysis carried out tried to detect possible natural selection affecting the nine DNA microsatellites studied. Both mutation models (infinite allele and step-wise) offered the same results with two out of nine microsatellites showing evidence of natural selection within the Alouatta genus. Diversifying selection was detected at D14S51 and constrictive selection was detected at D8S165. The remaining microsatellites were within the limits of a neutral behavior.

DISCUSSION The diverse central moments for the markers and for the species revealed noteworthy intraspecific differences between different markers and interspecific differences of a determined marker. For example, the first moment was extremely different for the four species considered at D5S111 and D5S117, and this trend was independent of the sample size studied. The D14S51 and D17S804 markers showed symmetrical distributions for the third and fourth central moment in A. palliata, while the opposite trend was determined for the three other South American Alouatta species analyzed. In fact, A. palliata was the species with the most divergent central moments compared to the other species studied, which concurs with its phylogenetic position as demonstrated by Cortés-Ortiz et al., (2003). There were similar differences (in quantity) of the central moments among the three South American species. Relevant differences were recorded between A. seniculus and A. macconnelli at D5S117 and between A. seniculus and A. caraya at D5S111 for the first central moment and there were conspicuous differences for several central moments at D5S117 and D6S260.

Table 6. Estimates of maximum likelihood of  with uni-step and a multi-step mutation models. This analysis was carried for the detection of DNA microsatellites that had significantly evolved following a multi-step model. Also the multi-step mutation proportions were calculated for each species

0

Uni-step Log likelihood 

Multi-step Multi-step mutation proportion



Log likelihood

-22.1792 -10.9069 -10.7159 -29.7882 -12.0983 -5.9213 -5.5851

10.7279 10.2025 0.8387 20.0856 5.1527 1.2733 1.1636

-22.0 778 -10.8703 -11.3512 -28.0743 -11.8396 -5.9037 -5.6529

P = 0.025 2 = 0.202 NS P = 0.000 2 = 0.073 NS P = 0.000 2 = 1.270 NS P = 0.075 2 = 3.427 SIG P = 0.175 2 = 0.517 NS P = 0.000 2 = 0.035 NS P = 0.000 2 = 0.134 NS P = 0.0392 ± 0.0659

1.8571 3.0967 7.0418 2.9913 68.2036 58.1060 14.0258 4.5513 18.2948

-13.2 689 -7.9361 -13.99 51 -13.2 204 -42.7 391 -15.6 952 -17.7 441 -23.6 177 -14.8 482

1.9657 3.0967 3.1449 3.3198 68.2036 36.4587 10.8021 4.5513 10.8080

-13.1854 -7.9301 -15.0506 -13.2881 -44.8431 -16.7864 -18.5809 -19.7163 -14.6334

P = 0.000 P = 0.000 P = 0.000 P = 0.000 P = 0.100 P = 0.100 P = 0.025 P = 0.325 P = 0.050 P = 0.0666 ± 0.1053

2 = 0.012 NS 2 = 2.111 NS 2 = 0.167 NS 2 = 0.135 NS 2 = 4.208 SIG 2 = 2.182 NS 2 = 1.673 NS 2 = 7.802 SIG 2 = 0.429 NS

1.1467

-2.0 428

1.1467

-2.0473

P = 0.000

2 = 0.009 NS

Species and markers Alouatta palliata AP68 AP74 D5S111 D5S117 D6S260 D14S51 D17S804

13.9320 4.8580 1.4460 17.4660 13.2120 0.6670 1.2120

6.7572 11.1256 1.9376 33.3595 6.4079 1.2733 0.8182

Alouatta seniculus AP40 AP68 AP74 D5S111 D5S117 D6S260 D8S165 D14S51 D17S804

1.1430 1.4750 2.7350 3.4580 88.5760 37.9780 5.6560 11.6700 11.2580

Alouatta macconnelli AP40

0.6670

Table 6. (Continued)

Species and markers AP68 AP74 D5S111 D5S117 D6S260 D8S165 D17S804

0 10.2440 21.8330 17.8000 44.6440 6.1330 4.8000 10.1430

Alouatta caraya AP40 AP74 D5S111 D5S117 D14S51

0.9330 0.9330 0.5330 3.5880 0.1430

Uni-step Log likelihood  15.6740 -10.3 792 39.6275 -5.86 51 33.9980 -12.1 119 13.1701 -17.3 892 15.2107 -6.0369 2.3280 -5.6465 7.8100 -8.9352

 23.4597 29.2567 44.1440 8.9289 14.0453 0.9600 7.8100

Log likelihood -10.3052 -6.2323 -10.7584 -13.9341 -6.1174 -5.3214 -8.8559

Multi-step Multi-step mutation proportion P = 0.000 2 = 0.148 NS P = 0.025 2 = 0.734 NS P = 0.000 2 = 2.706 NS P = 0.200 2 = 6.910 SIG P = 0.000 2 = 0.161 NS P = 0.475 2 = 0.650 NS P = 0.000 2 = 0.158 NS P = 0.0875 ± 0.1711

P = 0.475 2 = 0.037 NS P = 0.475 2 = 0.037 NS P = 0.000 2 = 0.013 NS P = 0.000 2 = 0.091 NS P = 0.000 2 = 0.004 NS P = 0.1583 ± 0.2452 Total Average of P for the four Alouatta species studied (P = 0.0879 ± 0.0509) o = Initial value to undertake the  maximum likelihood estimates. SIG = microsatellites which significantly departure from a uni-step mutation model. P = Average multi-step mutation proportions affecting the markers and the species studied. NS = No significant multi-step mutation proportion 0.7187 0.7187 0.8160 5.1491 0.3950

-6.0118 -6.0111 -2.5250 -11.5713 -2.6979

0.3640 0.3640 0.8160 5.4900 0.4086

-5.9931 -5.9931 -2.5184 -11.5259 -2.6961

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It is fundamentally important both from evolutionary and genetic conservation points of view, to have a thorough understanding of the possible differential dynamics concerning microsatellite central moments used in order to better highlight evolutionary entities that might support other postulated phylogenetic frameworks of the Alouatta genus (Cortés-Ortiz et al., 2003 or Ruiz-García et al., 2016a, in the current book). The molecular evolution of microsatellites and other molecular markers seems to be a better tool for the determination of evolutionary genetics relationships among Neotropical primates compared to past methods that relied on differences in pelage traits (such as color). For instance, Shedd and Macedonia (1991) and Jacobs et al., (1995) have cautioned against using metachronism inferences to determine evolutionary relationships among taxa of Neotropical primates due to varying evolutionary pathways that occur once gene flow is interrupted. Evidence suggest that there is no agreement between pelage-based and DNA marker-based taxonomy studies for Ateles (Collins, 2001 or Ruiz-García et al., 2016b, in the current book). Therefore the molecular results indicate that an Alouatta species’ definition based only on pelage color may lead to erroneous conclusions. This was further ratified by Schneider et al., (1991)’s study that found differences between two Alouatta belzebul populations living on the east and west banks of the Tocantins River in Brazil. These populations showed opposite patterns when morphological and molecular traits were compared. The color diversity on the east bank was the highest and the expected heterozygosity was lowest whereas the opposite was found on the west bank. In addition, some red howler monkeys were captured in the east bank of the Tocantins River that were similar to A. seniculus, not previously documented in that area, and living together in troops with A. belzebul. These rare animals presented no distinguishable difference in karyotype from that of A. belzebul (Lima and Seuánez, 1989). All of these studies emphasize that color descriptions are not sufficient to describe the evolutionary relationships among the Alouatta taxa. Therefore, microsatellite central moments (evolution and differences) may be a strong tool in the analysis of different Alouatta taxa as well as other species taxonomy problems.

Assignation Differentiation Power in the Microsatellite Evolution of Alouatta The assignment analyses revealed that the best procedure to correctly classify all the animals genotypified in their respective species was the Bayesian method with the “as is” technique (86.15%). In contrast, other procedures that relied on standard and the minimum Nei genetic distance (73.85%) and the Cavalli-Sforza and Edwards chord genetic distance (72.31%) with the “leave-one-out” technique had lower classification percentages. This is one indication that the classical genetic distances based on allele frequencies probably do not take into consideration the most primordial information of the microsatellite evolution, unlike some other techniques. Some of the most frequent incorrectly classified cases involve the A. seniculus-A. caraya pair, classifying animals of the first species in the second one. These taxonomic errors reveal a close genetic relationship between A. seniculus and A. caraya not previously found in other traditional studies. For example, Herkovitz (1949) used hyoid bone morphology to separate A. caraya in a different clade than A. seniculus. A. caraya has a smaller, more angular and less inflated hyoid bone compared to that within A. seniculus. The same separation result was also reported by Meireles et al. (1999), but based on globin pseudogene sequences. However, Cortés-Ortiz et al. (2003) sequenced five genes (three mitochondrial and two nuclear) and revealed that A. caraya was within the same clade as A. seniculus, which is the same as we determined with microsatellites. Some A. seniculus

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individuals were also incorrectly classified within A. macconnelli and A. palliata. This supports that the genetic variability found within other Alouatta species and their ancestors is contained within A. seniculus and that this species (or some direct ancestor) could be the origin of the other Alouatta species studied. It agrees quite well with the fact that A. seniculus was the species with the highest levels of heterozygosity and mean number of alleles per locus (shown elsewhere), although all of the animals in that study came from Colombia. Recall that the species, or populations, with the highest levels of gene diversity are frequently considered the original geographical centers of dispersion (Dobzhansky, 1971). In agreement with this idea, recall that the oldest Alouttinae fossil registered is Stirtonia tatacoensis from La Venta in Colombia, a form allied with Alouatta and dated to 12-16 million years (MY) (Feagle, 1988). Therefore, Alouatta could have originated in some part of the current Colombian territory, where A. seniculus lives today. In contrast, A. macconnelli and A. palliata individuals have probably not been misclassified because they have well delimited and particular gene pools which were likely derived from A. seniculus. In agreement with Cortés-Ortiz et al., (2003), our microsatellite data differentiated A. macconnelli from A. seniculus. Cortés-Ortiz et al., (2003) used mtDNA-based phylogenetic analyses to show that A. macconnelli and A. sara were phylogenetically distinct forms from A. seniculus. Some of our microsatellite results (unpublished) also strongly differentiated A. sara (from Bolivia) from A. seniculus, such as we observed with A. macconnelli.

Effective and Total Numbers, the Mutation Dynamics and Natural Selection of DNA Microsatellites in Howler Monkeys Several interesting features can be obtained from an analysis of effective and total numbers and mutation dynamics of the microsatellites studied in the four Alouatta species analyzed. First, the multi-step mutation model that was globally determined for the four species did not significantly differ from the uni-step mutation model. D5S117 was the unique marker which systematically presented a significant departure from the uni-step mutation model in three of the four species studied (A. seniculus, A. macconnelli, and A. palliata) and yielded the highest mutation rate in all four species. A. seniculus was the species, with the most cases of significant multi-step mutation percentages (22.22%). Nevertheless, this percentage was not significantly different from the 5% type I error. As it was commented before, the two species with the most divergent multi-step mutation proportion average percentages were A. caraya (15.8%-the highest) and A. palliata (3.9%- the lowest), whereas the two northern South American species presented similar percentages (A. seniculus – 8.7%and A. macconnelli – 6.7%-). Therefore, the two most peripheral species studied showed the highest degree of deviation in regard to the two northern South-American species, which revealed the highest degrees of gene diversity (not shown here) and the highest effective and historical numbers. It is possible that the speciation events with relevant founder effects could have influenced the levels of multi-step mutation percentages and therefore provide an explanation of why A. caraya and A. palliata are the species with the extreme values. The overall multi-step percentage averages for the four species was around 8%. Therefore, from an evolutionary point of view, the uni-step is more remarkable than the multistep mutation model, at least for the microsatellites we studied in Alouatta, in contradiction to that affirmed for humans by DiRienzo et al., (1994). Second, through the application of maximum likelihood tests we determined that most of the microsatellites analyzed presented different mutation rates within each one of the Alouatta species considered. A. macconnelli /A.

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seniculus was identified as the species pair with the greatest number of microsatellites that suffered from similar mutation rates. As previously mentioned, this could be of use as a phylogenetic signal. Third, as a consequence of no conspicuous differences among the unistep and the multi-step mutation models, the effective and the total historical numbers were very similar for both mutation models. Consequently, the use of the uni-step mutation model, when the maximum likelihood multi-step mutation model is not estimated, does not introduce an excessive bias, at least, for the howler monkeys with the microsatellites used in this study. Fourth, the arithmetic and harmonic effective and total numbers clearly revealed that A. seniculus was the species which presented the largest historical demographic populations. This agrees quite well with the fact that this is the species with the largest distribution range. Its total numbers ranged from 22,100 to 176,800 animals (arithmetic mean) or from 5,900 to 46,900 individuals (harmonic mean). The large effective numbers probably indicate that until recently, the gene flow has been elevated within populations of this species. On the other hand, the values obtained from the most peripheral Alouatta species were clearly lower (A. palliata and A. caraya), especially for A. caraya, which ranged from 5,900 to 47,000 (arithmetic mean) or from 990 to 7,900 (harmonic mean). The fact that A. macconnelli presented a gene diversity (not shown here),  estimates, historical effective and total numbers almost as high as A. seniculus is quite interesting, even though its sample size and its distribution range were small in comparison to A. seniculus. Indeed, the distribution range of A. macconnelli is considerable smaller than that of either A. palliata or A. caraya, but its estimates of effective and total numbers are higher than either of these two species. It could mean that the origin of the current Alouatta species studied was in northern South America, as we previously speculated. A. macconnelli could be a direct descendent of A. seniculus without loss of genetic diversity during speciation, while a relevant founder effect was effective in the appearance of A. palliata and A. caraya. A. belzebul was not included in this study, but it has been shown to have extremely high levels of protein gene diversity. It could be another good original-species candidate from which other current Alouatta species derived. For this reason microsatellites should be applied to this species to support or not support the proposed hypothesis. As we will demonstrate, some of our microsatellite mutation rates agree quite well with a divergence between A. seniculus and A. macconnelli around 3.3 MYA (CortésOrtiz et al., 2003). This suggests that they were isolated by the separation of the Guyana region from the Amazon basin and the emergence of the current Amazon River as the major drainage of this river basin more than 3 MYA. We believe that the pathway of speciation was from A. seniculus to A. macconnelli because several A. seniculus individuals were classified in the A. macconnelli group with the assignment analysis as previously quoted. This could mean that the original microsatellite genotypes of A. macconnelli were derived from individuals that resembled the multi-genotype of A. seniculus. Furthermore, this would demonstrate that A. macconnelli not only derived from A. seniculus but that no extreme founder effect occurred when both forms were isolated because A. macconnelli retained the major part of the original gene diversity of A. seniculus. The isolation of the Guyana region also affected other Atelidae primates such as Ateles paniscus (Collins and Dubach, 2000b). Ateles paniscus separated from the other Ateles species around 3.27 MYA, which correlates with this time of separation for A. macconnelli and A. seniculus. Thus, the same biogeographic event simultaneously affected both Ateles and Alouatta in that region of SouthAmerica. The trans-Andean Alouatta species (A. palliata) separated from the South American Alouatta species around 6.8 MYA. Meanwhile the Pleistocene refugia, with the Magdalena

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river valley fluctuations, could provide a relevant explanation to the appearance (by isolation) of several Ateles species, such as Ateles hybridus (1.41 MYA) or the Central American Ateles geoffroyi (2.0 MYA) (Collins and Dubach, 2000b), this explanation is not satisfactory for Alouatta. The South and the Central American Alouatta species had diverged previously and the climatic Pleistocene fluctuations seemed to have no noticeable impact on the microsatellite gene dynamics of Alouatta. Therefore the use of microsatellite evolutionary dynamics may therefore allow for better recreations of past biogeographic events mainly responsible of speciation by measuring levels of gene diversity in isolated populations and providing approximate dates for species ancestors that can be associated with previous climatic and geological changes. Molecular evolution studies of microsatellites in Neotropical primates may also lead to an increase of our evolutionary knowledge and to the development of new conservation ideas. Fifth, several fundamental questions however must be resolved in order to have more confidence in the obtained results. To calculate total historical numbers from the effective numbers estimated, we need to determine the ratio Ne/N from demographic data. By using the ecological and demographical studies of Baldwin and Baldwin (1976), Heltne et al., (1976), Milton (1982) and Estrada (1982) for A. palliata, of Neville (1972), Eisenberg (1979), Braza et al., (1981), Defler (1981), Terborgh (1983), Crockett (1984, 1985) and Crockett and Eisenberg (1987) for A. seniculus and of Thorington et al., (1984) for A. caraya, we calculated this ratio considering the mean number of adult males, the mean number of adult females, the mean troop size and the fraction of adult males that did not reproduce within their troops. This value was around 0.4 for the three species indicated. We assumed that this ratio was also feasible for A. macconnelli. With this in mind, we will try to answer three questions as follows: (1) From an evolutionary perspective what is the likely average microsatellite mutation rate per generation (5.6 x 10-4 or 7 x 10-5) for Alouatta? This is interesting not only from a microsatellite mutation evolution perspective, but also because it is important to know the most feasible effective and total numbers throughout the history of the species in question. Additionally, this is of interest to conservation geneticists. For example, the conservation measures to be implemented for the conservation of A. seniculus will not be the same if we consider the lowest estimate of the total number obtained, 5,855 animals (5.6 x 10-4 and harmonic averages), or the highest total number estimated of 176,815 animals (7 x 10-5 and arithmetic averages). The same could be mentioned for A. caraya, the species which showed the lowest number estimates. There is a difference in the conservation of 988 compared to 47,045 animals. (2) Which of the two methods used to obtain means (arithmetic or harmonic) fits more adequately with reality? (3) Several genetic heterogeneity statistics (FST and GST) could be employed to measure the degree of differences among the Alouatta species. One relevant question is, which of these two statistics is more consistent with the molecular evolution of the microsatellites? Herein, we can answer these three questions. Cortés-Ortiz et al., (2003) obtained a maximum likelihood tree of three mitochondrial genes (ATPase 8 and 6 and Cyt b) with an enforced molecular clock for different Alouatta species and estimated the divergence time between the Central and the South American species to be 6.8 MYA. By means of Slatkin (1995), we can employ the following equations: F = 4Ne (FST/(1 – FST)) and R = 4Ne (RST/(1 – RST)),

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where  is the number of generations elapsed from the separation of the taxa analyzed and Ne is the effective number. We obtained average FST values ranging from 0.189 to 0.259 and average RST values from 0.545 to 0.696, depending on the procedure used. To simplify we used the mean values of these ranges (FST = 0.224 and RST = 0.621, respectively) jointly with the effective numbers summed for all the species analyzed estimated throughout the two extreme mutation rates (5.6 x 10-4 and 7 x 10-5). Moreover, 6.6-6.8 MYA approximately represents 1,100,000 to 1,133,333 generations for Alouatta. Most red howler females first give birth at about age 5 whereas males actively reproduce through age 7 (Crockett and Eisenberg, 1987). Therefore, one generation in this genus is approximately 6 years. The combination of data (mutation rate of 7 x 10-5, the mean numbers obtained with the arithmetic procedure and the mean value of RST [= 0.621]) used to estimate the number of generation ( of 1,170,481) agrees quite well with the data of Cortes-Ortiz et al., (2003). Other combinations values were not compatible with this number of generations. For example, only 6,825 (41,000 years ago) elapsed using the 5.6 x 10-4 mutation rate, the harmonic effective number means, and FST (= 0.224). This is completely incompatible with the divergence times obtained by Cortés-Ortiz et al., (2003). These values are compatible with the fact that the diversification of the current howler monkey species was parallel to the formation of the northern Andes (Lundberg et al., 1998) approximately 6.8 MYA. The posterior diversification for the cis-Andean howler monkeys (5.1 MYA) is roughly parallel to the origin of the modern Amazon River. Also, our estimates (using microsatellites) with mutation rates of 7 x 10-5, arithmetic means, and the heterogeneity RST statistic offer a divergence time strikingly similar to that determined with mtDNA (Cortés-Ortiz et al., 2003). Thus, future works with these microsatellites and with Alouatta should use the 7 x 10-5 mutation rate, the arithmetic means to calculate overall effective and total numbers, and RST to more accurately reconstruct evolutionary relationships. It is interesting to note that the present results for RST disagree with that claimed by Gaggiotti et al., (1999). Another interesting conclusion is that these nuclear DNA microsatellites provided a good phylogenetic resolution with these parameters comparable to mtDNA genes. Other nuclear regions such as the sequences of the Calmodulin (CAL) and the Recombination activating (RAG1) genes did not because they presented very low levels of sequence heterogeneity, even between Alouatta and Ateles (Cortés-Ortiz et al., 2003). Hence, with the average mutation rate of 7 x 105, we were able to address theories of biogeographic mechanisms responsible for the production of Alouatta species patterns. We can affirm that the majority of the speciation events among the Alouatta species studied occurred during the middle to late Pliocene and very early Pleistocene. They were primarily the result of major events such as the final uplift of the Andes Cordillera and development of savannas in northern and middle-southern South America. There is little evidence to support the importance of Pleistocene refugia formation or current riverine barriers as primary mechanisms for the formation of the Alouatta species analyzed herein. Recall that the Pleistocene refugia has been used by many authors to try to explain speciation among many Neotropical animals (Bush, 1994; Haffer, 1997). Although some Neotropical primate species (and other species) were probably affected by the existence of smaller and less stable Pleistocene refugia among large and extensive primary rain forests, these smaller and less stable Pleistocene refugia were probably not an obstacle for the Alouatta dispersion. For example, Estrada and Coates-Estrada (1988) determined that Mexican Ateles populations

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have been absent from forest fragments as large as 250 ha for many decades and that howler monkeys are extremely efficient colonizers of any size Neotropical ecosystem. Perhaps only A. macconnelli, of the four Alouatta species that we studied, supports the riverine barrier hypothesis as a cause of speciation, because this species’ distribution is limited by the Amazon in southern Guyana and in northern Brazil and by several black water rivers in the northwest. Its distribution coincides within an area that houses multiple endemic species of vertebrates (Haffer, 1992), including A. paniscus (Collins and Dubach, 2000a). This last species diverged from the other Ateles lineages approximately 3.3-3.6 MYA, a value range extremely similar to the estimate of Cortés-Ortiz et al. (2003) and our estimation of around 3 MYA using microsatellites. These divergence times are probably too large to be explained by the original Pleistocene Refuge model (Haffer, 1969). Other authors such as Suemitsu et al., (2000) have claimed that several Alouatta populations (A. fusca clamitans) have specific cytotypes associated with geographical patterns in Brazil that suggests a north-south division due to possible reproductive isolation during the existence of quaternary refuges. However, our microsatellite data and mitochondrial data of Cortés-Ortiz et al.’s (2003) do not support quaternary refuges as important to the molecular gene evolution of the four Alouatta species studied herein. Lastly, the Beaumont and Nichols (1996) test, independent of the infinite allele or the step-wise mutation models, revealed two microsatellites (D14S51 and D8S165) inside the Alouatta genus that were outside neutral behavior. D14S51 seems to be affected by diversifying natural selection while D8S165 revealed the impact of constrictive natural selection. Although all the other microsatellite seem to be neutrally regulated, D14S51 could be impacted by hitchhiking of advantageous mutations (selective sweeps), whereas D8S165 could be affected by the continued removal of deleterious mutations by means of background selection (Schug et al., 1998). Therefore, the use of microsatellites in the genome of diverse Neotropical primates may reveal relevant selective patterns. The analysis of molecular microsatellite evolution is straightforward and interesting because it helps to determine the mutation mechanisms that underlie these repetitive segments of DNA. Furthermore, by coalescence this analysis helps to determine fundamental parameters (such as effective numbers) in the evolution of species as well as compares microsatellite phylogenetic signals with those of other molecular markers. Similar studies to that presented herein are ongoing in our laboratory with other Neotropical primate genera such as Ateles, Lagothrix, Cebus, Saimiri, Aotus, Callicebus, Pithecia and Saguinus.

ACKNOWLEDGMENTS We thank the Dean of the Faculty of Sciences and the Academic Vicerectory of the Pontificia Universidad Javeriana at Bogotá, Colombia for their financial support. Thanks also goes to the Instituto von Humboldt (IVH) at Villa de Leyva for the use of its facilities for providing the opportunity to sample integumentary tissues from its howler pelt collection for the extraction of DNA. These acknowledgments are mainly directed to the ex-curator of the mammalogy collection, Miss. Yaneth Muñoz-Sabas and the directors of IVH, Drs. Cristian Samper and Fernando Gast. Likely, we are indebted to many people throughout Colombia, who provided samples (hair) of howlers maintained in captivity or provided samples (teeth,

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bone or skin) from howler that had been hunted. Indeed, many thanks go to Luz Mercedes Borrero and Marcela Ramirez from Santa Fe zoo at Medellín who provided samples of red howler from the Antioquia region and to Dr. Francois Catzeflis who provided four of the seven DNA samples used of A. macconnelli from French Guiana. Similarly, many thanks to Dr. Liliana Cortés-Ortiz, who provided two DNA samples of A. palliata mexicana. Thanks go to the Huitoto, Ticuna and Jaguas Indian communities throughout the Colombian Amazon, and the Movima, Moxeño, Sirionó, Canichana, Cayubaba and Chacobo Indian communities in Bolivia.

REFERENCES Ayres, J. M. C. (1986). Uakaris and Amazonian flooded forest.” PhD diss., Cambridge University. Baldwin, J. D. and Baldwin, J. I. (1976). The vocalizations of howler monkeys (Alouatta palliata) in southwestern Panama. Folia Primatologia., 26, 81-108. Beumont, M. and Nichols, R. (1996). Evaluating loci for use in the genetics analysis of population structure. Proceedings of the Royal Society of London, Series B, 263, 16191626. Braza, F., Alvarez, F. and Azcarate, T. (1981). Behavior of the red howler monkey (Alouatta seniculus) in the llanos of Venezuela. Primates, 22, 459-473. Bruford, M. W. and Wayne, R. K. (1993). Microsatellites and their application to population genetics studies. Current Opinion in Genetics and Development, 3, 939-943. Bush, M. B. (1994). Amazonian speciation: a necessarily complex model. Journal of Biogeography, 21, 5-7. Cavalli-Sforza L. L. and Edwards, A. W. F. (1967). Phylogenetic analysis: models and estimation procedures. Evolution, 21, 550-570. Chakraborty, R. and Jin, L. (1993). A unified approach to study hypervariable polymorphisms: Statistical considerations of determining relatedness and population distances.” In Pena, S. D. J., Chakraborty, R., Epplen, T. J. and Jeffreys, A. J. DNA Fingerprinting: State of the Science, (pp. 153-175). Birkhauser Verlag: Basel. Collins, A. C. (2001). The importance of sampling for reliable assessment of phylogenetics and conservation among Neotropical Primates: a case study in spider monkeys (Ateles). Primate Report, 61, 9-30. Collins, A. C. and Dubach, J. M. (2000a). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology, 21, 381-420. Collins, A. C. and Dubach, J. M. (2000b). Biogeographic and Ecological forces responsible for speciation in Ateles. International Journal of Primatology, 21, 421-444. Cornuet, J. M., Piry, S., Luikart, G., Estoup, A. and Solignac, M. (1999). New methods employing multilocus genotypes to select or exclude populations as origins of individuals. Genetics, 153, 1989-2000. Cortés-Ortiz, L., Bermingham, E., Rico, C., Rodríguez-Luna, E., Sampaio, I. and RuizGarcía, M. (2003). Molecular systematics and biogeography of the Neotropical monkey genus, Alouatta. Molecular Phylogenetics and Evolution, 26, 64-81.

392

Manuel Ruiz-García, Pablo Escobar-Armel, Marta Mudry et al.

Crockett, C. M. (1984). Emigration by female red howler monkeys and the case for female competition. In Female primates: Studies by women primatologists. Edited by M. F. Small, 87-94. New York: Alan R. Liss. Crockett, C. M. (1985). Population studies of red howler monkeys (Alouatta seniculus). National Geographic Research, 1, 264-273. Crockett, C. M. and Eisenberg, J. F. (1987). Howlers: variation in group size and demography. In Smuts, B. B., Cheney, D. L., Seyfarth, R. M., Wrangham, R. W., and Struhsaker T. T. (Eds.). Primate Societies, (54-68). Chicago: University of Chicago Press. Defler, T. R. (1981). The density of Alouatta seniculus in the eastern llanos of Colombia. Primates, 22, 564-569. De Oliveira, E. H., M. Neusser, W. B. Figueiredo, C. Nagamachi, J. C. Pieczarka, I. Sbalqueiro, J., Wienberg, J. and Muller, S. (2002). The phylogeny of howler monkeys (Alouatta, Platyrrhini): Reconstruction by multicolor cross-species chromosome painting. Chromosome Research, 10, 669-683. Di Rienzo, A., Peterson, A., Garza, J., Valdés, A., Slatkin, M. and Freimer, N. (1994). Mutational processes of simple-sequence repeat loci in human populations Proceedings of the National Academy of Sciences USA, 91, 3166-3170. Dobzhansky, Th. (1971). Evolutionary oscillations in Drosophila pseudoobscura. In Creed, R. (Ed.) Ecological Genetics and Evolution., (109-133). Oxford and Edinburgh: Blackwell Scientific Publications. Eisenberg, J. F. (1979). Habitat, economy, and society: Some correlations and hypotheses for the Neotropical primates. In Bernstein, I., and Smith, E. O., Primate Origins and Human Evolution, (215-262). New York: Graland Press. Ellegren, H. (1995). Mutation rates at porcine microsatellite loci. Mammalian Genome, 6, 376-377. Estrada, A. (1982). Survey and census of howler monkeys (Alouatta palliata) in the rain forest of “Los Tuxtlas” Veracruz, Mexico. American Journal of Primatology, 2, 363-372. Estrada, A. and Coates-Estrada, R. (1988). Tropical rain forest conservation and perspectives in the conservation of wild Primates (Alouatta and Ateles) in Mexico. American Journal of Primatology, 4, 315-327. Feagle, J. G. (1988). Primate, adaptation and Evolution. San Diego: Academic Press, Inc. Gaggiotti, O. E., Lange, O., Rassmann, K. and Gliddon. C. (1999). A comparison of two indirect methods for estimating average levels of gene flow using microsatellite data. Molecular Ecology, 8, 1513-1520. Goldstein, D. B., Ruiz-Linares, A., Cavalli-Sforza, L. L. and Feldman, M. W. (1995). An evaluation for genetic distances for use with Microsatellite loci. Genetics, 139, 463-471. Griffiths, R. C. and Tavaré, S. (1994). Simulating probability-distributions in the coalescent. Theoretical Population Biology, 46, 131-159. Haffer, J. (1969). Speciation in Amazonian forest birds. Science, 165, 131-137. Haffer, J. (1992). On the “river effect” in some forest birds of southern Amazonia. Bol. Mus. Para. Emilio Goeldi, ser. Zool., 8, 127-245. Haffer, J. (1997.) Alternative models of vertebrate speciation in Amazonia: an overview. Biodiversity and Conservation, 6, 451-476. Heltne, P. G., Turner, D. C. and Jr. Scott, N. J. (1976). Comparison of census data on Alouatta palliata from Costa Rica and Panama. Pp. 116-131. In R. W. Thorington, Jr.,

Microsatellite DNA Analyses of Four Alouatta Species (Atelidae, Primates)

393

and P. G. Heltne. (Eds.). Neotropical Primates: Field studies and conservation. National Academy of Sciences. Washington, D.C., USA. Herskovitz, P. (1949). Mammals of northern Colombia: preliminary report No 4, Monkeys (Primates) with taxonomic revisions of some forms. Proceedings of the United States National Museum, 98, 323-427. Hernández-Camacho, J. and Cooper, R. W. (1976). The non human Primates of Colombia. In R. W. Thorington and P. G. Heltheydes. (Eds). Neotropical Primates: field studies and conservation. (pp. 35-69). National Academy of Sciences, Washington D.C. Jacobs, S. C., Larson, A. and Cheverud, J. M. (1995). Phylogenetic relationships and orthogenetic evolution of coat color among tamarins (genus Saguinus). Systematic Biology, 44, 515-532. Lima, de, M. M. C. and Seuánez, H. N. (1989). Cytogenetic characterization of Alouatta belzebul with atypical pelage coloration. Folia Primatologica, 52, 97-101. Lundberg, J. G., Maeshal, L. G., Guerrero, J., Horton, B., Malabarba, C. S. L. and Wesselingh, F. (1998). The stage of Neotropical fish diversification: a history of tropical South American Rivers. In L. R. Malabarba, R. E. Reis, R. P. Vari, Z. M. Lucena, and C. A. S. Lucena. (Eds). Phylogeny and classification of Neotropical fishes. (pp. 13-48). Porto Alegre, Brazil. Meireles, C. M., Czelusniak, J., Schneider, M. P. C., Muniz, J. A. P. C., Brigido, M. C., Ferreira, H. S. and Goodman, M. (1999). Molecular phylogeny of Ateline New World monkeys (Platyrrhini, Atelinae) based on -globin gene sequences: Evidence that Brachyteles is the sister group of Lagothrix. Molecular Phylogenetics and Evolution, 12, 10-30. Milton, K. (1982). Dietary quality and demographic regulation in a howler monkey population. Pp. 146-167. In E. G. Leigh, Jr., A. S. Rand and D. M. Windsor. (Eds.). The ecology of a tropical forest: Seasonal rhythmus and long-term changes. Smithsonian Institution Press. Washington, D. C. USA. Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89, 583-590. Neville, M. K. (1972). The population structure of red howler monkeys (Alouatta seniculus) in Trinidad and Venezuela. Folia Primatologica, 17, 56-86. Nielsen, R. (1997). A likelihood approach to populations samples of Microsatellite alleles. Genetics, 146, 711-716. Paetkau, D., Calvert, W., Stirling, I. and Strobeck, C. (1995). Microsatellite analysis of population structure in Canadian polar bears. Molecular Ecology, 4, 347-354. Rannala, B. and Mountain, J. L. (1997). Detecting immigration by using multilocus genotypes. Proceedings of the National Academy of Sciences USA, 94, 9197-9201. Ruiz-García, M., Cerón, A., Pinedo-Castro, M. and Gutierrez-Espeleta, G. (2016a). Which howler monkey (Alouatta, Atelidae, Primates) taxa is living in the Peruvian Madre de Dios River Basin (Southern Peru)? Results from mitochondrial gene analyses and some insights in the phylogeny of Alouatta. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Lichilín, N., Escobar-Armel, P., Rodríguez, G. E. and Gutierrez-Espeleta, E. (2016b). Historical Genetic demography and some insights into the systematics of Ateles (Atelidae, Primates) by means of diverse mitochondrial genes. In Phylogeny, Molecular

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Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Ruiz-García, M., Shostell, J. M. (Eds.). Nova Science Publishers, Inc. New York, USA. Rylands, A. B-, Rodríguez-Luna, E. and Cortés-Ortiz, L. (1997). Neotropical Primate conservation – The species and the IUCN/SSC Primate specialist group network. Primate Conservation, 17, 46-69. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schneider, H., Sampaio, M. I. C., Schneider, M. P. C., Ayres, J. M., Barroso, C. M. L., Hamel, A. R., Silva, B. T. F. and Salzano, F. M. (1991). Coat color and biochemical variation in Amazonian wild populations of Alouatta belzebul. American Journal of Physical Anthropology, 85, 85-93. Serikawa, T. (1992). Rat gene mapping using PCR-analyzed microsatellites. Genetics, 131, 701-721. Sheed, D. H. and Macedonia, J. M. (1991). Metachromism and its phylogenetic implications for the genus Eulemur. Folia Primatologica, 57, 221-231. Slatkin, M. (1995). A measure of population subdivision based on Microsatellite allele frequencies. Genetics, 139, 457-462. Suemitsu, E., da Silva, A. F., Sbalqueiro, I. J. and de Oliveira, E. H. C. (2000). Geographical variation of chromosomal number in Alouatta fusca clamitans (Primates, Atelidae). Caryologia, 53, 163-168. Terborgh, J. (1983). Five New World Primates. Princenton University Press, Princeton, NJ, USA. Thorington, R. W., Jr., Ruiz, J. C. and Eisenberg, J. F. (1984). A study of a black howler monkey (Alouatta caraya) population in northern Argentina. American Journal of Primatology, 6, 357-366. Vassart M, A. Guédant, J. C. Vié, J. Kéravec, Séguéla, A. and Volobouev, V. T. (1996). Chromosomes of Alouatta seniculus (Platyrrhini, Primates). Journal of Heredity, 87, 331334. Von Dornum, M. and Ruvolo, M. (1989). Phylogenetic relationships of the New World monkeys (Primates, Platyrrhini) based on nuclear G6PD DNA sequences. Molecular Phylogenetics and Evolution, 11, 459-476. Walsh, P. S., Metzger, D. A. and Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10, 506513. Weber, J. L. and May, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. American Journal of Human Genetics, 44, 388-396. Weber, J. L. and Wong, C. (1993). Mutation of human short tandem repeats. Human Molecular Genetics, 2, 1123-1128. Zhivotosky, L. A. and Feldman, M. W. (1995). Microsatellite variability and genetic distances. Proceedings of the National Academy of Sciences USA, 92, 11549-11552.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 12

WHICH HOWLER MONKEY (ALOUATTA, ATELIDAE, PRIMATES) TAXON IS LIVING IN THE PERUVIAN MADRE DE DIOS RIVER BASIN (SOUTHERN PERU)? RESULTS FROM MITOCHONDRIAL GENE ANALYSES AND SOME INSIGHTS IN THE PHYLOGENY OF ALOUATTA

Manuel Ruiz-García1,*, Angela Cerón1, Myreya Pinedo-Castro1 and Gustavo Gutierrez-Espeleta2 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva. Unidad de Genética. Departamento de Biología. Facultad de Ciencias. Pontificia Universidad Javeriana. Bogotá DC., Colombia 2 Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica

ABSTRACT We sequenced mitochondrial genes (COI, COII and Cyt-b) from 138 howler monkeys (Alouatta) representing six “a priori” species (A. pigra, A. palliata, A. coibensis trabeata, A. seniculus, A. sara and A. caraya). These data were used to address two main issues: 1- to determine which red howler monkey taxa were distributed in Southern Peru (four of the sequenced were from there) and 2- to provide new insights about the systematics of howler monkeys. Our data support six main findings. 1-There is strong molecular gene heterogeneity between A. seniculus and A. sara. This verifies that A. sara (a taxon currently living in Southern Peru) is a full species; 2- A. seniculus seniculus is present in Colombia as well as across the northern and middle Peruvian Amazon. The alleged full species A. juara and A. puruensis seem to be molecularly un-differentiable from A. s. seniculus. Therefore, until these potential taxa are studied from a karyological point of view, only two red howler species are considered to be living in Peru: A. seniculus and A. sara; 3- The cis and trans-Andean howler monkeys are clearly *

Corresponding author: [email protected], [email protected].

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Keywords: Alouatta, mitochondrial genes (COI, COII and Cyt-b), population expansion, molecular phylogeny, Southern Peru

INTRODUCTION The howler monkey (Alouatta) is the unique living genus within the subfamily Alouattinae (or Mycetinae) (Rylands et al., 2000; Groves, 2001) and family Atelidae. Howler monkeys are among the largest Neotropical primates and are similar in size to individuals within the Ateles, Brachyteles and Lagothrix genera. Alouatta is also the most widely distributed Neotropical primate genus ranging from Veracruz, Mexico (Estrada and CoatesEstrada, 1984) to Corrientes, Argentina (Cabrera, 1939). Howler monkeys are an ecologically flexible pioneer species that can live in many types of diversified habitats from primary rain forests to scrub and savanna woodlands (Crockett and Eisenberg, 1987; Pope, 1992). The systematics of Alouatta is complex due to the phenotypic similarity among many taxa of this genus. The taxa also have extensive chromosomal rearrangements, probably a result of chromosomic speciation (parapatric and stasipatric; Lewis, 1966; White, 1978; Reig, 1980; King, 1993). Within the A. seniculus complex (red howler monkeys), many classifications have been registered. Cabrera (1958) determined four subspecies [A. s. arctoidea (mainly in Venezuela), A. s. sara (Bolivia), A. s. seniculus (in Colombia, Northwestern Venezuela, and Peru) and A. s. stramineus (south of the Orinoco River and between the Negro and Branco rivers, mainly in the Northern Brazilian Amazon)]. Hill (1962) elevated the number of subspecies within A. seniculus to nine (the four previously cited plus A. s. amazonica [in a small area near to the mouth of the Purus River within the Amazon River], A. s. insularis [in the Caribbean island of Trinidad], A. s. juara [in the Juara River, mainly in the Brazilian Amazon], A. s. macconnelli [mainly in the Guiana area] and A. s. puruensis [in the Purus River, mainly in the Brazilian Amazon]). This classification was accepted by Stanyon et al., (1995). However, four of the forms Hill presented are misleading since he considered A. s. macconnelli and A. s. amazonica to be synonymous to A. s. stramineus and A. s. juara and A. s. puruensis to be synonymous of A. s. seniculus. Rylands et

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al., (1995 and 1996/1997) recognized seven of these forms as valid subspecies but included another subspecies as A. s. sp following Bodini and Pérez-Hernández (1987), who claimed an undescribed subspecies in the Venezuelan Llanos, north of the Orinoco River. Two previous subspecies were elevated to the species category by Rylands et al., (1995), A. arctoidea and A. sara. A. sara was elevated to full species because Minezawa et al., (1985) demonstrated that this Bolivian taxon (46 chromosomes) has a very different karyotype from A. seniculus (44 chromosomes) found in Colombia. They proposed that A. sara had a unique X1X1X2X2/X1X2Y sex chromosome pattern. As we will discuss in this work, the unusual chromosome mechanisms to determine sex in the howler monkeys have also been seen in other Neotropical primates such as the Aotus (Ma el al., 1976, 1980; Pieczarka and Nagamachi, 1988) and Cacajao (Dutrillaux el al., 1981). Other mammals including rodents such as Lemnus (Bull and Bulmer, 1981) and insectivores like Sorex araneus (Ford et al., 1957) also can have unusual sexual chromosomes. Stanyon et al., (1995) and Consigliere et al., (1996) used G banding chromosomal analyses to compare the Bolivian animals to the red howlers from Venezuela (A. s. arctoidea). Both Alouatta taxa differed at least in 16 chromosomal rearrangements. However, these last authors found a sex-chromosome pattern in A. sara identical to that found in A. macconnelli and A. s. stramineus as we will explain. A. sara possessed microchromosomes as did A. s. seniculus and other red howler monkeys in contrast to that observed in other Alouatta taxa. The first comprehensive molecular genetics work with Alouatta included the majority of Alouatta taxa and determined A. sara to be a full species (Cortés-Ortiz et al., 2003). Villalobos et al., (2004) also accepted A. sara as a full species. Groves’s seminal book on primate taxonomy only recognized three subspecies (A. s. arctoidea, A. s. juara and A. s. seniculus) with A. macconnelli and A. sara as full species (Groves, 2001). A. macconnelli was elevated to the full species category because Lima and Seúanez (1989) and Lima et al., (1990) showed that this taxon has a X1X1X2X2/X1X2Y1Y2 sex chromosome pattern with the Y chromosome being translocated onto an autosome. These chromosomes are similar to that found by Lima and Seúanez (1989) for A. belzebul, but with differences regarding this taxon in the extra number of nuclear organizing regions (NORs). The karyotype of A. macconnelli has a 2N = 47 to 49 chromosomes with three microchromosomes (Bonvicino et al., 1995). Thirty years earlier, the first karyological study with A. s. seniculus showed 44 chromosomes (Bender and Chu, 1963). Later, Yunis et al., (1976) studied 23 specimens and found a range of 43 to 45 chromosomes and from three to five microchromosomes (four being the most frequent number). There were pericentric inversions in chromosome 13 with an XX/XY pattern. Torres and Leibocini (2001) analyzed another 12 Colombian A. s. seniculus specimens. All of these animals had 44 chromosomes. Additionally, males in this study had a translocation from the Y chromosome to autosomal chromosome 3 (X1X2Y1Y2). These results disagree with those of Yunis et al., (1976). Vassart et al., (1996) studied 42 exemplars from French Guiana and determined that they were very similar (47-49 chromosomes, 1-3 microchromosomes) to those animals classified as A. macconnelli from the Jari River (Brazil) by Lima et al., (1990). One individual even showed 50 chromosomes, with trisomy in chromosome 11. The sex chromosome pattern in A. macconelli was also found in A. s. stramineus (Uatama River, Brazil; Lima and Seuánez, 1991). However, these two taxa showed differences and their karyotypes can be derived from each other by a reciprocal translocation on chromosome 2 (Lima and Seúanez, 1991). Two molecular genetics studies also ratified A. macconnelli as a full species (Cortés-Ortiz et al., 2003; Ruiz-García et al.,

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2007). Groves (2001) considered A. s. amazonica and A. s. puruensis to be the same as A. s. juara and A. s. insularis to be the same as A. macconnelli. Gregorín (2006) accepted A. macconnelli as a full species and elevated A. juara and A. puruensis to full species as well. The author also classified A. nigerrima, a traditional subspecies classified within A. belzebul within the A. seniculus complex (Cabrera, 1958; Hill, 1962; Rylands et al., 1995; Groves, 2001). However, Lima and Seuánez (1991) did not find any relevant chromosomal differences between A. s. seniculus and A. juara (following the Gregorin’s terminology). This variety of classifications should be observed in foundational books about Neotropical primates. Here we list a few examples. For example, Mittermeier et al., (1988) affirmed that almost certainly, within A. seniculus, a number of subspecies were included but they preferred to not list the subspecies until further studies had been conducted. Auricchio (1995) recognized two A. seniculus taxa in Brazil, A. s. seniculus (including within it, A. s. juara and A. s. puruensis) and A. s. stramineus. Dos Reis et al., (2008) recognized four species of red howler monkeys in Brazil: A. juara, A. macconnelli, A. puruensis and A. seniculus. This last one was not recognized by Gregorin (2006) in Brazil. In the book edited by Garber et al., (2009), Rylands and Mittermeier (2009) accepted six full species out of the previously unique species, A. seniculus. They are: A. seniculus, A. arctoidea, A. macconnelli, A. juara, A. puruensis and A. sara. This confusing taxonomic situation regarding red howler monkeys also carries over into Peru. Soini et al., (1989) and Aquino and Encarnación (1994) reported the existence of red howler monkeys through many Peruvian Amazon areas, such as Manu National Park (Southern Peruvian Amazon) as well as in the Pacaya-Samiria National Perk and in the Tamshiyacu-Tahuayo Reserve (Northern Peruvian Amazon). They classified all of these animals as A. s. seniculus. Groves (2001) on the other hand claimed the presence of two red howler monkeys, A. seniculus juara and A. sara, in Peru. Pacheco et al., (2009) accepted the classification of Gregorin (2006) and affirmed that A. juara’s and A. puruensis’s distributions reached the mid and Southeastern section of Peru (Purús area). As they accepted A. juara for Peru, then the presence of A. seniculus should be rejected. Also, they commented on the possible existence of A. sara in Peru. Nevertheless, Aquino et al., (2009) cited A. seniculus (not A. juara) as being in the central area of the Peruvian Amazon (forests of the Contamana Sierra). In 2011 Pacheco and others claimed that A. juara, A. puruensis and A. sara were the three possible red howler monkey species in Peru (MINAM, 2011). However, they doubted the validity of the alleged new species of Alouatta and concluded it better to consider that the unique red howler species in Peru was A. seniculus sensu Hill (1962). For example, Voss and Fleck (2011) claimed, that due to the absence of determinant information, it would be preferable to not use juara as a specific name. Also, they commented that the presence of A. sara in Southern Peru is only assumed because this taxon has been cited in Bolivia very near the Peruvian frontier by Wallace and Rumiz (2010) and Mercado and Wallace (2010). The presence of A. sara is well documented in La Paz Department (Apolobamba and Madidi National Park). Indeed, these authors commented that in the eastern limit of Bolivia (Noel Kempff Mercado National Park), A. puruensis could be present following Rylands and Brandon-Jones (1999). However, MINAM (2011) claimed that since there is no voucher specimen of this taxon from Peru, the presence of A. sara is not sustained for this country. In contrast, Gregory et al., (2012) cited A. sara in the Lower Urubamba Region of Peru. The web page Primate Info Net (2014) (pin.primate.wisc.edu) cited the two following red howler monkey taxa for Peru: A. s. seniculus and A. s. juara.

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Boubli et al., (2008) speculated about the geographical distribution of A. puruensis in Peru and Bolivia and whether it was the form in Northwestern Bolivia and extreme Southeastern Peru in the basins of the upper Purus, Madre de Dios and Tampopata rivers. They also speculated that the Inuya River may be the northern limit to A. puruensis in Peru. Due to this uncertainty, we analyzed the sequences for three mitochondrial genes (cytochrome b, Cyt-b; sub-unity cytochrome oxidase I, COI; sub-unity cytochrome oxidase II, COII) for four red howler monkeys from the southern area of Peru (Madre de Dios and Tambopata rivers) and six exemplars from the northern and middle Peruvian Amazon (Javari, Napo, Ucayali, Tapiche and Pachitea rivers). The mitochondrial genes are interesting markers for phylogenetic tasks because they lack introns and include a rapid accumulation of mutations, rapid coalescence time, a negligible recombination rate, and haploid inheritance (Avise et al., 1987). For all of these reasons, mitochondrial gene trees are more precise in reconstructing the divergence history among species than other molecular markers (Moore, 1995). Supporting this, Cummings et al., (1995) showed that mitochondrial genomes have higher information content per base than nuclear DNA. The Cyt-b gene is commonly used among the molecular markers relevant for phylogeography, biosystematics, and genetic structure studies in mammalian populations (Lavergne et al., 2010; Ruiz-García et al., 2015a). The COI gene has emerged as the standard barcode region for animals, including mammals (www.mammaliabol.org) (Hebert et al., 2003, 2004) and the COII gene has been employed extensively in the phylogenetics of different mammalian groups, including primates (Ashley and Vaughn, 1995; Ruiz-García et al., 2010, 2014). Therefore, the main aims of the current work are to clarify the systematics of the red howler monkeys in Peru, to describe several molecular population genetics statistics (including possible demographic changes) within and among different Alouatta taxa, as well as to obtain insights about the phylogeny of the howler monkeys.

MATERIAL AND METHODS A total of 138 howler monkeys of different taxa were sampled across several countries of Latin-America. Here we provide a breakdown of number and taxonomic type per sampled location. There was one A. caraya (from Santa Ana de Yacumo, Mamore River, Bolivia), three A. pigra (from San Miguel, Peten area in Guatemala) and five A. sara. One of the five was from the Mamore River, Beni Department, Bolivia. The other four individuals, later classified as A. sara, were from Chonta and La Torre, Tambopata River and Taricaya and Madre de Dios River in Peru. There were also 11 A. seniculus (two from Santa Rita, Javari River at the Peruvian side; one from Vencedores del Zapote, Napo River, Peru; one from Puerto Inca, Pachitea River, Peru; two from Requena, Ucayali-Tapiche rivers, Peru; three from the Antioquia Department in Colombia and two from the Valle del Cauca Department in Colombia). Finally, there were 115 A. palliata. Of these, 103 were sampled from across Costa Rica. One was from Los Katios National Park in the Choco Department and two were from the Cordoba Department in Colombia. Other nine were from four different locations within the Pacific area in Ecuador. Three Panamanian individuals belonged to the A. coibensis trabeata taxon.

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DNA were obtained from hair (all samples with the exception of A. palliata from Costa Rica) and blood (the samples of A. palliata from Costa Rica) sampled from living howler monkeys within Central and South America.

Molecular Procedures The DNA from blood was extracted using the phenol-chloroform procedure (Sambrook et al., 1989), while DNA samples from hairs were extracted with 10% Chelex resin (Walsh et al., 1991). The 138 howler monkey individuals sampled were sequenced for three mt genes (COI, COII, and Cyt-b). For the mt COI amplification (polymerase chain reaction, PCR), we used the forward primer LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’), and the reverse primer HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (657 base pairs, bp) (Folmer et al., 1994) under the following PCR profile: 94°C for 5 min, followed by 39 cycles at 94°C for 30 s, 44°C for 45 s, 72°C for 45 s, and a final cycle of 72°C for 5 min. For the amplification of the mt COII gene (located in the lysine and asparagine tRNAs) we used the forward primer L6955 (5’ -AACCATTTCATAACTTTGTCAA-3’) and the reverse primer H7766 (5’ -CTCTTAATCTTTAACTTAAAAG-3’) (720 bp) (Ashley and Vaughn, 1995; Collins and Dubach, 2000; Ruiz-Garcia et al., 2010, 2012a,b). We used the following temperatures: 95° C for 5 min, 35 cycles for 45 s at 95° C, 30 s at 50° C and 30 s at 72° C and a final extension time for 5 min at 72° C. For both genes, the PCRs were performed in a 25 l volume with reaction mixtures including 4 l of 10 x buffer, 6 l of 3 mM MgCl2, 2 l of 5 mM dNTPs, 2l (8 mM) of each primer, 2 units of Taq DNA polymerase, 5 l of ddH2O and 2 l (20–80 ng/l) of DNA. The mt Cyt-b was amplified by PCR using the procedure of Montgelard et al., (1997) (1,140 bp). The conditions for Cyt-b amplification were performed in 25 l reactions including: 2 l of DNA, 2 l of 10 x buffer, 13 l ddH20, 2 l (25 mM) MgCl2, 1 l (10 mM) each of forward and reverse primers, 2l (10 mM) dNTPs, and 2 units of Taq DNA polymerase. The standard thermal cycling program consisted of 10 min at 95°C, 35 cycles of 35 s at 94°C, 35 s at 55°C and 30 s at 70°C and a final extension time for 10 min at 72°C. The total length of the sequences studied for the three genes was 2,517 base pairs (bp). All amplifications, including positive and negative controls, were checked in 2% agarose gels. The gels were visualized in a Hoefer UV Transilluminator. Both mtDNA strands were sequenced directly using BigDye Terminator v3.1 (Applied Biosystems, Inc.). We used a 377A (ABI) automated DNA sequencer. The samples were sequenced in both directions to ensure sequence accuracy.

Data Analysis Molecular Population Analyses Two kinds of procedures were carried out to estimate genetic heterogeneity, and theoretical gene flow estimates, among the diverse Alouatta species detected by the

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phylogenetic analyses. We applied procedures to haplotypic frequencies (GST statistic) and to nucleotide sequences (ST, NST and FST statistics, Hudson et al., 1992). To determine possible differences among the Alouatta species pairs considered, we used the FST statistic (Weir and Hill, 2002). The statistics used to determine the genetic diversity within the diverse Alouatta species considered were: the number of polymorphic sites (S), the haplotypic diversity (Hd), the nucleotide diversity (), the average number of nucleotide differences (k) and the  statistic by sequence. These genetic heterogeneity and gene diversity statistics were undertaken in the DNAsp 5.10 program (Librado and Rozas, 2009). We used two procedure sets to determine possible historical population changes for the different Alouatta species analyzed. First, the mismatch distribution (pairwise sequence differences) was obtained following the method of Rogers and Harpending (1992) and Rogers et al., (1996). We used the raggedness rg statistic (Harpending et al., 1993; Harpending 1994) to determine the similarity between the observed and the theoretical curves. Second, we used the Fu and Li D* and F* tests (Fu and Li, 1993), the Fu FS statistic (Fu, 1997), the Tajima D test (Tajima 1989) and the R2 statistic of Ramos-Onsins and Rozas (2002) to determine possible population size changes in the Alouatta species we analyzed (Simonsen et al., 1995; Ramos-Onsins and Rozas, 2002).

Phylogenetic Analyses The sequence alignments were carried out manually and with the DNA alignment program (Fluxus Technology Ltd.). The Modeltest Software (Posada and Crandall, 1998) and the Mega 6.05 Software (Tamura et al., 2013) were applied to determine the best evolutionary mutation model for the sequences analyzed of the three gene sequences concatenated. Akaike information criterion (AIC; Akaike, 1974) and the Bayesian information criterion (BIC; Schwarz, 1978) were used to determine the best evolutionary nucleotide model. Additionally, we obtained maximum likelihood estimates of transition/transversion bias as well as maximum likelihood estimates of the gamma parameter for site rates for the best evolutionary nucleotide model obtained (Tamura et al., 2013). The phylogenetic trees were obtained using three different procedures for the concatenated genes: Neighbor-joining tree, Maximum likelihood tree, and Bayesian analysis (NJ; Saitou and Nei, 1987; ML; Felsenstein, 1981). The Neighbor-joining tree utilized the Kimura 2P genetic distance (Kimura, 1980). The Maximum likelihood tree used the HKY + G model (Hasegawa-Kishino-Yano, 1985) and a discrete Gamma distribution (+ G) with five rate categories. Both trees were constructed with the PAUP*4.0b8 program (Swofford, 2002) and MEGA 6.05. The Bayesian analysis was performed using a HYK + G model with the gamma distributed rate varying among sites, because it was determined to be the better model using the FindModel program. This Bayesian analysis was completed with the BEAST v. 1.8.1 program (Drummond et al., 2012). Two separate sets of analyses were run, assuming a Yule speciation model and a relaxed molecular clock with an uncorrelated log-normal rate of distribution (Drummond et al., 2006). Results from the three independent runs (30,000,000 generations with the first 3,000,000 discarded, 10%, as burn-in and parameter values sampled every 1000 generations) were combined with LogCombiner v1.8.0 Software (Rambaut and Drummond, 2013a). Posterior probability values provide an assessment of the degree of

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support of each node on the tree. The effective sample size (ESS) for the parameter estimates and convergence were checked using the Tracer Program (version 1.6) (Rambaut et al., 2013). The lower and upper 95% highest posterior densities (HPD) of these parameters as well as the means, geometric means, medians, marginal densities and traces were estimated with the same program. To determine the reality of the values of these parameters, we obtained the autocorrelation tree (ACT) and ESS for parameter estimates. Sampled trees were summarized with TreeAnnotator v1.8.0 (Rambaut and Drummond, 2013b) and visualized in the FigTree Program (version. 1.4) (Rambaut, 2012). This program was run to estimate the time to most recent common ancestor (TMRCA) for the different Alouatta species analyzed. We used two priors. One was around 16.13 ± 2.0 MYA (95% HPD: 12.21-19.42 MYA) for Alouatta and the Atelinae (Ateles, Brachyteles and Lagothrix). The second was around 11.25 ± 2.0 MYA (95% HPD: 7.96-14.54 MYA) for the split between Ateles and Lagothrix (Meireles et al., 1999; Opazo et al., 2006; Perelman et al., 2011; Springer et al., 2012; and Von Dornum and Ruvolo, 1999). We constructed a Median Joining Network (MJ) (Bandelt et al., 1999) using Network 4.2.0.1 Software (Fluxus Technology Ltd) to estimate possible divergence times among the haplotypes found in Alouatta. Additionally, the  statistic (Morral et al., 1994) and its standard deviation (Saillard et al., 2004) were estimated and transformed into years. The  statistic is unbiased and highly independent of past demographic events. We took the mutation rate of the mt COII gene as an average for the three mitochondrial genes used. Ruvolo et al., (1991) determined a mutation rate of 0.85% per million years per lineage for Hominoidea at the mt COII gene. This represents one mutation each 199,402 years. This mutation rate was almost identical to that determined by Ruiz-García and Pinedo-Castro (2010) for Lagothrix at the same gene (one mutation every 191,000 years). Ashley and Vaughn (1995) and Ruiz-García et al., (2011) determined one mutation every 199,000 years for Aotus. Herein we used a rate of one mutation every 195,000 years for Alouatta.

RESULTS Molecular Population Genetics Clearly, the genetic heterogeneity among the different Alouatta taxa studied was very significant (Table 1). This agrees with the fact that they are different species. Similarly, the theoretical gene flow estimates among the taxa were extremely small (Nm = 0.09-0.13), which supports with the existence of different species. The highest degree of genetic heterogeneity was between A. seniculus-A. palliata (FST = 0.938) and between A. seniculus-A. pigra (FST = 0.936). These values were highly significant especially between one cis-Andean taxon and the two trans-Andean taxa (Table 2). The A. seniculus-A. sara pair was also highly significant (FST = 0.641), which indicates that they are different species and both live in Peru. Table 3 lists the gene statistics of the 11 A. seniculus and the five A. sara individuals. Gene diversity was considerably higher for A. sara ( = 0.0322 and k = 23) than for A. seniculus ( = 0.0057 and k = 4.15).

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Table 1. Overall genetic heterogeneity and gene flow (Nm) statistics for different Alouatta species for three concatenated mitochondrial genes. *Significant Probability (P < 0.05) Genetic differentiation estimated  = 411.000 df = 105 HST = 0.0825 KST = 0.7758 KST* = 0.4524 ZS = 3,674.6439 ZS* = 8.0124 Snn = 1.0000

P

Gene flow

0.0000* 0.0000* 0.0000* 0.0000* 0.0000* 0.0000* 0.0000*

γST = 0.7895 NST = 0.8429 FST = 0.8387

Nm = 0.13 Nm = 0.09 Nm = 0.10

Table 2. Population FST (below) and NST (above) pairs among the different Alouatta species studied for three concatenated mitochondrial genes Alouatta species 1 2 3 4 1 = A. palliata; 2 = A. were significant

1 0.9275 0.9387 0.7928 pigra; 3 =

2 0.9293 0.9359 0.7720 A. seniculus;

3 0.9414 0.9388 0.6407 4 = A. sara.

4 0.7992 0.7782 0.6449 All the values

Table 3. Gene diversity statistics for the different Alouatta species studied for three concatenated mitochondrial genes. The statistics estimated were the number of haplotypes (NH), the haplotypic diversity (Hd), the nucleotide diversity (), the average number of nucleotide differences (K) and the  statistic (= 2Ne; Ne = effective female population size;  = mutation rate per generation) by sequence Alouatta taxa A. palliata A. pigra A. seniculus A. sara

NH

Hd



K



22 2 7 5

0.662 ± 0.045 0.667 ± 0.114 0.909 ± 0.066 1.000 ± 0.126

0.0036 ± 0.0011 0.0037 ± 0.0024 0.0058 ± 0.0014 0.0321 ± 0.0093

2.573 ± 1.389 2.667 ± 1.919 4.145 ± 2.232 23.00 ± 12.26

17.78 ± 4.503 2.667 ± 1.919 4.097 ± 1.937 27.36 ± 0.999

We analyzed for possible historical demographic changes in A. seniculus, A. sara and A. palliata and analyzed for possible historical demographic changes (Table 4 and Figure 1). For A. seniculus, neither test nor the mismatch distribution clearly showed any demographic change during the evolutionary history of this species. In the case of A. sara, three of the tests employed showed significant evidence of population expansion (Tajima D test, Fu and Li D* and Fu and Li F*). All the tests we used for A. palliata were statistically significant indicating population expansion. Thus, the two cis-Andean howler species showed less evidence of

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population expansions than the trans-Andean howler species, which showed a very striking population expansion. Table 4. Demographic statistics applied to three different Alouatta species (A. palliata, A. seniculus, A. sara) Tajima D A. palliata

A. seniculus

A. sara

P[D ≤ 2.789] = 0.001** P[D ≤ 0.051] = 0.553 P[D ≤ 1.205] = 0.026*

Fu and Li D* P[D* ≤ 8.811] = 0.001** P[D* ≤ 0.071] = 0.426 P[D* ≤ 1.432] = 0.038*

Fu and Li F* P[F* ≤ 7.438] = 0.001** P[F* ≤ 0.122] = 0.444 P[F* ≤ 1.306] = 0.036*

Fu’s Fs P[Fs ≤ 8.928] = 0.004** P[Fs ≤ 0.815] = 0.327 P[Fs ≤ 0.683] = 0.386

raggedness rg

R2

P[rg ≤ 0.0527] = 0.296

P[R2 ≤ 0.038] = 0.039* P[R2 ≤ 0.146] = 0.332 P[R2 ≤ 0.218] = 0.521

P[rg ≤ 0.103] = 0.460 P[rg ≤ 0.140] = 0.229

* P < 0.05; ** P < 0.01, significant population expansions

Phylogenetic Inferences The BIC and the Akaike information criterion showed that HKY + G (9,749.833 and 6,902.338, respectively) was the nucleotide substitution model which best fit with the three concatenated genes. The maximum likelihood estimate of transition/transversion bias was 2.07 (maximum log likelihood was -3,160.23). The Kimura 2P genetic distances (Table 5) showed values ranging from around 6% to 9% among the different Alouatta taxa studied. These are typical values of different species (Ascunce et al., 2003).

(A)

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(B)

(C) Figure 1. Mismatch distributions (pairwise sequence differences) at the three concatenated mitochondrial DNA genes (COI, COII and Cyt-b) for the different Alouatta taxa considered: A. palliata (A), A. seniculus (B) and A. sara (C).

Table 5. Kimura (1980) 2P genetic distances among the different Alouatta taxa considered. Below, genetic distance values in percentages (%); above, standard errors in percentages (%) Alouatta 1 2 3 4 5 6 pecies 1 0.8 1.1 1.0 1.2 1.7 2 5.3 1.3 1.0 1.2 1.9 3 8.4 8.0 0.8 0.9 1.6 4 9.3 8.4 5.5 1.7 1.6 5 8.0 7.2 5.5 7.6 1.5 6 15.1 16.0 14.3 16.4 13.6 1= A. palliata; 2 = A. pigra; 3 = A. seniculus; 4 = A. sara; 5 = A. caraya; 6 = Lagothrix lagotricha cana

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Figure 2. (Continued)

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Figure 2. (Continued).

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Figure 2. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 138 howler monkeys (Alouatta) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are bootstrap percentages.

The Kimura 2P genetic NJ tree clearly showed two main groups of howler monkeys for the three mitochondrial genes (Figure 2). One main clade consisted of three cis-Andean howler monkeys taxa (A. caraya, A. seniculus and A. sara). The other main clade contained the two trans-Andean howler taxa we analyzed (A. pigra and A. palliata). The splits of these five Alouatta taxa were correlated with high bootstrap percentages. The four Peruvian howler monkeys sampled in the Madre de Dios River Basin belonged to A. sara. On the other hand, the other six Peruvian howler monkey sampled at the Javari River (frontier with Brazil), at the Napo River, at the Ucayali-Tapiche rivers and at the Pachitea River were closely related to

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the howler monkeys sampled in different areas of Colombia, which traditionally have been designated as A. s. seniculus. Therefore, the molecular data detected at least two different Alouatta species in the Peruvian Amazon: one (A. seniculus) in the northern and middle Peruvian Amazon (from the Napo River until the middle Ucayali River), and another (A. sara) in the Southern Peruvian Amazon (Madre de Dios River basin). This tree also showed that no differentiated subspecies within A. palliata were detected in Costa Rica, Panama, Colombia or Ecuador (A. palliata palliata and A. p. aequatorialis).

Figure 3. (Continued).

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Figure 3. Maximum likelihood tree with the 138 howler monkeys (Alouatta) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are bootstrap percentages.

The ML tree (Figure 3) showed the same phylogenetic relationships as the previous tree with one exception: A. caraya was the most differentiated of the Alouatta taxa employed in this study.

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Figure 4. (Continued).

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Figure 4. Bayesian tree with the 138 howler monkeys (Alouatta) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are posterior probabilities higher than 0.5, the median temporal split and the interval of 95% High Posterior Density (HPD).

The Bayesian tree (BI tree; Figure 4) showed a split of the ancestor of Alouatta from the other Atelidae around 15.93 MYA (95% HPD: 14.93-16.85 MYA; P = 1). The split of the ancestors of Lagothrix and Ateles occurred around 11.18 MYA (95% HPD: 10.3-12.23 MYA; P = 1). The diversification within Alouatta began around 7.21 MYA (95% HPD: 6.20-8.01 MYA; P = 1), with the ancestor of A. seniculus being the first to diverge. Within A. seniculus, the mitochondrial diversification began around 3.94 MYA (95% HPD: 1.93-5.86 MYA; P = 1). The next ancestral branch to diverge within the remaining Alouatta taxa was the ancestor of A. sara around 6.61 MYA (P = 0.28). The mitochondrial diversification in A. sara began around 2.27 MYA (95% HPD: 1.2-4.72 MYA; P = 1). The split of the ancestors of the two trans-Andean howler monkey species (A. pigra and A. palliata) was around 6.06 MYA (P = 0.47) with the diversification within A. pigra around 2.12 MYA (95% HPD: 1.7-3.89 MYA; P = 1) and within A. palliata around 4.58 MYA (95% HPD: 4.04-7.21 MYA; P = 0.82). Remarkably different to that obtained in the two previous trees, A. caraya was more related to A. pigra than to the other cis-Andean howler monkey species. The mitochondrial diversification within Alouatta obtained with this procedure clearly overestimates the temporal splits obtained by Cortes-Ortiz et al., (2013). Nevertheless, in an identical fashion to the other two trees, six Peruvian howler monkeys were clearly identified as A. seniculus and the four animals from the Madre de Dios River basin were clearly related to A. sara. This ratifies the existence, at least, of two different species of Alouatta in the Peruvian Amazon.

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Figure 5. Median Joining Network (MJN) with haplotypes found at the three concatenated mitochondrial genes (COI, COII and Cyt-b) gene for the 138 howler monkeys (Alouatta) analyzed. Black circles = Lagothrix lagotricha cana; pink circle = A. caraya; blue circles = A. seniculus; green circles = A. sara; yellow circles = A. pigra; brown circles = A. palliata. Red circles indicate missing intermediate haplotypes.

The Median Joining Network (MJN) procedure (Figure 5) clearly differentiated the haplotypes of Lagothrix and Alouatta with an estimated temporal split around 18.33 ± 1.69 MYA ( = 94 ± 8.72), which agrees quite well with the temporal splits estimated by many authors between Alouatta and Atelinae (Meireles et al., 1999; Opazo et al., 2006; Perelman et al., 2011; Springer et al., 2012; Von Dornum and Ruvolo, 1999). Of all the haplotypes, A. caraya was the one most related to the original mitochondrial diversification of this genus. This is also supported by the ML tree. The temporal split between this more ancestral cisAndean howler monkey haplotype and the first differentiated trans-Andean howler monkey haplotype (one of A. pigra) was around 6.73 ± 0.35 MYA ( = 34.5 ± 1.80). This agrees with the temporal split between both Alouatta groups estimated by Cortes-Ortiz et al., (2003). The temporal splits between A. caraya-A. seniculus and A. caraya-A. sara were around 3.41 ± 0.28 MYA ( = 17.5 ± 1.41) and 3.99 ± 0. 28 MYA ( = 20.5 ± 1.41), respectively. Clearly, this analysis also differentiated the haplotypes of A. seniculus and A. sara with a time division of around 4.51 ± 0.84 MYA ( = 23.12 ± 4.32). The temporal splits between A. seniculus and A. sara with reference to the two trans-Andean species (A. pigra and A. palliata) were 4.88 ± 0.35 MYA (A. seniculus-A. pigra:  = 25 ± 1.80), 5.36 ± 0.41 MYA (A. seniculus-A. palliata:  = 27.5 ± 2.12), 4.29 ± 0.35 MYA (A. sara-A. pigra:  = 22 ± 1.80) and 4.78 ± 0.41 MYA (A. sara-A. palliata:  = 24.5 ± 2.12), respectively. The mitochondrial diversification within A. sara (around 2.22 ± 0.29 MYA;  = 11.4 ± 1.54) was older than in A. seniculus (around 0.86 ± 0.30 MYA;  = 4.29 ± 1.53) for this procedure. This disagrees with that obtained for the BI tree, which showed an older mitochondrial diversification for A. seniculus than for A. sara. On the other hand, the ancestor of A. pigra seems to be older than that of A. palliata. The time split between both trans-Andean Alouatta species was around 3.12 ± 0.41 MYA

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( = 16 ± 2.12) and the mitochondrial diversification within A. palliata was around 1.29 ± 0.23 MYA ( = 6.59 ± 1.18).

DISCUSSION How Many Taxa of Red Howler Monkeys are in Peru? Clearly our study reveals a striking and significant genetic heterogeneity among the five “a priori” Alouatta taxa we analyzed, which is related to the relevant and noteworthy karyological differences found by many studies for this Neotropical primate genus. We begin this section with a discussion about the number of red howler monkey species in Peru. It’s clear that at least two different species of red howler monkeys are present in Peru: A. seniculus and A. sara. The main subspecies of A. seniculus is A. s. seniculus, but other subspecies could be conserved within this species, distributed in the Northern Peruvian Amazon (Napo, Javari, Tapiche and lower Ucayali rivers) and also in the middle Peruvian Amazon (Pachitea River). A. sara was previously referred to as A. s. sara in the Southern Peruvian Amazon along the Tambopata and Madre de Dios rivers. This is the first study which unquestionably shows the existence of A. sara in Peru. Our results also show indisputable arguments in favor of A. sara as a different species from A. seniculus, coinciding with Cortés-Ortiz et al., (2003), using several different mitochondrial gene sequences. It’s interesting to note that mitochondrial genes are extremely more useful to discriminate different Alouatta species (and also in many other organisms) than nuclear gene sequences. For instance, Cortés-Ortiz et al., (2003) showed that neither RAG1 nor CAL (nuclear genes) data were capable of resolving the phylogenetic relationships among different Alouatta species. These nuclear genes could only discriminate between Alouatta and Ateles species. The same was found for other Neotropical primate phylogenetic studies (Collins and Dubach, 2001; Cropp and Boinski, 2000). Minezawa et al., (1985) showed 2N = 50 for females (with X1X1X2X2) and 2N = 49 for males (X1X2Y), with a range of 2-6 microchromosomes (six microchromosomes were more frequent) for A. sara. Later, Stanyon et al., (1995) determined 2N = 50 for both females and males (with X1X1X2X2 and X1X2Y1Y2) and 28 acrocentric chromosomes. Clearly, A. sara is chromosomically differentiated from A. seniculus in Colombia (2N = 44 also with X1X1X2X2 and X1X2Y1Y2, 26 acrocentric chromosomes with 3-4 microchromosomes following Torres and Leibocini, 2001, contrasting with the erroneously XX and XY system determined by Yunis et al., 1976). Consinglieri et al., (1998) showed numerous interchromosomal rearrangements in Alouatta. They demonstrated that A. belzebul had six unique apomorphic associations and that A. sara and A. seniculus arctoidea shared seven derived associations. Additionally A. sara had four apomorphic associations and A. seniculus arctoidea had seven. Indeed, Consiglieri et al. (1996) showed, at least, 16 chromosome rearrangements including numerous Robertsonian and tandem translocations and intra-chromosomal rearrangements between A. sara and A. s. arctoidea (same karyological characteristics as A. s. seniculus; Consiglieri et al., 1996; Torres and Ramírez, 2003). These karyotypic differences can in fact be considered high enough to ensure reproductive isolation of A. sara from other red howler monkeys (Stanyon et al., 1995; Consigliere et al., 1996).

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Within A. s. seniculus, there was also internal polymorphism. Torres and Leibocini (2001) showed that chromosomes 1, 2, 3, 6 and X showed heteromorphism with variability in size and placement of the constitutive heterochromatin. In chromosome 1, there was a polymorphism not having centromeric heterochromatin, which was presented as a heterozygote condition in four of the eleven animals. Several hypotheses should be proposed to explain the split between A. sara and A. seniculus. Minezawa et al., (1985) claimed this to be the result of A. sara being peripherally isolated in the Yungus 2 refugia. Brown (1982) proposed this refugia on the basis of butterfly endemism data presented in the context of the general forest refugia model (Haffer, 1969, 1982, 1997, 2008). However, Cortés-Ortiz et al., (2003) estimated a late Pliocene divergence between A. seniculus and A. sara (2.4 MYA). For this reason, Cortés-Ortiz et al., (2003) argued against this split because it was too old to be explained by the Pleistocene refugia hypothesis. However, we must not forget that Haffer (1997, 2008) explained that the dry-wet cycles were not exclusive to the Pleistocene and therefore these cycles should also be important during the Pliocene. Cortés-Ortiz et al., (2003) speculated that the appearance of the Madeira River could be essential to the split between these two red howler monkey species, because the river forms the eastern boundary of A. seniculus and A. sara. We estimated a temporal split somewhat higher than Cortés-Ortiz et al., (2003) (4.5 MYA with the MNJ procedure). This age agrees quite well with a very important geological change in the Amazon that had impacts on the rivers of the region. Espurt et al., (2007, 2010) demonstrated that the Nazca Ridge subduction imprint had a significant influence on the eastern side of the Andes by means of the Fitzcarrald Arch. This uplift is responsible for the atypical three-dimensional shape of the Amazon’s foreland basin. Related with the Nazca Ridge subduction, arc volcanism in the Peruvian Andes ceased around 4 MYA (Rosenbaum et al., 2005), thus the older time estimate of the Fitzcarrald Arch uplift is around 4 MYA (Pliocene). This could explain changes in the relationship of the Mamore-Beni Rivers (or Beni Lake) with other drainage river systems, which divided A. sara and A. seniculus. This event could also isolate the Bolivian dolphins from other Amazonian pink river dolphins (Ruiz-García et al., 2016). Another question is which of the two taxa could be first. The levels of gene diversity were considerably higher in A. sara than in A. seniculus (3.22% vs. 0.57%), although the geographical range extension of the second is considerably higher than that of the first. Dobzhansky (1971) showed that original populations have the greatest levels of gene diversity. However, if there were different molecular lineages of A. sara (from other Alouatta taxa), they should artificially increase the gene diversity of A. sara. The MNJ procedure also showed an older mitochondrial diversification in A. sara (around 2.22 MYA) than in A. seniculus (0.86 MYA), which also coincides with the ancestors of A. sara as older than the ancestors of A. seniculus. Some karyotypic results also agree with this possibility. Consiglieri et al., (1998), starting from the hypothetical ancestral karyotype, estimated the number of derived associations for each of three howler monkeys taxa. There were six for A. belzebul, 11 for A. sara, and 14 for A. seniculus arctoidea. It is clear then from the hybridization data alone that the two red howler taxa have more derived karyotypes than A. belzebul and that A. s. arctoidea is more derived than A. sara. Therefore, A. sara is more primitive than one A. seniculus subspecies. The phylogenetic tree presented by Villalobos et al., (2004) also yielded the ancestors of A. sara as more ancestral than that of A. seniculus. In contrast, other proofs agree better with A. seniculus as an older taxon than A. sara. Our BI tree showed that the

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ancestors of A. seniculus diverged before the ancestors of A. sara (thus both MNJ and BI procedures did not agree in this aspect). Generally, the most ancestral populations show less evidences of population expansions than the most modern populations. No tests, for A. seniculus, showed population expansions, whilst three tests showed population expansions in A. sara, which could be in agreement with a young origin for this last taxon. Although Bovicino et al., (2001), did not enclose sequences of A. sara, they showed neighbor joining and parsimony trees with mt Cyt-b gene sequences. They grouped A. belzebul and A. fusca as sister branches in one clade. In a separate clade, A. seniculus was the most basal offshoot, followed by A. nigerrima as a sister lineage of the most derived A. macconnelli and A. stramineus. The grouping of A. belzebul and A. fusca as sister branches was also reported with γ1-globin pseudogene sequence data, with A. caraya as the most basal offshoot followed by A. seniculus and, subsequently, by the most derived A. belzebul/A. fusca clade (Meireles et al., 1999). Thus, this question required further analyses. What is clear is that the red howler monkeys are more related among themselves than with other Alouatta taxa. A. sara, A. seniculus artoidea (Consigliere et al., 1996, 1998; Minezawa et al., 1985), A. seniculus seniculus (Yunis et al., 1976; Torres and Levobivi, 2001), A. macconelli (Lima et al., 1990; Vassart et al., 1996) and A. stramineus (Lima and Seuánez, 1991) all showed microchromosomes (or B-chromosomes). However, microchromosomes have not been reported for A. palliata (Ma et al., 1975; Torres and Ramírez, 2003), A. caraya (Egozcue and Egozcue, 1966; Mudry et al., 1994, 1998), A. belzebul belzebul (Armada et al., 1987), A. nigerrima (Armada et al., 1987) and A. fusca (Koiffman, 1982; Koiffman and Saldanha, 1974; De Oliveira, 1996; De Oliveira et al., 1995, 1998, 2000, 2002). Following the typological nomenclature of Gregorín (1996, 2006) and based on geographical distribution, the animals sampled at the Javari and Ucayali rivers should be A. juara. Furthermore, the animal from the Pachitea River (middle Ucayali River) should be A. puruensis. Nevertheless, they were clearly related (with very small genetic distances) with the A. s. seniculus individuals from Colombia. Therefore, our molecular results don’t agree with A. juara and A. puruensis as full species. They are taxa within A. seniculus. Only, karyological analyses could show if these taxa are really different from A. seniculus because from a molecular point of view they were undifferentiated from A. s. seniculus. The morphological characteristics used by Gregorin (1996, 2006) are probably not good characters for reconstructing evolutionary histories. Their genetic basis are unknown and positive natural selection could affect them. Thus, everything seems to indicate that there are two different species of red howler monkeys, A. seniculus and A. sara, in the Peruvian Amazon.

Molecular Phylogenetic Insights into the Systematics of Alouatta Our BI split estimate for the initial diversification of Alouatta was a litter higher to that obtained by Cortés-Ortiz et al., (2003). Our estimate was around 7.21 MYA, whilst the estimate of Cortés-Ortiz et al., (2003) was 6.8 MYA. Nevertheless, our MJN estimate was 6.73 MYA, which is nearly identical to that estimated by the quoted authors. This temporal split coincides with the formation of the Northern Andes (Lundberg et al., 1998). Similarly, our BI temporal estimate for the diversification within the trans-Andean (Central American;

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A. palliata and A. pigra) howler monkeys was higher (6.06 MYA) than the 3 MYA estimate of Cortés-Ortiz et al., (2003) and our own MNJ estimate of 3.12 MYA. It’s important to note the similarity of these last two estimates. In this work, as well as in previous studies we carried out regarding Neotropical mammalian species the MNJ temporal splits are systematically lower than those obtained by BI trees (Ruiz- García et al., 2010, 2012a,b, 2014, 2015b, 2016; Ruiz-García and Pinedo, 2010). We suspect that the MJN procedure (borrowed molecular clock) produces more realistic temporal splits than do the BI estimations (fossil-calibrated DNA phylogeny). This is especially true when the mutation rates are well estimated and substantiated as those in the current work. Another advantage of the MJN procedures compared to traditional trees is that they explicitly allow for the co-existence of ancestral and descendant haplotypes, whereas traditional trees treat all sequences as terminal taxa (Posada and Crandall, 2001). Use of the MJN procedures allows us to observe which current taxa began to evolve first and also to identify the more recently derived taxa. From our MJN, it seems clear that the South American (cis-Andean) howler monkeys began their mitochondrial evolution much earlier than the trans-Andean howler monkeys. This conclusion is quite logical. The clear population expansion detected for A. palliata (for all the statistics employed) revealed that this species has a younger population than the two cis-Andean species (A. seniculus and A. sara). Recall, there was scant evidence of any population expansions in the latter two species. Furthermore, haplotypes of A. pigra, derived from cis-Andean haplotypes generated the haplotypes of A. palliata. Indeed, the traditional morphological subspecies, A. p. palliata and A. p. aequatorialis, were not molecularly differentiable throughout Costa Rica, Colombia and Ecuador. Therefore, although Cortés-Ortiz et al., (2003) conjectured that the minor phylogeographic break separating northern and southern A. palliata could be located near Panama’s Sona Peninsula (Bermingham and Martin, 1998), we didn’t find any geographical point break. Even the three Panamanian exemplars sampled (A. coibensis trabeata) could not be molecularly differentiated from A. palliata. Our own MJN estimate and that of Cortés-Ortiz et al., (2003) (both around 3 MYA) for the differentiation of trans-Andean howler monkeys agree quite well with the completion date of the Panama Isthmus. This stresses the importance of the Panama Isthmus in the speciation of Central American howler monkeys. The data of Cortés-Ortiz et al., (2003) correlated very well with our affirmation that the molecular evolution of the cis-Andean howler monkeys preceded the evolution of the trans-Andean. These authors estimated that the diversification of the South American howler monkey species occurred around 4.8–5.1 MYA, near the end of the Miocene. They also estimated that the temporal split between A. belzebul and A. guariba occurred approximately 3.9–4.0 MYA. This is opposite to that claimed by Villalobos et al., (2004). These authors claimed that their results demonstrated that A. palliata was the most basal taxon for the genus and the sister taxon to all other Alouatta species. Furthermore, they specified that this agreed with previously reported topologies (Meireles et al., 1999; Bonvicino et al., 2001). However, none of these cited works included sequences of A. palliata and therefore their conclusions are misleading. To support their hypothesis, they claimed that A. palliata had been described as having an XY system for males and XX for females, a claim based on a population studied on the Barro Colorado Island in Panama (Ma et al., 1975). This is the original sexual chromosome system in the vast majority of mammals and primates, but the sexual chromosome system is more complex in many Alouatta taxa. Therefore, any “normal” XX/XY system could be considered

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the original one. The findings of Torres and Ramírez (2003) did show some Colombian A. palliata to have the highest diploid number for the genus (2N = 56 and XX /XY sex chromosome system). However the findings of Ma et al., (1975) indicated a different sex chromosome system for the Panamanian animals. The system is not the XX/XY one claimed by Villalobos et al., (2004). Instead, females have 2N = 54 and X1X1X2X2 and males have 2N = 53 and X1X2Y (30 acrocentric chromosomes versus 34 acrocentric chromosomes in the Colombian animals). The karyotype comparisons of the Colombian and Panamanian individuals showed that the A4 pair of the Panamanian individuals was replaced in the Colombian individuals by two acrocentric pairs of different sizes (B15 and B24; the second pair is the result of a Robertsonian fussion of pairs 15 and 24). Additionally, the B19 pair of the Colombian male did not show the heteromorphism observed in the Panamanian males. The remaining chromosomes were similar. However, the molecular results showed that no important differences were found among Panamanian and the Colombian individuals. Our molecular results, as well as those of Cortés-Ortiz et al., (2003), don’t validate A. coibensis trabeata as a separate species from the formerly known A. palliata panamensis, as stipulated by Froehlich and Froehlich (1987) who analyzed dermal ridge patterns in hands and feet. Our results, as well as that of Cortés-Ortiz et al., (2003) clearly differentiated A. pigra from A. palliata. This aligns with Smith’s earlier work (1970). Cortés-Ortiz et al., (2003) detected a 5.7% difference between A. palliata and A. pigra. Similarly, our calculated percent was 5.3. Our molecular results also permit some evaluation of the two hypotheses presented by Smith (1970) to account for the origins of A. palliata and A. pigra. Smith’s first model posits a single colonization of Central America from South America. This is followed by the separation of A. palliata in the Talamancan region of Costa Rica from A. pigra in the north. His second hypothesis recognizes two sequential invasions of Central America by the transAndean Alouatta ancestor in South America. Our results more strongly agree with the first than with the second hypothesis. The explanations of Cortés-Ortiz et al., (2003) are highly plausible: 1- Both of these trans-Andean howler monkey species would have been separated by forest reduction in Central America during the last Pliocene-Pleistocene dry-wet glacial periods (Haffer, 1969, 1982, 1997, 2008). They would have had isolated populations persisting in the highlands of Guatemala, Mexico, and Belize and in Costa Rica’s Talamanca Mountains. 2- Also, parapatric speciation could be important here across the ecotone that separates the drier and lower forest of the Mexican Yucatan peninsula occupied by A. pigra, from the moist, tall forests inhabited by A. palliata. The A. pigra-A. palliata contact zone is placed between the Grijalva and Usumacinta rivers, with the possibility that either or both of these rivers might have acted as barriers to gene flow between the two howler monkey species. However, Cortés-Ortiz et al., (2007), in Tabasco (Mexico), reported hybridization of individuals with a mosaic of morphological characteristics between A. palliata and A. pigra. These included individuals living in various grades of disturbed vegetation and that had characteristics of both species. However, this seems to be an exception rather than the rule. Other cases of hybridization for different Alouatta taxa were those reported by Aguiar et al., (2007), who described hybridization between A. caraya and A. fusca clamitans in a group of eight individuals observed near the Paraná River in Brazil, in the ecotone between rain forest and the Cerrado, showing intermediate morphological variation. Also, hybridization has been documented to occur in captivity between A. caraya and A. fusca (De Souza et al., 2010). Nevertheless, the chromosome complement of Alouatta fusca damitans showed a wide variation in the diploid number, with 2N = 45, 46 and 52 (De Oliveira et al., 2000) and the

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diploid number for A. caraya is 2N = 52 (Mudry et al., 1984, 1998). Thus, perhaps animals with 52 chromosomes should hybridize without a problem. Indeed, Zúñiga-Leal and Defler (2013) confirmed sympatry between A. seniculus and A. palliata along the west bank of the Atrato River (Chocó Department in Colombia) but they did not find hybridization between the two species. According to local information, sympatry between the two species continues to an undetermined point up the Atrato River, but no hybrids have been discovered between these two taxa. This could be related with the fact of the important chromosome differences between the two species (Torres and Leibocini, 2001; Torres and Ramírez, 2003). Molecularly and karyotypically speaking, there is clear evidence that A. macconnelli is a full species. From a molecular perspective, Cortés-Ortiz et al., (2003) showed that this taxon, A. sara and A. seniculus formed monophyletic groups (with 100% of bootstrap) and had a level of gene divergence among them similar to that observed between A. palliata and A. pigra. Ruiz-García et al., (2007) also showed that the dynamics of DNA microsatellites were different between A. s. seniculus and A. macconnelli. Although, we don’t enclose mitochondrial sequences of A. macconnelli in this study, in a parallel study, we also show the differentiation of both red howler taxa. From a chromosomal perspective, A. macconnelli was recognized as a full species by Lima and Seuánez (1991) and Vassart et al., (1996). Also, De Oliveira et al., (2002) revealed that A. macconnelli and A. s. arctoidea differ by multiple translocations which may further contribute to reproductive isolation between A. macconnelli and A. seniculus. However, the degree of differentiation between A. macconnelli and A. stramineus is not very clear. Bonvicino et al., (1995) proposed that A. stramineus (west Trombetas River) and A. macconnelli (east Trombetas River) should be considered as separate species basing their results in the analysis of quantitative cranial traits. Armada et al., (1987) and Lima and Seuánez (1991) showed that A. macconnelli and A. stramineus, although sharing the same diploid number (47, 48 or 49) and sex chromosome system (X1X2Y1Y2/X1X1X2X2), differed in two chromosome pairs. It was a result of a translocation event (reciprocal homozygote translocation between chromosomes 2 and 20). This is why genetic introgression between them was considered unlikely and they were considered valid species by Bonvicino et al., (1995). But other authors have revealed minor differences between the two red howler taxa. Figueiredo et al., (1998), using the mitochondrial COII gene (one of the genes that we analyzed here), presented a strong genetic homogeneity among the three populations studied. There was a low number of nucleotide differences, phylogenetic continuity and the complete absence of any geographic partitioning of the sequences. Sampaio et al., (1996) using biochemical data (20 protein loci; genetic divergence of 0 to 0.2%) and cytogenetics showed that A. macconelli and A. stramineus were related closely enough to be considered a single species. Bonvicino et al., (2001) used karyotypic data to confirm the molecular topology of the tree that they obtained from the mitochondrial Cyt-b gene. Their finding showed a closer relationship between A. macconnelli and A. stramineus than either to A. nigerrima. They showed 2N = 50 in the female, with nine pairs of biarmed autosomes against 11 pairs in A. macconnelli and A. stramineus. Indeed, very low genetic distance values (average 0.3%; around a divergence of 300,000 years) were observed between A. macconnelli and A. stramineus. These values were lower than divergence estimates within other species such as A. caraya (temporal diverge between haplotypes of this species is around 0.6 MYA) or A. belzebul (temporal diverge between haplotypes of this species is around 0.8 MYA). Other studies of G-banding data from A.

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macconnelli individuals collected from the Uatuma and Jari rivers revealed two distinct karyotypes due to two independent Robertsonian translocations or a centric fission-fusion event (De Oliveira et al., 2002; Lima and Seuánez, 1991). The Uatuma River karyotype showed chromosome forms 2p/2q and 16 and the Jari karyotype 2q and 2p/16. For these authors, however, it is unclear whether one chromosomal difference has the potential to act as a profound post-mating isolating mechanism. Therefore, it is highly probably that both of these red howler forms belong to the same species that contains two subspecies: A. stramineus stramineus and A. stramineus macconnelli. However, these red howler monkeys should be studied in more detail to determine the real status of these forms. The most external cis-Andean howler monkey that we found was A. caraya, with a temporal split of around 3.4-4 MYA with reference to the two red howler taxa we studied. Bonvicino et al., (2001) estimated this divergence to have occurred around 4.2 MYA. Another estimate by Cortés-Ortiz et al., (2003) of 4.0 MYA is almost the same. Therefore, there is clear similarity among these three estimates for the split between A. caraya and the red howler monkey species. Also, the three works showed A. caraya as the sister clade of the red howler monkeys. However, other authors claimed a different relationship status for A. caraya. Hershkovitz (1949) divided the five species that were accepted at that time into three different Alouatta groups, based on morphological studies of the hyoid bone. The three groups are: Alouatta seniculus (A. seniculus, A. belzebul and A. fusca); A. palliata (A. palliata; two further species, A. pigra and A. coibensis, were recognized and included in the A. palliata group by Mittermeier et al., 1981); and A. caraya (A. caraya). Mittermeier et al., (1981) and Gregorín (1996) placed A. palliata as the most basal genus. We previously discussed the uncertainty of this claim. This genus was followed by A. caraya and next the clade formed by A. fusca and the Amazonian species, A. seniculus and A. belzebul. Also, Meireles et al., (1999) used globin pseudogene phylogeny to determine A. caraya as a monotypic clade separated from the rest of the South American Alouatta species. Other authors, however, showed that the position of several Alouatta taxa did not correspond with Hershkovitz’s (1949) scheme. For instance, De Oliveira (1996) used cytogenetical data to propose the placement of A. belzebul nigerrima within the A. seniculus group. Sampaio et al., (1996), using biochemical and chromosomal data, found that A. seniculus and A. belzebul were not the closest species to the A. seniculus group. Their compiled chromosomal data supported A. belzebul and A. fusca to have the closest relationship. De Oliveira et al., (2002) used chromosomal data to propose another classification. Alouatta can be divided into two distinct species groups, the first with A. caraya and A. belzebul and the second with A. macconnelli, A. sara, A. seniculus arctoidea and A. fusca. This classification agrees with the fact that eight out of 28 ancestral Neotropical primate chromosomes are conserved in A. macconnelli, 11 in A. fusca clamitans, 14 in A. fusca fusca and 15 in A. caraya. Thus, taking this data into account, A. macconnelli was the most derived taxa and A. caraya was the most ancestral one. In addition, De Oliveira et al., (2002) observed high level of species-specific (autapomorphic) rearrangements, reflecting the extensive karyological variation within this genus. The number varies between two in A. caraya to 10 in A. macconnelli. The variation in number should stress A. macconnelli as the most derived taxa and A. caraya as the most ancestral one. This coincides with Mudry et al., (1998), who showed a very stable karyotype for A. caraya (data from 25 males and 22 females from different geographical origins; Mudry et al., 1994). Thus, while chromosomal speciation mechanisms should be very important in

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the red howler monkeys, there is no evidence that these mechanisms are present in A. caraya. But, as we mentioned, the three mitochondrial genes support A. caraya as the sister clade of the red howler monkey clade. Bonvicini et al., (2001) estimated the temporal split between the clades to have occurred around 4.7 MYA whereas Cortés-Ortiz et al., (2003) estimated the split to have occurred a little earlier (5.1 MYA). Cortés-Ortiz et al., (2003) explained that the current distribution of A. caraya is not a result of the presence any clear geographical barriers. It is parapatric with A. sara to the west, with A. nigerrima and A. belzebul to the north, and with A. fusca to the east. It is true however that the Goia refugia (Brown, 1982, for butterflies) in southeast Matto Grosso (Brazil) may help to explain the distribution of A. caraya. However, Cortés-Ortiz et al., (2003) discarded this idea because their estimated temporal split of A. caraya from the red howler clade (4 MYA) suggested a Pliocene origin for this species and not a Pleistocene refugia model of speciation. Nevertheless, Cortés-Ortiz et al., (2003) forget that Haffer (1997, 2008) expanded the dry-wet cycles also to the Pliocene. Additionally, Espurt et al., (2007, 2010) demonstrated that the Nazca Ridge subduction imprint had a significant influence by means of the Fitzcarrald Arch uplift around 4 MYA (Pliocene). This could have been decisive in the origination of the Madeira River, a separation barrier to the ancestor of A. caraya. Bonvicino et al., (2001) commented that the X1X2Y1Y2/X1X1X2X2 sex chromosomal system found in A. sara and A. s. arctoidea consists of shared chromosomes that are to identical Y1, Y2, and X2 chromosomes (Stanyon et al. 1995; Consiglieri et al., 1998). They did not know whether they were the same as the Y1, Y2, and X2 of A. macconnelli/A. stramineus. They concluded that if this were the case, the rearrangements that originated this sex chromosome system must have occurred in the common ancestor of the red howler species. Alternatively, these rearrangements could have occurred independently (and involving different autosomes) in two separate ancestors, one in A. macconnelli/A. stramineus and another in A. sara and A. s. arctoidea. Nevertheless, De Oliveira et al., (2002) showed that in all A. sara, A. seniculus arctoidea, A. macconnelli and A. caraya, the autosome involved in this rearrangement corresponded to a 3c/15b association. However, one further rearrangement involving the human chromosome 1b homologous segment was observed in A. fusca. These results agree quite well with the fact that A. caraya is more closely related to the red howler monkey species than A. fusca should be. This is also highly correlated with the observation made by Cortés-Ortiz et al., (2003) but it disagrees with the tree proposed by De Oliveira et al., (2002). As we did not include sequences of A. belzebul or A. fusca/A. clamitans in this study, we cannot clarify which is the oldest ancestor of the current cis-Andean howler monkey taxa. De Oliveira et al., (2002) concluded that the ancestral sex chromosome system for the Alouatta genus should consist of X1X2/Y1Y2 chromosomes, where X1 would correspond to the homolog in humans (X, X2 to 3c/15b, Y1 to Y/15b, and Y2 to 3c). Alouatta shows more common fragile sites in their chromosomes than do other Neotropical primates. Fundia et al., (2000), for instance, showed that Saimiri boliviensis presented a low level of spontaneous breakage (six spontaneous events), whereas A. caraya (39 spontaneous events) showed elevated frequencies with 1q23, 2q13 and 11q19 as hot-spots for spontaneous breakage. Even, in A. caraya, one fragile site (1q31) correlated with a breakpoint involved in a pericentric inversion (Mudry et al., 1990). Therefore, chromosomal speciation in Alouatta could be very more frequent than in other mammals. Wienberg et al., (1997) showed that karyotype evolution in mammals is generally assumed to be highly

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conservative. For example the number of translocations between man and cat has been estimated to be on the order of one every 10 million years. The most extreme karyological diversity in Alouatta is found A. fusca. De Oliveira et al., (1995, 1998, 2000) showed, at least, four different karyomorphic groups: 2N = 52 with XY/XX, 2N = 48 or 50 with XY/XX, 2N = 49 with X1X2Y/50 with X1X1X2X2 and 2N = 45 with X1X2Y/ 46 with X1X1X2X2. A. belzebul also presented a similar sex chromosome system of 50 with X1X1X2X2/49 with X1X2Y (Armada et al., 1987). De Oliveira et al., (2000) showed that the different karyotypes described revealed a clinal distribution for the chromosomal variation in A. fusca clamitans. This could suggest that there are populations in different stages of speciation, and probably reproductively isolated, due to meiotic disturbance. Stasipatric speciation by chromosomal mutations would have been more easily fixed due to inbreeding among small populations with low effective numbers. In Alouatta, the dominant male copulates with the great majority of females in small groups (Wolheim, 1983), increasing the frequency of inbreeding. De Oliveira et al., (2002) showed that A. f. fusca and A. f. clamitans differ by two Robertsonian translocations. A hybrid between the respective karyotypes, 2N = 49/50 and 2N = 45/46, would require the formation of two trivalents, which could have serious problems during meiosis. Most likely, A. fusca and A. clamitans are both full species. With the exception of one population of A. clamitans (Espiritu Santo state), XY/XX (De Oliveira et al., 2000), and the Colombian A. palliata individuals studied by Torres and Ramírez (2003) (56 with XY/XX), all the remaining Alouatta taxa show the Y chromosome translocated to an autosomal chromosome. The alleged XY/XX karyotypes for A. s. seniculus (Yunis et al., 1976) and for A. caraya (Mudry et al., 1984) were validated by more recent studies with improved resolution (Mudry et al., 1998; Torres and Leibovici, 2001). Therefore, the Alouatta genus should experience speciation with relative frequency by means of chromosomal changes (parapatric and stasipatric speciation). These striking karyotypical changes plus molecular genetics analyses should help to establish an accurate account of the phylogeny and systematics of this Neotropical primate genus more easily than in other organisms where chromosomal stability is higher.

ACKNOWLEDGMENTS Thanks to Dr. Diana Alvarez, Pablo Escobar-Armel, Nicolás Lichilín, Armando Castellanos, Andrés Laguna, Fernando Nassar, Luz Mercedes Botero, Marcela Ramírez, Connie Stelle and Hugo Gálvez for their respective help in obtaining howler monkey samples during the last 20 years. Thanks to Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Muñoz), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de Extracción y Procesamiento Pesquero), Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales from Peru, and to the Colección Boliviana de Fauna (Dr. Julieta Vargas) and to CITES Bolivia for their role in facilitating the obtainment of the collection permits in Colombia, Peru and Bolivia. The Costa Rican howler monkeys were sampled with the collection permits conceded by the Costa Rican government to Dr. Gustavo Gutiérrez-Espeleta. Thanks also goes to ARCAS (Guatemala) for providing hair samples of Alouatta pigra. The first author also thanks the help of many people of diverse Indian tribes

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in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana and Alamas), Bolivia (Sirionó, Canichana, Cayubaba and Chacobo) and Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna) by their facilities in obtaining samples of howler monkeys.

REFERENCES Aguiar, L., Mellek, D., Abreu, K., Boscarato, T., Bernardi, I., Miranda, J. and Passos, F. (2007). Sympatry of Alouatta caraya and A. clamitans and the rediscovery of freeranging potential hybrids in Southern Brazil. Primates, 48, 245–248. Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on Automatic Control, AC, 19, 716–723. Aquino, R. and Encarnación, F. (1994). Primates of Peru. Primate Report, 40, 1-127. Aquino, R., Terrones, W., Navarro, R., Terrones, C. and Cornejo, F. M. (2009). Caza y estado de conservación de primates en la cuenca del río Itaya, Loreto, Perú. Revista Peruana de Biología, 15, 33-39. Armada, J. L., Barroso, C. M., Lima, M. C., Muñiz, J. A. and Seuánez, H. N. (1987). Chromosome studies in Alouatta belzebul. American Journal of Primatology, 13, 283296. Ascunce, M. S., Hasson, E. and Mudry, M. D. (2003). COII: a useful tool for inferring phylogenetic relationships among New World monkeys (Primates, Platyrrhini). Zoologica Scripta, 32, 397-406. Ashley, M. V. and Vaughn, T. A. (1995). Owl monkeys (Aotus) are highly divergent in mitochondrial cytochrome c oxidase (COII) sequences. International Journal of Primatology, 5, 793–807. Auricchio, P. (1995). Primatas do Brasil. Terra Brasilis Editora Ltda. Sao Paulo, Brazil. pp. 1-168. Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, J. E., Reeb, C. A. and Saunders, N. C. (1987). Intraspecific phylogeographic: the mitochondrial DNA bridge between population genetics and systematics. Annual Review of Ecology, Evolution, and Systematics, 18, 489-522. Bandelt, H-J., Forster, P. and Rohl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution, 16, 37-48. Bender, M. A. and Chu, E. H. Y. (1963). The chromosomes of primates. Vol. 1. In BuettnerJanusch (Ed.). Evolutionary and genetic biology of primates., Academic Press. New York. pp. 261-310. Bermingham, E. and Martin, A. P. (1998). Comparative mtDNA phylogeography of neotropical freshwater fishes: testing shared history to infer the evolutionary landscape of lower Central America. Molecular Ecology, 7, 499–517. Bodini, R. and Pérez-Hernández, R. (1987). Distribution of the species and subspecies of Cebids in Venezuela. Fieldiana Zoology, 39, 231-244. Bonvicino, C. R., Fernandes, M. E. B. and Seuánez, H. N. (1995). Morphological analysis of Alouatta seniculus species group (Primates, Cebidae). A comparison with biochemical and karyological data. Journal of Human Evolution, 10, 169–176.

Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon ...

425

Bonvicino, C. R., Lemos, B. and Seuánez, H. N. (2001). Molecular phylogenetics of howler monkeys Alouatta. Platyrrhini. A comparison with karyotypic data. Chromosoma, 110, 241-246. Boubli, J.-P., Di Fiore, A., Rylands, A. B. and Mittermeier, R. A. (2008). Alouatta seniculus ssp. puruensis. The IUCN Red List of Threatened Species. Version 2015.2. . Downloaded on 26 July 2015. Brown, K. S. (1982). Paleoecology and regional patterns of evolution in Neotropical forest butterflies. In: Prance GT (Ed.) Biological diversification in the tropics. Columbia University Press. Bull, J. J. and Bulmer, M. G. (1981). Evolution of XY females in mammals. Heredity, 4, 347360. Cabrera, A. (1939). Los monos de Argentina. Physis (Rev. Soc. Argentina Cienc. Natur), 329. Cabrera, A. (1957). Catálogo de los mamíferos de América del Sur. I (Metatheria, Unguiculata, Carnívora). Revista del Museo Argentino de Ciencias Naturales “Bernardo Rivadavia,” Zoología, 4, 1-307. Collins, A. C. and Dubach, J. M. (2000). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology, 21, 381–420. Collins, A. C. and Dubach, J. M. (2001). Nuclear DNA variation in spider monkeys (Ateles). Molecular Phylogenetics and Evolution, 19, 67–75. Consigliere, S., Stanyon, R., Koehler, U., Agoramoorthy, G. and Wienberg, J. (1996). Chromosome painting defines genomic rearrangements between red howler monkeys subspecies. Chromosome Research, 4, 264–270. Consigliere, S., Stanyon, R., Koehler, U., Arnold, N. and Wienberg, J. (1998). In situ hybridization (FISH) maps chromosomal homologies between Alouatta belzebul (Platyrrhini, Cebidae) and other primates and reveals extensive interchromosomal rearrangements between howler monkeys genomes. American Journal of Primatology, 46, 119–133. Cortés-Ortiz, L., Bermingham, E., Rico, C., Rodriguez-Luna, E., Sampaio, I., Ruiz-García, M. (2003). Molecular systematics and biogeography of the Neotropical monkey genus Alouatta. Molecular Phylogenetics and Evolution, 26, 64–81. Cortés-Ortiz, L., Duda, Jr., T. F., Canales-Espinosa. D., García-Ordun, F., Rodríguez Luna, E. and Bermingham, E. (2007). Hybridization in large-bodied New World primates. Genetics, 176, 2421-2425. Crockett, C. M. and Eisenberg, J. F. (1987). Howlers: variations in group size and demography. In: Smuts, B.B., Cheney, D.L., Seyfarth, R.M., Wrangham, R.W., Struhsaker, T.T. (Eds.). Primate Societies. (pp. 54-68). University of Chicago Press, Chicago. Cropp, S. and Boinski, S. (2000). The Central American squirrel monkey (Saimiri oerstedii): introduced hybrid or endemic species. Molecular Phylogenetics and Evolution, 16, 350– 365. Cummings, M. P., Otto, S. P. and Wakeley, J. (1995). Sampling properties of DNA sequence data in phylogenetic analysis. Molecular Biology and Evolution, 12, 814–822.

426

Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro et al.

De Oliveira, E. H. C. (1996). Estudos citogenéticos e evolutivos nas espécies brasileiras e argentinas do gênero Alouatta Lacépède, 1799 (Primates, Atelidae). MSc Dissertation. Universidade Federal do Paraná. Curitiba. Brazil. De Oliveira, E. H. C., Lima, M. C. and Sbalqueiro, I. J. (1995). Chromosomal variation in Alouatta fusca. Neotropical Primates, 3, 181-183. De Oliveira, E. H. C., Lima, M. C., Sbalqueiro, I. J. and Pissinatti, A. (1998). The karyotype of Alouatta fusca clamitans from Rio de Janeiro State. Brazil: evidence for a Y autosome translocation. Genetics and Molecular Biology, 31, 361-364. De Oliveira, E. H. C., Suemitsu, E., da Silva, A. F. and Sbalqueiro, I. J. (2000). Geographical variation of chromosomal number in Alouatta fusca clamitans (Primates, Atelidae). Caryologia, 53, 163-168. De Oliveira, E. H. C., Neusser, M., Figueiredo, W. B., Nagamachi, C., Pieczarka, JC, Sbalqueiro, I. J., Wienberg, J. and Miller, S. (2002). The phylogeny of howler monkeys (Alouatta, Platyrrhini): Reconstruction by multicolor cross-species chromosome painting. Chromosome Research, 10, 669-683. De Souza Jesus, A., Schunemann, H. E., Müller, J. and da Silva, M. A. (2010). Hybridization between Alouatta caraya and Alouatta guariba clamitans in captivity. Primates, 51, 227– 230. Dobzhansky, T. (1971). Evolutionary oscillations in Drosophila pseudoobscura. In: Creed, R. (Eds.). Ecological genetics and evolution. Blackwell Scientific, Oxford, UK. pp. 109– 133. Dos Reis, N. R., Peracchi, A. L. and Andrade, F. R. (2008). Primatas Brasileiros. Technical Books Editora. Londrina, Brazil. Pp. 1-260. Drummond, A. J., Ho, S. Y. W., Phillips, M. J. and Rambaut, A. (2006). Relaxed phylogenetics and dating with confidence. PLOS Biology, 4, e88. Drummond, A. J., Suchard, M. A., Xie, D. and Rambaut, A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29, 1969–1973. Dutrillaux, B., Descaileaux, J., Viegas-Péquignot, E. and Couturier, J. (1981). Y-Autosome translocation in Cacajao calvus rubicundus (Platyrrhini). Annales Génetique, 24, 197201. Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W., Regard, V., Antoine, P. O., Salas-Gismondi, R. and Bolanos, R. (2007). How does the Nazca Ridge subduction influence the modern Amazonian foreland basin? Geology, 35, 515–518. Espurt, N., Baby, P., Brusset, S., Roddaz, M., Hermoza, W. and Barbarand, J. (2010). The Nazca Ridge and uplift of the Fitzcarrald Arch: implications for regional geology in northern South America. In Hoorn, C., and Wesselingh, F. (Eds.). Amazonia, Landscape and Species Evolution: A Look into the Past. (pp. 89-102). Wiley-Blackwell, Oxford. Estrada, A. and Coates-Estrada, R. (1984). Some observations on the present distribution and conservation of Alouatta and Ateles in southern Mexico. American Journal of Primatology, 7, 133-137. Egozcue, J. and Egozcue, M. V. (1966). The chromosome complement of the howler monkey (Alouatta caraya Humboldt, 1812). Cytogenetics, 5, 20–24. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution, 17, 368–376.

Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon ...

427

Figueiredo, W. B., Carvalho-Filho, N. M., Schneider, H. and Sampaio, I. (1998). Mitochondrial DNA sequences and the taxonomic status of Alouatta seniculus populations in Northeastern Amazonia. Neotropical Primates, 6, 73-77. Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294-299. Ford, C. E., Hamerton, J. L. and Sharman, G. B. (1957). Chromosome polymorphism in the common shrew. Nature, 180, 392-394. Frohelich, J. W. and Frohelich, P. H. (1987). The status of Panama’s endemic howling monkeys. Primate Conservation, 8, 58–62. Fu, Y-X. (1997). Statistical tests of neutrality against population growth, hitchhiking and background selection. Genetics, 147, 915-925. Fu, Y. and Li, W. (1993). Statistical Tests of Neutrality of Mutations. Genetics, 133, 693-709. Fundia, A., Gorostiaga, M. and Mudry, M. (2000). Expression of common fragile sites in two Ceboidea species: Saimiri boliviensis and Alouatta caraya. (Primates: Platyrrhini). Genetics Selection Evolution, 32, 87-97. Garber, P. A., Estrada, A., Bicca-Marques, J. C., Heymann, E. W. and Strier, K. B. (2009). South American Primates. Comparative perspectives in the study of behavior, ecology and conservation. Springer Science+Business Media, New York, USA. pp. 1-564. Gregorin, R. (2006). Taxonomia e variação geográfica das espécies do gênero Alouatta Lacépède (Primates, Atelidae) no Brasil. Revista Brasileira de Zoologia, 23, 64-144. Gregory, T., Carrasco Rueda, F., Deichmann, J. L., Kolowski, J. and Alonso, A. (2012). Primates of the lower Urubamba region, Peru, with comments on other mammals. Neotropical Primates, 19, 16-23. Groves, C. P. (2001). Primate taxonomy. Washington, DC: Smithsonian Institution Press. Haffer, J. (1969). Speciation in Amazonian forest birds. Science, 165, 131-137. Haffer, J. (1982). General aspects of the refuge theory. In Prance, G. T., (Ed.). Biological diversification in the tropics. (pp. 6-24). New York: Columbia University. Haffer, J. (1997). Alternative models of vertebrate speciation in Amazonia: an overview. Biodiversity Conservation, 6, 451-476. Haffer, J. (2008). Hypotheses to explain the origin of species in Amazonia. Brazilian Journal of Biology, 68, 917-947. Harpending, H. C. (1994). Signature and ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Human Biology, 66, 591-600. Harpending, H. C., Sherry, S. T., Rogers, A. R. and Stoneking, M. (1993). Genetic structure of ancient human populations. Current Anthropology, 34, 483-496. Hasegawa, M., Kishino, H. and Yano, T. (1985). Dating of human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution, 22, 160-174. Hebert, P. D. N., Ratnasingham, S. and de Waard, J. R. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B, 270 (Suppl.), S96-9. Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. and Francis, C. M. (2004). Identification of birds through DNA barcodes. PLoS Biology, 2, 1657-63. Hershkovitz, P. (1949). Mammals of northern Colombia. Preliminary report no. 4: monkeys (primates), with taxonomic revisions of some forms. Proceedings of the United States National Museum, 98, 323–427.

428

Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro et al.

Hill, W. C. O. (1962). Primates Comparative Anatomy and Taxonomy. V. Cebidae. Part. B. Edinburgh University Press, Edinburgh. Hudson, R. R., Boss, D. D. and Kaplan, N. L. (1992). A statistical test for detecting population subdivision. Molecular Biology and Evolution, 9, 138-151. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111-120. King, M. (1993). Species Evolution. Cambridge University Press, Cambridge. Koiffmann, C. P. (1982). Variabilidadi cromossomica em Cebidae. In P. H (Ed.). Genética comparada de primates Brasileiros. (pp. 113-132). Koiffmann, C. P. and Saldhana, P. H. (1974). Cytogenetics of Brazilian monkeys. Journal of Human Evolution, 3, 275–282. Lavergne, A., Ruiz-García, M., Catzeflis, F., Lacote, S., Contamin, H., Mercereau-Puijalon, O., Lacaste, A. and Thoisy B. (2010). Taxonomy and phylogeny of squirrel monkey (genus Saimiri) using cytochrome b genetic analysis. American Journal of Primatology, 72, 242-253. Lewis, H. (1966). Speciation in flowering plants. Science, 152, 167-172. Librado, P. and Rozas, J. (2009). DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-1452 | doi: 10.1093/bioinformatics/btp187. Lima, M. C. and Seuánez, H. N. (1989). Cytogenetic characterization of Alouatta belzebul with atypical pelage coloration. Folia Primatologica, 52, 97 -101. Lima, M. C., Sampaio, M. I., Schneider, M. P., Scheffrahn, W., Schneider, H. and Salzano, F. M. (1990). Chromosome and protein variation in red howler monkeys. Revista Brasileira de Genética, 13, 789 - 802. Lima, M. M. C. and Seuánez, H. N. (1991). Chromosome studies in the red howler monkey, Alouatta seniculus stramineus (Platyrrhini, Primates): description of an X1X2Y1Y2/X1X1X2X2 sex-chromosome system and karyological comparisons with other subspecies. Cytogenetics and Cell Genetics, 57, 151–156. Lundberg, J. G., Marshall, L., Guerrero, J., Horton, B., Malabarba, C. and Wesselingh, F. (1998). The stage for neotropical fish diversification: history of a tropical South American river. In Malabarba, L.R., Reis, R., Vari, R., Lucena, Z., and Lucena, C.A., (Eds.). Phylogeny and classification of neotropical fishes. (pp. 13-48). Porto Alegre, Brazil: Pontificia Universidade Católica Do Rio Grande Do Sul. Ma, N. S., Jones, T. C., Thorington, R. W., Miller, A. and Morgan, L. (1975). Y-autosome translocation in the howler monkey, Alouatta palliata. Journal of Medical Primatology, 4, 299-307. Ma, N. S., Jones, T. C., Miller, A. C., Morgan, L. M. and Adams, E. A. (1976). Chromosome polymorphism and banding patterns in the owl monkey (Aotus). Laboratory Animal Science, 26, 1022-1036. Ma, N. S. F., Renquist, D. M., Hall, R., Sehgal, P. K., Simeone, T., Jones, T. C. (1980). XX/XO sex determination systems in a population of Peruvian owl monkeys, Aotus. Journal of Heredity, 71, 336–342. Meireles, C. M., Czelusniak, J., Schneider, M. P., Muniz, J. A. P. C., Brigido, M. C., Ferreira, H. S. and Goodman, M. (1999). Molecular phylogeny of Ateline New World Monkeys (Platyrrhini, Atelinae) based on -globin gene sequences: evidence that Brachyteles is the sister group of Lagothrix. Molecular Phylogenetics and Evolution, 12, 10–30.

Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon ...

429

Mercado, N. I. and Wallace, R. B. (2010). Distribución de primates en Bolivia y áreas prioritarias para su conservación. Tropical Conservation Science, 3, 200-217. MINAM. (2011). Informe Final del Estudio de Especies CITES de Primates Peruanos. Ministerio del Medio Ambiente, Lima, Perú. pp. 1-219. Minezawa, M., Harada, M., Jordan, O. C. and Borda, C. J. V. (1985). Cytogenetics of Bolivian endemic red howler monkeys (Alouatta seniculus sara): accessory chromosomes and Y-autosome translocation related numerical variations. Kyoto Univ. Overs. Res. Rep. N. W. Monkeys, 5, 7–16. Mittermeier, R. A. and Coimbra-Filho, A. F. (1981). Systematics: species and subspecies. In Coimbra-Filho, A. F., and Mittermeier, R. A. (Eds.). Ecology and behavior of Neotropical Primates., Vol 1. (pp. 29-109). Mittermeier, R. A., Rylands, A. B., Coimbra-Filho, A. and Fonseca, G. A. B. (1988). Ecology and behavior of Neotropical Primates., Volume 2. World Wildlife Fund, Washington, D. C., USA. pp. 1-610. Montgelard, C., Catzeflis, F. M. and Douzery E. (1997). Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Molecular Biology and Evolution, 14, 550–559. Moore, W. (1995). Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution, 49, 718–726. Morral, N., Bertrantpetit, J. and Estivill, X., et al. (1994). The origin of the major cystic fibrosis mutation (delta F508) in European populations. Nature Genetics, 7, 169-175. Mudry, M. D., Laval, M. L., Colillas, O. J. and Brieux, S. (1984). Banding patterns of Alouatta caraya. Revista Brasileira de Genética, 2, 373 - 379. Mudry, M. D., Slavutsky, I. and Vinuesa, M. L. (1990). Chromosome comparison among five species of Platyrrhini (Alouatta caraya, Aotus azarae, Callithrix jacchus, Cebus apella, and Saimiri sciureus). Primates, 31, 415–420. Mudry, M., Ponsa, M., Borell, A., Egozcue, J. and Garcia, M. (1994). Prometaphase chromosomes of the howlermonkey (Alouatta caraya): G, C, NOR and restriction enzyme (Res) banding. Am. J. Primatol., 33, 121-134. Mudry, M. D., Rahn, M., Gorostiaga, M., Hick, A., Merani, M. S. and Solari, A. J. (1998). Revised karyotype of Alouatta caraya (Primates: Platyrrhini) based on synaptonemal complex and banding analyses. Hereditas, 128, 9-16. Opazo, J. C., Wildman, D. E., Prychitko, T., Johnson, R. M. and Goodman, M. (2006). Phylogenetic relationships and divergence times among New World monkeys (Platyrrhini, Primates). Molecular Phylogenetetics and Evolution, 40, 274-280. Pacheco, V., Cadenillas, R., Salas, E., Tello, C. and Zeballos, H. (2009). Diversidad y endemismo de los mamíferos del Perú. Revista Peruana de Biología, 16, 5-32. Perelman, P., Johnson, W. E., Roos, C., Seuanez, H. N., Horvath, J. E., Moreira, M. A. M., Kessing, B., Pontius, J., Roelke, M., Rumpler, Y., Schneider, M. P. C., Silva, A., O’Brien, S. J. and Pecon-Slattery, J. (2011). A molecular phylogeny of living primates. PLoS Genetics, 7, e1001342. Pieczarka, J. C. and Nagamachi, C. Y. (1988). Cytogenetic studies of Aotus from eastern Amazonia Y autosome rearrangement. American Journal of Primatology, 14, 255–264. Pope, T. R. (1992). The influence of dispersal patterns and mating system on genetic differentiation within and between populations of the red howler monkeys (Alouatta seniculus). Evolution, 46, 1112–1128.

430

Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro et al.

Posada D, Crandall KA. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817-818. Posada, D. and Crandall, K. A. (2001). Intraspecific gene genealogies: trees grafting into networks. Trends Ecology and Evolution, 16, 37–45. Rambaut, A. (2012). FigTree v1.4. http://tree.bio.ed.ac.uk/software. Rambaut, A. and Drummond, A. J. (2013a). LogCombiner v1.8.0. http://beast.bio.ed.ac.uk/. Rambaut, A. and Drummond, A. J. (2013b). TreeAnnotator v1.8.0. http://beast.bio.ed.ac.uk/. Rambaut, A., Suchard, M. A., Xie, W. and Drummond, A. J. (2013). Tracer v1.6. http://tree.bio.ed.ac.uk/software/tracer/. Ramos-Onsins, S. E. and Rozas, J. (2002). Statistical Properties of New Neutrality Tests Against Population Growth. Molecular Biology ad Evolution, 19, 2092–2100. Reig, O. (1980). Modelos de especiación cromosómica en las casiraguas (Género Proechimys) de Venezuela. In Reig, O (Ed.). Ecología y Genética de la especiación animal. pp. 149-190). Equinoccio, Editorial de la Universidad Simón Bolívar, Caracas, Venezuela. Rogers, A. R. and Harpending, H. C. (1992). Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution, 9, 552569. Rogers, A. R., Fraley, A. E., Bamshad, M. J., Watkins, W. S. and Jorde, L. B. (1996). Mitochondrial mismatch analysis is insensitive to the mutational process. Molecular Biology and Evolution, 13, 895-902. Rosenbaum, G., Giles, D., Saxon, M., Betts, P. G., Weinberg, R. F. and Duboz, C. (2005). Subduction of the Nazca Ridge and the Inca Plateau: Insights into the formation of ore deposits in Peru. Earth Planetary Science Letters 239, 18–32. Ruiz-García, M. and Pinedo-Castro, M. (2010). Molecular systematics and phylogeography of the genus Lagothrix (Atelidae, Primates) by means of mitochondrial COII gene. Folia Primatologica, 81, 109–128. Ruiz-García, M., Escobar-Armel, P., Alvarez, D., Mudry, M., Ascunce, M., GutierrezEspeleta, G. and Shostell, J. M. (2007). Genetic variability in four Alouatta species measured by means of nine DNA microsatellite markers: Genetic structure and recent bottlenecks. Folia Primatologica, 78, 73–87. Ruiz-García, M., Castillo, M. I., Vásquez, C., Rodriguez, K. and Pinedo-Castro, M. et al. (2010). Molecular phylogenetics and phylogeography of the white-fronted capuchin (Cebus albifrons; Cebidae, Primates) by means of mtCOII gene sequences. Molecular Phylogenetics and Evolution, 57, 1049-1061. Ruiz-García, M., Castillo, M. I., Lichilin, N. and Pinedo-Castro, M. (2012a). Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, C. xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene sequences. Folia Primatologica, 83, 100-125. Ruiz-Garcia, M., Castillo, M. I., Ledezma, A., Leguizamon, N. and Sánchez, R. et al. (2012b). Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of Primatology, 74, 366-380. Ruiz-García, M., Pinedo-Castro, M. and Shostell, J. M. (2014). How many genera and species of woolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular

Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon ...

431

analysis of Lagothrix flavicauda, an endemic Peruvian primate species. Molecular Phylogenetics and Evolution, 79, 179–198. Ruiz-García, M., Vásquez, C., Sandoval, S., Kaston, F., Luengas-Villamil, K. and Shostell, J. M. (2015a). Phylogeography and spatial structure of the lowland tapir (Tapirus terrestris, Perissodactyla: Tapiridae) in South America. Mitochondrial DNA, 26, 1-9, on line DOI: 10.3109/19401736.2015.1022766. Ruiz-García, M., Luengas, K., Leguizamón, N., Thoisy, B. and Gálvez, H. (2015b). Molecular Phylogenetics and Phylogeography of all the Saimiri species (Cebidae, Primates) inferred from mt COI and COII gene sequences. Primates, 56, 145-161. Ruiz-García, M., Escobar-Armel, P., Thoisy, B., Martínez-Agüero, M., Pinedo-Castro, M. and Shostell, J. M. (2016). Biodiversity in the Amazon: origin hypotheses, intrinsic capacity of species colonization, and comparative phylogeography of river otters (Lontra longicaudis and Pteronura brasiliensis, Mustelidae, Carnivora) and pink river dolphin (Inia sp, Iniidae, Cetacea). Journal of Mammalian Evolution (in press). Ruvolo, M., Disotell, T. R., Allard, M. W., Brown, W. M. and Honeycutt, R. L. (1991). Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence. Proceedings of the National Academy of Sciences of the USA, 88, 1571–1574. Rylands, A. B., Mittermeier, R. A. and Rodríguez-Luna, E. (1995). A species list for the New World primates (Platyrrhini): distribution by country, endemism, and conservation status according to the Mace-Land system. Neotropical Primates, 3, 113–160. Rylands, A. B., Rodríguez-Luna, E. and Cortés-Ortiz, L. (1996/1997). Neotropical primate conservation-The species and the IUCN/SSC primate specialist group network. Primate Conservation, 17, 46-69. Rylands, A. B. and Brandon-Jones, D. (1998). Scientific nomenclature of the red howlers from the northeastern Amazon in Brazil, Venezuela and the Guianas. International Journal of Primatology, 19, 879-905. Rylands, A. B., Schneider, H., Langguth, A., Mittermeier, R. A., Groves, C. P. and Rodríguez-Luna, E. (2000). An assessment of the diversity of New World primates. Neotropical Primate, 8, 61–93. Rylands, A. B. and Mittermeier, R. A. (2009). The diversity of the New World primates (Platyrrhini): An annotated taxonomy. In South American Primates. Comparative perspectives in the study of behavior, ecology and conservation. Garber, P. A., Estrada, A., Bicca-Marques, J. C., Heymann, E. W., Strier, K. B. (Eds.). Springer Science+Business Media, New York, USA. Pp. 23-54. Saillard, J., Forster, P., Lynnerup, N., Bandelt, H-J. and Norby, S. (2000). mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion. American Journal of Human Genetics, 67, 718-726. Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 405–425. Sambrock, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Laboratory manual. 2nd edition. V1. Cold Spring Harbor Laboratory Press. New York. Sampaio, I., Schneider, M. P. and Schneider, H. (1996). Taxonomy of the Alouatta seniculus group: biochemical and chromosome data. Primates, 37, 65–73. Schwarz, G. E. (1978). Estimating the dimension of a model. Annals of Statistics, 6, 461-464. Simonsen, K., Churchill, G. and Aquadro, C. (1995). Properties of Statistical Tests of Neutrality for DNA Polymorphism Data. Genetics, 141, 413-429.

432

Manuel Ruiz-García, Angela Cerón, Myreya Pinedo-Castro et al.

Smith, J. D. (1970). The systematic status of the black howler monkey, Alouatta pigra Lawrance. Journal of Mammalogy, 51, 358–369. Soini, P., Aquino, R., Encarnación, F., Moya, L. and Tapia, J. (1989). Situación de los primates en la Amazonía Peruana. In Saavedra, C. J., Mittermeier, R. A., and Santos, I. B. (Eds.). La Primatología en Latinoamérica. Symposio de Primatología. Arequipa, Perú, 1983. (pp. 13-21). World Wildlife Fund, Littera Maciel Ltda. Minas Gerais, Brazil. Springer, M. S., Meredith, R. W., Gatesy, J., Emerling, C., Park, J., Rabosky, D. L., Stadler, T., Steiner, C., Ryder, O., Janecka, J. E., Fisher, C. and Murphy, W. J. (2012). Macroevolutionary dynamics and historical biogeography of primate diversification inferred from a species supermatrix. PLoS One, 7, e49521. Stanyon, R., Tofanelli, S., Morescalchi, M. A., Agoramoorthy, G., Ryder, O. A. and Wienberg, J. (1995). Cytogenetic analysis shows extensive genomic rearrangements between red howler (Alouatta seniculus, Linnaeus) subspecies. American Journal of Primatology, 35, 171–183. Swofford, D. L. (2002). PAUP*. Phylogenetic analysis using parsimony and other methods. http://paup.csit.fsu.edu. pp. 1-142. Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585-595. Tamura K., Stecher G., Peterson D., Filipski A. and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 27252729. Torres, O. M. and Leibovici, M. (2001). Caracterización del cariotipo del mono aullador colorado Alouatta seniculus que habita en Colombia. Caldasia, 23, 537-548. Torres, O. M. and Ramírez, C. (2003). Estudio citogenético de Alouatta palliata (Cebidae). Caldasia, 25, 193–198. Vassart, M., Guédant, A., Vié, J. C., Kéravec, J., Séguéla, A. and Volobouev, V. T. (1996). Chromosomes of Alouatta seniculus (Platyrrhini, Primates) from French Guiana. The Journal of Heredity, 87, 331-334. Villalobos, F., Valerio, A. A. and Retana, A. P. (2004). A phylogeny of howler monkeys (Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data. Revista de Biología Tropical, 52, 665-677. Von Dornum, M., Ruvolo, M. (1999). Phylogenetic relationships of the New World monkeys (Primates, Platyrrhini) based on nuclear G6PD DNA sequences. Molecular Phylogenetics and Evolution, 11, 459–476. Voss, R. S. and Fleck, D. W. (2011). Mammalian Diversity and Matses Ethnomammalogy in Amazonian Peru. Part 1: Primates. Bulletin of the American Museum of Natural History, 351, 1-81. Wallace, R., B. and Rumiz, D. I. (2010). Atelidae. In Wallace, R. B., Gómez, H., Porcel, Z. R., and Rumiz, D. I. (Eds.). Distribución, Ecología y Conservación de los Mamíferos medianos y grandes de Bolivia. (pp. 1-884). Centro de Ecología Difusión. Fundación Simón I. Patiño. Walsh, P. S., Metzger, D. A. and Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10, 506513. Weir, B. S. and Hill, W. G. (2002) Estimating F-statistics. Annual Revue of Genetics, 36, 721–750.

Which Howler Monkey (Alouatta, Atelidae, Primates) Taxon ...

433

Wienberg, J., Stanyon, R., Nash, W. G., O’Brien, P. C. M., Yang, F., O’Brien, S. J. and Ferguson-Smith, M. A. (1997). Conservation of human vs. feline genome organization revealed by reciprocal chromosome painting. Cytogenetics and Cell Genetics, 77, 211– 217. Wolfheim, J. H. (1983). Primates of the New World: Distribution, Abundance and Conservation. Seattle, University of Washington Press. Yunis, E. J., Torres, O. M., Ramírez, C. and Ramírez, E. (1976). Chromosomal variations in the primate Alouatta seniculus seniculus. Folia Primatologica, 25, 215 - 224. Zuñiga Leal, S. A. and Defler, T. R. (2013). Sympatric distribution of two species of Alouatta (A. seniculus and A. palliata: Primates) in Chocó, Colombia. Neotropical Primates, 20, 111.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 13

HISTORICAL GENETIC DEMOGRAPHY AND SOME INSIGHTS INTO THE SYSTEMATICS OF ATELES (ATELIDAE, PRIMATES) BY MEANS OF DIVERSE MITOCHONDRIAL GENES Manuel Ruiz-García1,*, Nicolás Lichilín1, Pablo Escobar-Armel1, Geven Rodríguez1 and Gustavo Gutiérrez-Espeleta2 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética. Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá DC., Colombia 2 Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica

ABSTRACT We sampled 283 spider monkeys (Ateles) and sequenced three of their mitochondrial genes (Cyt-b, COI and COII). This was the largest molecular genetics sample set of Ateles ever analyzed and included all of the morphological taxa described for this genus. There were six main findings: 1.

2.

*

All the species, or main taxa studied, showed elevated mitochondrial gene diversity levels, with the exception of A. paniscus. The hybridus taxa, which showed considerably low gene diversity levels for nuclear DNA microsatellites (Ruiz-García et al., 2006), but also showed relatively elevated levels of mitochondrial gene diversity. Changes in population size: The taxa chamek and belzebuth showed strong evidence of population expansions during the Pleistocene. Also, we detected population expansions in fusciceps and geoffroyi. The demographic history of marginatus was ambiguous with some of our analyses indicating population expansion while others suggesting a declination in females. This was probably due to small sample size.

Corresponding author: [email protected], [email protected].

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3.

4.

5. 6.

Similarly, paniscus didn’t show clear evidence of demographic changes, but hybridus did show a declination trend in its female population. Genetic heterogeneity: Taxa pairs with A. paniscus showed the highest values of genetic differentiation—suggesting A. paniscus is the most differentiated taxon within Ateles. Genetic heterogeneity values were only statistically significant when all taxa were analyzed together. The Kimura 2P genetic distance model showed maximum values around 4% for the different taxa pairs. Many of these were around 2-3%, clearly lower than those of other Neotropical primate species within genera. This indicates that the number of Ateles species proposed by Groves (2001) is probably an overestimation (taxonomic inflation) caused by a very typological use of the Phylogenetic species concept. Our molecular phylogenetic results support the existence of two (A. paniscus and A. belzebuth) or three (A. paniscus, A. belzebuth and A. geoffroyi) species. We (both mitochondrial and microsatellites) detected a very strong phylogenetic relationship between hybridus and some fusciceps individuals. These results don’t agree with the view of Collins and Dubach (2000a,b) and Nieves et al., (2005) that hybridus is a full and differentiated species.

Keywords: Ateles, mitochondrial gene sequences, demographic changes, IUCN classifications, phylogenetic insights, two or three Ateles species

INTRODUCTION The spider monkeys, Ateles genus, are among the largest primates in the Neotropics along with Alouatta, Lagothrix and Brachyteles (Strier, 1992). Unfortunately, Ateles populations are currently being threatened by many factors. They are intensively hunted for food by indigenous people throughout Central and South America. There is also the traditional demand for spider monkeys as attractive animals in zoos and as pets. These demands have led to the development of intense commercial trafficking (legal and illegal) of this genus. As large primates, spider monkeys have low reproductive rates and therefore, even a low hunting pressure, could extirpate extensive populations throughout their distribution range (Collins, 1999). Konstant et al., (1985) and Rosenberger and Strier (1989) revealed that Ateles is extremely environmentally sensitive and they are the first primates to disappear after small environmental changes disturb the rain forest. Spider monkeys live in undisturbed areas within primary rain forests and they are highly arboreal. Furthermore, Ateles is primarily frugivorous and feeds largely on the mature, soft parts of a wide variety of fruits that provide more energy than leaves. Van Roosmalen (1980) estimated that 82.9 to 90% of the Ateles’s diet is composed of fruit. This diet requires Ateles to have extensive territories to encompass the non-uniform distributions of fruit. Thus, high sensitivity to environmental change, low reproductive rate, and large territories help to explain why many Ateles populations are now seriously threatened and need to be targeted for conservation. Indeed, after Brachyteles and Leontopithecus, Ateles is considered the most endangered genus of the New World Primates (Mittermeier et al., 1989). In Table 1, we show the evolution of the classification of IUCN in reference to the different Ateles taxa. Practically, all the taxa have worsened their situation throughout the last

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decades. Here we provide a brief update as to the situation of each Ateles taxon—using the terms vulnerable, endangered, and critically endangered. Vulnerable means that this taxon declined by at least 30% over the past 45 years (three generations) due primarily to hunting and habitat loss. Endangered means the same but with declination of at least 50%, while critically endangered implies a declination of at least 80%. Table 1. UICN classifications in 1991, 1996, 2000, 2003 and 2008 for the different Ateles taxa defined in each period Ateles taxa paniscus

-

UICN 1991

UICN 1996 Low Risk

UICN 2000 Least Concern

UICN 2003 Least Concern

chamek

-

Low Risk

Least Concern

Least Concern

belzebuth

Vulnerable

Vulnerable

Vulnerable

Vulnerable

marginatus

Critical Risk

Endangered

Endangered

Endangered

fusciceps fusciceps

Critical Risk

Critical Risk

Critical Risk

Critical Risk

fusciceps rufiventris

Endangered

Vulnerable

Vulnerable

Vulnerable

hybridus hybridus

Endangered

Endangered

Endangered

Critically endangered

hybridus brunneus

-

Endangered

Endangered

Critically endangered

geoffroyi

-

-

-

Least Concern

geoffroyi geoffroyi

Endangered

Low Risk

-

-

geoffroyi azuerensis

Critical Risk

Critical Risk

Critical Risk

Critical Risk

geoffroyi frontatus geoffroyi grisescens geoffroyi panamensis geoffroyi ornatus geoffroyi vellerosus

Endangered

Vulnerable

Vulnerable

Least Concern

-

Endangered

Endangered

-

UICN 2008 Vulnerable A2cd Endangered A2cd Endangered A2cd Endangered A2cd + 3cd Critically endangered A2cd Critically endangered A2cd Critically Endangered A2cd + 3cd Critically Endangered A2cd + 3cd Endangered A2c Critically Endangered A4c Critically endangered A2c Vulnerable A2c Deficit Data

Endangered

Endangered

-

-

-

-

Vulnerable

Vulnerable

Endangered

Vulnerable

Low Risk

-

Critically endangered

geoffroyi yucatanensis

-

Vulnerable

Vulnerable

Vulnerable

Endangered A4c Critically endangered A4c Endangered A4c

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The Ateles taxa have become increasingly vulnerable and their classifications have in many cases moved from vulnerable to endangered or even to critically endangered. For example, A. belzebuth was classified as vulnerable in 1991 and then later reclassified as endangered A2cd in 2008. Similarly, A. chamek was classified as low risk in 1996 and as endangered A2cd in 2008. A. fusciceps fusciceps was classified as critically endangered in 1991 and as critically endangered A2cd in 2008. A. fusciceps rufiventris was classified as endangered in 1991 and vulnerable in 1996 and as critically endangered A2cd in 2008. A. hybridus was classified as endangered in 1991 and as critically endangered A2cd + 3cd in 2008. A. hybridus brunneus was classified as endangered in 1996 and as critically endangered A2cd + 3cd in 2008. A. marginatus was classified as critically endangered in 1991 and as endangered A2cd + 3cd in 2008. A. paniscus was classified as low risk in 1996 and as vulnerable A2cd in 2008. A. geoffroyi, taken as a whole, was classified as least concern in 2003 and as endangered A2cd in 2008. Here we also provide updates on the subspecies of A. geoffroyi. A. g. geoffroyi was classified as endangered in 1991, low risk in 1996 and critically endangered A4c in 2008. A. g. azuerensis was listed as critically endangered in 1991 and critically endangered A2c in 2008. A. g. frontatus was listed as endangered in 1991 and vulnerable A2c in 2008. A. g. grisescens was considered endangered in 1996 and there was insufficient data to report on it in 2008. A. g. panamensis was listed as endangered in 1991 and there were no data reported for this subspecies in 2008. A. g. ornatus was vulnerable in 1996 and endangered A4c in 2008. A. g. vellerosus was vulnerable in 1991 and critically endangered A4c in 2008. Finally, A. g. yucatanensis was listed as vulnerable in 1996 and endangered A4c in 2008. The only cases, where the classification situation did not get worse, were for A. marginatus and for A. g. frontatus. In fact, Mittermeier et al., (2009) enclosed A. hybridus as one of the 25 most endangered primates in the world (2008-2010), together with two other Neotropical primates (Saguinus oedipus and Lagothrix flavicauda). For A. hybridus, only 0.67% of the current range is protected. However, the IUCN and other classifications did not contain genetic historical demographic information of these species. In many cases, the current demographic situation of the Ateles species could be a consequence of their own evolution without human intervention. For example, see the case of the Andean bear (Ruiz-García, 2003a, 2007, 2013; Ruiz-García et al., 2003, 2005). In contrast, if a taxon shows elevated levels of gene diversity and population expansions are detected throughout its evolution but its current censuses sizes are small, this would be a clear symptom of a negative human intervention. This would probably cause a severe bottleneck. For this reason it is extremely important to reconstruct the historical genetic demography of a taxon and to compare it with its classification by IUCN and other institutions. The case of Ateles is interesting from this point of view. Nevertheless, the systematics of the taxa under study could negatively affect these kinds of comparisons. There is disagreement in the literature over the systematics of Ateles, making it difficult to construct and present an effective conservation proposal for this genus. The most traditionally used classification scheme recognizes four different species (Kellog and Goldman 1944; Hill 1962): A. geoffroyi, A. belzebuth, A. fusciceps, and A. paniscus. Each of these species has their own distinct subspecies. A. geoffroyi has nine subspecies in Central America: A. g. ornatus (Costa Rica), A. g. panamensis (Costa Rica, Panama), A. g. azuarensis (Panama), A. g. geoffroyi (Costa Rica, Nicaragua), A. g. frontatus (Costa Rica, Nicaragua), A. g. yucatanensis (Mexico, Belize, Guatemala), A. g. vellerosus (Mexico,

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Guatemala, Honduras, El Salvador), A. g. pan (Guatemala), and A. g. grisescens, (Colombia, Panama). A. belzebuth has three geographical discontinuous subspecies: A. b. belzebuth (Colombia, Peru, Ecuador, Venezuela, Brazil), A. b. hybridus (Colombia, Venezuela), and A. b. marginatus (Brazil). The third species, A. fusciceps, is found in the Pacific coast of Colombia and Ecuador and has two subspecies: A. f. fusciceps (Ecuador) and A. f. rufiventris (= robustus; Colombia, Panama). The fourth species, A. paniscus, has two discontinuous subspecies A. p. paniscus (Guyana, Suriname, French Guiana, Brazil) and A. p. chamek (Peru, Bolivia, Brazil). Tallying all subspecies across the four species there are 16 taxa. Mittermeier and Coimbra-Filho (1977) and Konstant et al., (1985) used the same classificatory scheme, although Konstant et al., (1985) noted the difficulty in discriminating several of the subspecies identified by Kellog and Goldman (1944). This included the challenge of differentiating A. g. vellerosus and A. g. yucatanensis and the validity of A. g. azuerensis. The authors also claimed that marginatus might need to be classified within A. paniscus because of its close relationship with A. p. chamek. Differences between species and subspecies of this classification were based almost entirely upon pelage characteristics. Conversely, Hershkovitz (1968, 1969, 1972), Hernández-Camacho and Cooper (1976) and Wolfheim (1983) supported a second classification scheme and have considered all Ateles to belong to one, wide-ranging variable polytypic species, A. paniscus. The authors based this scheme on the varying coat color patterns within this genus. Nevertheless, Shedd and Macedonia (1991) and Jacobs et al., (1995) have not supported the use of metachromatism to infer phylogenetic relationships among diverse Neotropical primates because the genetic and developmental systems that underlie the phenotypic expression of pelage traits may be different across primate species. In a third classificatory scheme based on cytogenetic analyses, diverse authors (García et al., 1975; Kunkel et al., 1980) postulated that differences in the morphological chromosome pairs 5, 6 and 7 could support the Ateles taxonomy proposed by Kellog and Goldman (1944). This could also be interpreted as intraindividual heteromorphism and also support the view of one unique Ateles species as suggested by Hershkovitz (1968, 1969, 1972), and HernándezCamacho and Cooper (1976). Pieczarka et al., (1989) and DeBoer and DeBruijn (1990) determined that A. paniscus possessed 32 chromosomes, while all the other Ateles taxa had 34 chromosomes, suggesting the first as a separate species from the others (A. paniscus and A. belzebuth). A fourth scheme was proposed by Medeiros et al., (1997) from the analysis of karyotypes from four taxa: 1- A. geoffroyi (including geoffroyi and hybridus); 2- A. fusciceps (fusciceps and rufiventris); 3- A. belzebuth (including belzebuth, chamek and marginatus) and 4- A. paniscus. The authors did not discard the notion of more than the existence of two species (A. paniscus and A. belzebuth). Froehlich et al., (1991) used a fifth classificatory scheme based on discriminant analysis of 76 cranial and dental characters. These authors classified Ateles in three different species, A. paniscus, A. belzebuth (which included A. belzebuth, A. chamek and A. marginatus), and A. geoffroyi (which included A. geoffroyi, A. fusciceps and A. hybridus). Differently, Groves (2001) stated that there were seven different species of Ateles. These were A. paniscus, A. belzebuth, A. chamek, A. hybridus, A. marginatus, A. fusciceps (with two subspecies, A. f. fusciceps and A. f. rufiventris) and A. geoffroyi (with 5 susbspecies, A. g. yucatanensis, A. g.

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vellerosus, A. g. geoffroyi, A. g. ornatus, A. g. griscesens). However, Groves (1989) had previously considered A. belzebuth and A. hybridus to be of the same species. Collins and Dubach (2000a,b; 2001) and Collins (2008) were the first studies to use molecular markers to try and resolve the systematics of Ateles. They used two mitochondrial genes, the hypervariable I portion of the mitochondrial control region and the Cytochrome c Oxidase subunit II gene, COII, and the nuclear Aldolase A Intron V gene. They concluded that four species existed: A. paniscus, A. belzebuth, A. geoffroyi, and A. hybridus. For A. belzebuth they only included samples of chamek and marginatus. Later, Collins (2008), included one sample of belzebuth and thus proposed three subspecies forms of A. belzebuth. For A. geoffroyi, they mentioned three subspecies: A. g. fusciceps, the Northern Central America population (yucatanensis, and vellerosus) and the Southern Central American population. We sequenced three mitochondrial genes (Cytochrome b, Cyt-b; sub-unity cytochrome oxidase I, COI; sub-unity cytochrome oxidase II, COII) from 283 individuals to provide insights on the demographic evolution and systematics of Ateles taxa. This is the largest number of Ateles ever sampled (217 higher than Collins study conducted in 2008). The mitochondrial genes are interesting markers for phylogenetic tasks because they lack introns and include a rapid accumulation of mutations, rapid coalescence time, a negligible recombination rate, and haploid inheritance (Avise et al., 1987). For all of these reasons, mitochondrial gene trees are more precise in reconstructing the divergence history among species than other molecular markers (Moore, 1995). Supporting this, Cummings et al., (1995) showed that mitochondrial genomes have higher information content per base than nuclear DNA. The Cyt-b gene is commonly used among the molecular markers relevant for phylogeography, biosystematics, and genetic structure studies in mammal populations, including primates (Lavergne et al., 2010). The COI gene has emerged as the standard barcode region for animals, including mammals (www.mammaliabol.org) (Hebert et al., 2003, 2004) and the COII gene has been employed extensively in the phylogenetics of different mammalian groups, including primates (Ashley and Vaughn, 1995; Plautz et al., 2009; Ruiz-García et al., 2010, 2011, 2014).

MATERIAL AND METHODS We sequenced three mitochondrial genes in 283 Ateles individuals. The specimens and their geographical origins are as follows. 1-There were a total of 89 A. fusciceps rufiventris individuals sampled in diverse departments of Colombia (28 from Antioquia, 23 from Choco, 10 from Atlantico, one from Sucre, 13 from Cordoba, five from Valle del Cauca and Cauca and nine from Nariño). 2- Five specimens of A. fusciceps fusciceps were sampled from diverse locations of the Pacific area of Ecuador. 3- We sampled 62 A. geoffroyi specimens— representing all the taxa described in Meso-America (seven A. g. ornatus from Costa Rica; nine A. g. panamensis and A. g. azuarensis from Panama and Costa Rica; 16 A. g. geoffroyi from Costa Rica and Nicaragua; 11 A. g. yucatanensis, A. g. vellerosus and A. g. pan from Mexico, Guatemala and Honduras; 18 A. g. frontatus from Costa Rica, and one A. g. grisescens from Choco in Colombia). 4- There were also 24 A. hybridus. This included wild specimens of A. h. hybridus and A. h. brunneus, four animals from Santander, three from Magdalena, one from Norte de Santander, one from Arauca, and three from Antioquia. All of

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these locations are within Colombia. There was also one individual from Maracaibo, Venezuela. Some samples were also collected from European zoos. Three were from the Barcelona Zoo (Catalonia), two from the Erfurt Zoo (Germany), one from the Twycross Zoo (England) and five from Romagne Zoo (France). 5- We sampled a total of 45 A. chamek. Of these, 24 were from diverse areas of Peru. However, two samples were obtained in the Ecuadorian Amazon and another two were obtained in Leticia, within the Colombian Amazon. Another 18 of the 45 were from diverse areas of Bolivia and three were from Brazil. 6- There were also a total of 35 A. belzebuth (15 from Peru, 14 from Ecuador, five from Brazil and one from Colombia). 7- We also sampled 10 A. marginatus (all near Santarem, Pará in Brazil). 8- Additionally, we sampled 13 A. paniscus (one from Surinam, six from French Guiana and six from Brazil). In the early part of this chapter we use the “classical” terminology to name the different Ateles taxa. Later, in the discussion, we revisit the names and validate or reject them pending on our phylogenetic results. Samples were obtained in two ways. In all the countries sampled, with the exception of Costa Rica, we visited different indigenous communities living along major rivers. We requested permission to collect biological materials from either carcasses or live animals that were already present in the community. We sampled small pieces of tissue (muscle) or teeth from hunted animals that were discarded during the cooking process, or hairs with bulbs plucked from live pets. Communities were visited only once, all sample donations were voluntary, and no financial or other inducement was offered for supplying specimens for analysis. Second, in the case of Costa Rica, blood samples were obtained by darting wild monkeys. Five Lagothrix lagotricha individuals from Colombia were used as an out-group. These sampling procedures complied with all relevant Colombian, Peruvian, Ecuadorian, Bolivian, Brazilian, French Guiana, Panamanian, Costa Rican, Guatemalan and Mexican laws. These sampling procedures complied with all the protocols approved by the Ethical Committee of the Pontificia Universidad Javeriana (No. 45677) and the laws of the Ministerio de Ambiente, Vivienda y Desarrollo Territorial (R. 1252) from Colombia. This research also adhered to the American Society of Primatologists’ Principles for the Ethical Treatment of Primates.

Molecular Procedures The DNA from blood and muscle was extracted using the phenol-chloroform procedure (Sambrook et al., 1989), while DNA samples from hairs and teeth were extracted with 10% Chelex resin (Walsh et al., 1991). The 283 spider monkey individuals sampled were sequenced for three mt genes (COI, COII, and Cyt-b). For the mt COI amplification (polymerase chain reaction, PCR), we used the forward primer LCO1490 (5’GGTCAACAAATCATAAAGATATTGG-3’), and the reverse primer HCO2198 (5’TAAACTTCAGGGTGACCAAAAAATCA-3’) (657 base pairs, bp) (Folmer et al., 1994) under the following PCR profile: 94°C for 5 min, followed by 39 cycles of 94°C for 30 s, 44°C for 45 s, 72°C for 45 s, and a final cycle of 72°C for 5 min. For the amplification of the mt COII gene (located in the lysine and asparagine tRNAs) we used the forward primer L6955 (5’ -AACCATTTCATAACTTTGTCAA-3’) and the reverse primer H7766 (5’ CTCTTAATCTTTAACTTAAAAG-3’) (720 bp) (Ashley and Vaughn, 1995; Collins and Dubach, 2000a; Ruiz-Garcia et al., 2010, 2012a,b). The temperatures employed were as

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follows: 95°C for 5 min, 35 cycles of 45 s at 95°C, 30 s at 50°C and 30 s at 72°C and a final extension time for 5 min at 72°C. For both genes, the PCRs were performed in a 25 l volume with reaction mixtures including 4 l of 10 x buffer, 6 l of 3 mM MgCl2, 2 l of 5 mM dNTPs, 2l (8 mM) of each primer, 2 units of Taq DNA polymerase, 5 l of ddH2O and 2 l (20–80 ng/l) of DNA. The mt Cyt-b was amplified by PCR using the procedure of Montgelard et al., (1997) (1,140 bp). The conditions for Cyt-b amplification were performed in 25 l reactions including 2 l of DNA, 2 l of 10 x buffer, 13 l ddH20, 2 l (25 mM) MgCl2, 1 l (10 mM) of each forward and reverse primers, 2l (10 mM) dNTPs, and 2 units of Taq DNA polymerase. The standard thermal cycling program consisted of 10 min at 95°C, 35 cycles of 35 s at 94°C, 35 s at 55°C and 30 s at 70°C and a final extension time for 10 min at 72°C. The total length of the sequences studied for the three genes was 2,517 base pairs (bp). All amplifications, including positive and negative controls, were checked in 2% agarose gels. The gels were visualized in a Hoefer UV Transilluminator. Both mtDNA strands were sequenced directly using BigDye Terminator v3.1 (Applied Biosystems, Inc.). We used a 377A (ABI) automated DNA sequencer. The samples were sequenced in both directions to ensure sequence accuracy.

Data Analysis Molecular Population Analyses We used the following statistics to determine the genetic diversity within the Ateles taxa: number of polymorphic sites (S), haplotypic diversity (Hd), nucleotide diversity (), average number of nucleotide differences (k) and  statistic by sequence. These gene diversity statistics were undertaken in the program DNAsp 5.10 (Librado and Rozas, 2009). To determine possible historical population changes we relied on three procedures we describe here. 1- The mismatch distribution (pairwise sequence differences) was obtained following the method of Rogers and Harpending (1992) and Rogers et al., (1996). We compared the curves obtained assuming constant and non-constant sizes to the empirically observed distribution. We used the raggedness rg statistic (Harpending et al., 1993; Harpending 1994) to determine the similarity between the observed and the theoretical curves. 2- We used the Fu and Li D* and F* tests (Fu and Li, 1993), the Fu FS statistic (Fu, 1997), the Tajima D test (Tajima, 1989), the Strobeck’s S statistic (Strobeck, 1987) and the R2 statistic (Ramos-Onsins and Rozas, 2002), to determine possible population size changes in the fusciceps, geoffroyi, hybridus, chamek, belzebuth, marginatus and paniscus taxa (Simonsen et al., 1995; Ramos-Onsins and Rozas, 2002). Confidence interval at the 95% and probabilities were obtained with 1,000 coalescence permutations. 3- A Bayesian skyline plot (BSP) was obtained for the concatenated mitochondrial sequences by means of the BEAST v. 1.6.2 and Tracer v1.5 software. The Coalescent-Bayesian skyline option in the tree priors was selected with four steps and a piecewise-constant skyline model with 40,000,000 generations (the first 4,000,000 discarded as burn-in). In the Tracer v1.5, the marginal densities of temporal splits were analyzed and the Bayesian Skyline reconstruction option was selected

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for the trees log file. A stepwise (constant) Bayesian skyline variant was selected with the maximum time as the upper 95% high posterior density (HPD) and the trace of the root height as the treeModel.rootHeigh. To determine the time ranges for possible demographic changes of each Ateles taxa, we employed the time splits estimated by Collins and Dubach (2000b). We estimated possible demographic changes for fusciceps (in last 1.5 MY), geoffroyi (2 MY), hybridus (1.5 MY), chamek (2.5 MY), belzebuth (2.5 MY), marginatus (1.5 MY) and paniscus (3 MYA).

Phylogenetic Analyses The sequence alignments were carried out manually and with the DNA alignment program (Fluxus Technology Ltd.). Modeltest Software (Posada and Crandall, 1998) and Mega 6.05 Software (Tamura et al., 2013) were applied to determine the best evolutionary mutation model for the sequences analyzed for the three concatenated gene sequences. Akaike information criterion (AIC; Akaike, 1974) and the Bayesian information criterion (BIC; Schwarz, 1978) were used to determine the best nucleotide evolutionary model. Upon selection of the best model we obtained maximum likelihood estimates of transition/transversion bias as well as maximum likelihood estimates of the gamma parameter for site rates (Tamura et al., 2013). Two kinds of procedures were carried out to estimate genetic heterogeneity, and theoretical gene flow estimates, among the diverse Ateles taxa. They were applied to haplotypic frequencies (GST statistic) and to nucleotide sequences (ST, NST and FST statistics, Hudson et al., 1992; Weir and Hill, 2002). Only one phylogenetic tree was obtained by means of a neighbor-joining tree (NJ; Saitou and Nei, 1987) with the Kimura 2P genetic distance (Kimura, 1980) and is presented here. A future publication will provide additional phylogenetic results of Ateles (Ruiz-García et al., 2016a). Bootstrap analyses (1,000) were performed to determine the more consistent clades. Table 2. Gene diversity statistics for all the Ateles taxa defined and for the mitochondrial genes studied. The statistics estimated were the number of haplotypes (NH), the haplotypic diversity (Hd), the nucleotide diversity (), the average number of nucleotide differences (K) and the  statistic (= 2Ne; Ne = effective female population size;  = mutation rate per generation) by sequence Ateles taxa paniscus chamek belzebuth marginatus fusciceps hybridus geoffroyi

NH 5 24 28 8 38 10 17

Hd 0.628 ± 0.143 0.957 ± 0.014 0.970 ± 0.020 0.933 ± 0.077 0.901 ± 0.023 0.783 ± 0.070 0.883 ± 0.023

 0.0014 ± 0.0001 0.0095 ± 0.0012 0.0142 ± 0.0009 0.0101 ± 0.0003 0.0132 ± 0.0005 0.0147 ± 0.0008 0.0075 ± 0.0002

K 0.974 ± 0.706 6.636 ± 3.192 9.911 ± 4.644 7.044 ± 3.615 9.231 ± 4.281 10.297 ± 1.41 5.239 ± 2.567

 1.289 ± 0.768 18.295 ± 5.48 28.653 ± 8.82 9.544 ± 4.186 19.159 ± 5.01 23.833 ± 8.02 12.137 ± 3.54

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RESULTS Gene Diversity and Historical Demographic Evolution in Ateles Taxa Paniscus yielded the lowest levels of gene diversity (Hd = 0.628,  = 0.0014; k = 0.974) of all the Ateles taxa. A large fraction of remaining taxa showed high levels of gene diversity (Table 2). One taxon (geoffroyi) had intermediate gene diversity values (Hd = 0.883,  = 0.0076; k = 5.239). The hybridus taxon, one of the 25 most endangered primates of the world, did not show especially low levels of mitochondrial gene diversity. Historical demographic analyses by taxon revealed seven trends (Table 3 and Figures 1 and 2): 1. Analyses of paniscus indicated demographic change but it was inconsistent across tests. The Tajima’s D, Fu and Li’s D*, Fu and Li’s F* and the Strobeck’s S statistics did not reveal any appreciable demographic change in this taxon. The BSP analysis showed a constant, but moderate increase in female population size within the last 2.25 MY. However, the Fu’s FS, R2 statistics together with the mismatch pairwise distribution and its associated rg statistic showed a significant population expansion in paniscus. 2. chamek showed multiple indications of population expansion. Five statistics (Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS) showed significant evidence of population expansion as well as the mismatch pairwise distribution and its associated rg statistic did. Also, the BSP analysis yielded a population expansion in the last 0.225 MY. Only the R2 statistic did not reveal any demographic change trend in chamek. 3. belzebuth practically showed the same demographic dynamics as did chamek. Five statistics (Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS) showed significant evidence of population expansion, as did the mismatch pairwise distribution and its associated rg statistic. Also the BSP analysis yielded a population expansion, although in a more complex pattern than chamek did. A first population expansion episode was detected around 2.2 MYA with a moderate increase in the number of females up until about 1.5 MYA. Then, a striking and exponential growth began and lasted until about 1 MYA, when a very slight increase was detected that continued until the present. Only the R2 statistic did not reveal any change of demographic trend in belzebuth. 4. For marginatus the mismatch pairwise distribution and its associated rg statistic as well as the R2 statistic indicated population expansion. The other five statistics also supported population expansion but they were not statistically significant. A. marginatus had the lowest sample size of all the Ateles taxa we sampled and it may have been insufficient to reach statistical significance. However, its demographical evolution could be similar to that observed in chamek and belzebuth. Nevertheless, the BSP analysis for marginatus was different to those of both previously mentioned Amazon Ateles taxa. Its female size was constant until around 50,000 YA where the female population decreased until present day.

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Table 3. Tests for possible historical demographic expansions or contractions in the different Ateles taxa studied by means of three concatenated mitochondrial genes

Tajima D paniscus

chamek

belzebuth

marginatus

fusciceps

hybridus

geoffroyi

P[D ≤ 0.829] = 0.231 P[D ≤ 2.313] = 0.001** P[D ≤ 2.526] = 0.001** P[D ≤ 1.251] = 0.113 P[D ≤ 1.714] = 0.019* P[D ≤ 2.267] = 0.004** P[D ≤ 1.949] = 0.006**

Fu and Li D* P[D* ≤ 0.509] = 0.269 P[D* ≤ 3.812] = 0.002** P[D* ≤ 4.189] = 0.001** P[D* ≤ 1.406] = 0.115 P[D* ≤ 3.646] = 0.005** P[D* ≤ 3.983] = 0.001** P[D* ≤ 2.895] = 0.009**

(A) Fu and Li F* P[F* ≤ 0.672] = 0.265 P[F* ≤ 3.897] = 0.003** P[F* ≤ 4.291] = 0.001** P[F* ≤ 1.543] = 0.095 P[F* ≤ 3.406] = 0.005** P[F* ≤ 4.042] = 0.001** P[F* ≤ 3.032] = 0.007**

Fu’s Fs P[Fs ≤ 1.82] = 0.041* P[Fs ≤ 7.414] = 0.019* P[Fs ≤ 12.675] = 0.001** P[Fs ≤ 1.113] = 0.232 P[Fs ≤ 9.318] = 0.019* P[Fs ≤ 2.537] = 0.863 P[Fs ≤ 1.520] = 0.335

raggedness rg P[rg ≤ 0.0281] = 0.0032** P[rg ≤ 0.038] = 0.330 P[rg ≤ 0.046] = 0.522 P[rg ≤ 0.018] = 0.026* P[rg ≤ 0.031] = 0.627 P[rg ≤ 0.043] = 0.395 P[rg ≤ 0.057] = 0.257

R2 P[R2 ≤ 0.108] = 0.0043** P[R2 ≤ 0.189] = 0.530 P[R2 ≤ 0.174] = 0.458 P[R2 ≤ 0.082] = 0.028* P[R2 ≤ 0.121] = 0.719 P[R2 ≤ 0.123] = 0.335 P[R2 ≤ 0.117] = 0.638

* P < 0.05 and ** P < 0.01, significant population expansions. For the statistics of Tajima D, Fu and Li D*, Fu and Li F*, Fu’s Fs, rg and R2 (A); for the statistic of Strobeck S (B)

Ateles taxa paniscus chamek belzebuth marginatus fusciceps hybridus geoffroyi

(B) Strobeck S P = 0.101 P = 0.0001** P = 0.0001** P = 0.174 P = 0.0001** P = 0.085 P = 0.073

5. A. hybridus only showed three statistics that indicated a possible population expansion (Tajima’s D, Fu and Li’s D* and Fu and Li’s F*). However, the other statistics (Strobeck’s S, Fu’s FS and R2) did not detect any relevant population change. Neither the mismatch pairwise distribution nor its associated rg statistic revealed any significant population expansion. The BSP analysis detected a continuous but slight increase in the female population size from 1 MY to 0.25 MY. Since that time the female population has either been constant or has slightly decreased as it has in the last 15,000-20,000 years. 6. Five different statistical tests supported fusciceps as having population expansions (Tajima’s D, Fu and Li’s D*, Fu and Li’s F*, Strobeck’s S and Fu’s FS). In contrast,

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. the mismatch pairwise distribution and its associated rg statistic and the R2 statistic did not reveal any significant population expansion. The BSP analysis revealed a constant, or very slight decrease, in the last 1.5 MY up until about 30,000 YA when a very striking increase in the size of the female population began and continued until today. 7. geoffroyi showed a similar situation with that observed in fusciceps. Four statistics revealed population expansion (Tajima’s D, Fu and Li’s D*, Fu and Li’s F* and Strobeck’s S), but the mismatch pairwise distribution and its associated rg statistic, the Fu’s F and the R2 statistics did not. The BSP analysis was practically identical to that of fusciceps with more or less a constant female population up until 30,00040,000 YA. After this point the female population size hardly increased.

Therefore, we can distinguish four different demographic trajectories in these Ateles taxa (1- paniscus, 2- chamek, belzebuth and marginatus, 3- hybridus and 4- fusciceps and geoffroyi).

(A)

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(D)

(E) Figure 1. (Continued).

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(F)

(G) Figure 1. Mismatch distributions (pairwise sequence differences) at the three concatenated mitochondrial DNA genes (COI, COII and Cyt-b) for the different Ateles taxa considered: paniscus (A), chamek (B), belzebuth (C), marginatus (D), hybridus (E), fusciceps (F) and geoffroyi (G).

Molecular Phylogenetic Inferences The BIC and the Akaike information criterion highlighted the best nucleotide substitution model for the three concatenated genes we sequenced. For BIC, the best model was HKY + G (16,404.049), whilst for the Akaike information criterion it was GTR + G (10,479,003). The maximum likelihood estimate of transition/transversion bias was 3.01 (maximum log likelihood = 4,731.25).

Historical Genetic Demography...

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(B)

(C) Figure 2. (Continued).

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(G) Figure 2. Bayesian skyline plot analysis (BSP) to determine possible demographic changes across the natural history of the different Ateles taxa considered: paniscus (A), chamek (B), belzebuth (C), marginatus (D), hybridus (E), fusciceps (F) and geoffroyi (G). On the x-axis, time in millions of years; on the y-axis, effective population size of females.

Table 4. Genetic heterogeneity and gene flow (Nm) statistics among all the Ateles taxa considered Genetic differentiation estimated  = 1629.633 df = 756 HST = 0.0860 KST = 0.5858 KST* = 0.3944 ZS = 8,130.8037 ZS* = 8.3433 Snn = 0.9198

P NS 0.0000* 0.0000* 0.0000* 0.0000* 0.0000* 0.0000*

Gene flow γST = 0.5938 NST = 0.6747 FST = 0.6729

Nm = 0.34 Nm = 0.24 Nm = 0.24

*Significant Probability (P < 0.05); NS = No significant

All of the genetic heterogeneity tests that simultaneously compared the “a priori” Ateles taxa were significant with the exception of the contingency 2 table (Table 4). The heterogeneity statistics NST (= 0.675) and FST (= 0.673) showed considerable sequence differentiation among the Ateles taxa. Indirect historical gene flow estimates were lower than 1 (Nm = 0.24 for both statistics), and thus low enough to consider these taxa as different species. However, the haplotype genetic heterogeneity statistic, GST = 0.089, was considerably lower and the associated gene flow estimate (Nm = 5.10) showed an appreciable genetic connectivity among these Ateles taxa. Indeed, if we look for genetic heterogeneity by Ateles taxa pairs, two considerations can be made. First, all comparisons implicating paniscus were those which showed the highest levels of genetic heterogeneity (FST values higher than 0.75), which means that paniscus is the most differentiated Ateles taxon. Second, there were some comparisons with FST values lower than 0.50 (medium value of genetic differentiation). These were the cases, geoffroyi-fusciceps (both Northern Ateles taxa) and chamek-belzebuth, chamek-marginatus and belzebuth-marginatus (all Amazonian Ateles taxa).

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The Kimura 2P genetic distances among Ateles taxa are shown in Table 5. The largest genetic distances were around 4% and were between the following pairs: fusciceps fuscicepsbelzebuth (4.3%), fusciceps fusciceps-hybridus (4.0%), geoffroyi-hybridus (4.0%) and belzebuth-hybridus (3.9%). The smallest values (around 2%) were between belzebuthmarginatus (2.4%), geoffroyi-fusciceps rufiventris (2.1%), chamek-belzebuth (1.9%), fusciceps rufiventris-fusciceps fusciceps (1.9%) and chamek-marginatus (1.7%). We generated an NJ tree with the Kimura 2P genetic distances (Figure 3) of the 283 Ateles individuals analyzed. Clearly, the 13 individuals of paniscus (Surinam, French Guiana and two different areas from northern Brazilian Amazon) formed a monophyletic clade (100% bootstrap) well differentiated from the remaining Ateles clades. Two other large clades were found (58%). The first differentiated clade (56%) mainly contained three Amazon Ateles taxa: chamek, belzebuth and marginatus. The first two morphological taxa were intermixed across all the clades. There were eight sub-clades with bootstrap percentages higher than 70%. 1- One chamek group contained eight individuals from Bolivia and Peru (70%). 2- A chamekbelzebuth group had five individuals from Peru (86%). 3- One little chamek cluster contained two individuals from the Nanay River (Peru) (70%). 4- Another chamek group included three individuals from Peru and Bolivia (88%). 5- A small belzebuth cluster contained two individuals from Ecuador (88%). 6- A belzebuth clade consisted of seven individuals from Ecuador, Peru and Brazil (86%). 7- A small belzebuth cluster contained two individuals from Peru (76%). 8- Finally, one chamek group had five individuals from Bolivia and Brazil (74%). Within this large clade, there was a monophyletic clade consisting of 10 marginatus individuals although the bootstrap was only 38% (within this clade, there was a cluster of three marginatus individuals from the area of Santarem, Brazil, with 72%). Another interesting fact is that this large Amazonian clade contained three individuals, which belonged to the northern Caribbean Colombia and Venezuela and trans-Andean Ateles populations. One geoffroyi grisescens individual was sampled in the Colombian Choco and appeared within the chamek haplotypes. Also, there were two fusciceps rufiventris individuals (one from Monte Libano, Cordoba, Colombia and other from Antioquia, Colombia) which presented haplotypes related to belzebuth haplotypes. Table 5. Kimura (1980) 2P genetic distances among the different Ateles taxa considered. Below, genetic distance values in percentages (%); above, standard errors in percentages (%) Ateles taxa 1 2 3 4 5 6 7 8 9

1 3.0 3.5 2.9 3.1 3.7 3.7 3.0 11.5

2 0.7 1.9 1.7 3.4 3.8 3.7 3.2 11.8

3 0.7 0.3 2.4 3.8 4.3 3.9 3.7 12.8

4 0.6 0.4 0.5 3.4 2.4 3.6 3.4 12.7

5 0.6 0.6 0.7 0.6 1.9 3.7 2.1 12.4

6 0.6 0.7 0.7 0.7 0.3 4.0 2.6 13

7 0.7 0.7 0.7 0.7 0.6 0.6 4.0 12.9

8 0.6 0.6 0.7 0.7 0.4 0.4 0.7 12.1

1 = A. paniscus; 2 = A. chamek; 3 = A. belzebuth; 4 = A. marginatus; 5 = A. fusciceps rufiventris; 6 = A. fusciceps fusciceps; 7 = A. hybridus; 8 = A. geoffroyi; 9 = Lagothrix lagotricha

9 1.4 1.5 1.6 1.6 1.5 1.6 1.5 1.5 -

Historical Genetic Demography... Ateles fusciceps rufiventris 99 Uraba COL Ateles fusciceps rufiventris 100 Uraba COL 56 Ateles fusciceps rufiventris 75 Antioquia COL 64 Ateles fusciceps rufiventris 15 Risaralda COL Ateles fusciceps rufiventris 47 Antioquia COL 36 Ateles fusciceps rufiventris 35 Valle Cauca COL 6 Ateles fusciceps rufiventris 59 Nariño COL Ateles fusciceps fusciceps 122 Manabí Ecuador 22 Ateles fusciceps rufiventris 38 ValleCauca COL 40 10 Ateles fusciceps rufiventris 23 Choco COL 76 Ateles fusciceps rufiventris 39 ValleCauca COL Ateles fusciceps rufiventris 42 Antioquia COL 14 Ateles fusciceps rufiventris 21 Cordoba COL 40 6 Ateles fusciceps rufiventris 27 Cordoba COL Ateles fusciceps fusciceps 119 Manabí Ecuador Ateles fusciceps fusciceps 124 Santa Elena Ecuador 56 Ateles fusciceps fusciceps 121 Manabí Ecuador Ateles fusciceps fusciceps 120 Manabí Ecuador Ateles fusciceps rufiventris 14 Choco COL Ateles fusciceps rufiventris 63 Cordoba COL 68 Ateles fusciceps rufiventris 98 Uraba COL 28 Ateles fusciceps rufiventris 76 Antioquia COL Ateles fusciceps rufiventris 22 Chocó COL Ateles fusciceps rufiventris 20 Chocó COL Ateles fusciceps rufiventris 11 Chocó COL Ateles fusciceps rufiventris 36 ValleCauca COL Ateles fusciceps rufiventris 54 Nariño COL Ateles fusciceps rufiventris 77 Antioquia COL Ateles fusciceps rufiventris 93 Chocó COL Ateles fusciceps rufiventris 29 Darien COL Ateles fusciceps rufiventris 94 PNLosKatios COL 40 Ateles fusciceps rufiventris 19 Cordoba COL Ateles chamek 116 Yarinacocha PER 66 Ateles fusciceps rufiventris 91 Atlantico COL Ateles fusciceps rufiventris 4 Atlantico COL Ateles fusciceps rufiventris 9 Chocó COL Ateles fusciceps rufiventris 16 Chocó COL Ateles fusciceps rufiventris 19 Choco COL Ateles fusciceps rufiventris 21 Chocó COL Ateles fusciceps rufiventris 24 Chocó COL 40 Ateles fusciceps rufiventris 26 Chocó COL Ateles fusciceps rufiventris 27 Atlántico COL Ateles fusciceps rufiventris 29 Atlántico COL Ateles fusciceps rufiventris 30 Atlántico COL Ateles fusciceps rufiventris 31 Atlántico COL Ateles fusciceps rufiventris 32 Atlántico COL Ateles fusciceps rufiventris 34 Atlántico COL Ateles fusciceps rufiventris 40 Antioquia COL Ateles fusciceps rufiventris 41 Antioquia COL Ateles fusciceps rufiventris 51 Nariño COL Ateles fusciceps rufiventris 52 Nariño COL Ateles fusciceps rufiventris 53 Nariño COL 74 Ateles fusciceps rufiventris 55 Nariño COL Ateles fusciceps rufiventris 56 Nariño COL Ateles fusciceps rufiventris 57 Nariño COL Ateles fusciceps rufiventris 58 Nariño COL Ateles fusciceps rufiventris 62 SahagunCordoba COL Ateles fusciceps rufiventris 66 LosCordobas Cordoba COL Ateles fusciceps rufiventris 68 Montelibano Cordoba COL Ateles fusciceps rufiventris 74 Antioquia COL 66 62

Figure 3. (Continued).

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fusciceps rufiventris 44 Antioquia COL fusciceps rufiventris 50 Antioquia COL 54 fusciceps rufiventris 10 Choco COL fusciceps rufiventris 43 Antioquia COL fusciceps rufiventris 45 Antioquia COL 34 fusciceps rufiventris 46 Antioquia COL fusciceps rufiventris 48 Antioquia COL fusciceps rufiventris 49 Antioquia COL fusciceps rufiventris 60 Choco COL fusciceps rufiventris 12 Choco COL 54 fusciceps rufiventris 13 Choco COL fusciceps rufiventris 17 Choco COL fusciceps rufiventris 18 Choco COL fusciceps rufiventris 25 Choco COL 22 fusciceps rufiventris 37 Cauca COL fusciceps rufiventris 61 Montilibano Cordoba COL fusciceps rufiventris 64 LosCordobas Cordoba COL fusciceps rufiventris 65 LosCordobas Cordoba COL fusciceps rufiventris 96 Antioquia COL Ateles fusciceps rufiventris 86 Turbo Antioquia COL 92 Ateles fusciceps rufiventris 87 Choco COL Ateles geoffroyi ornatus 51 CostaRica 82 Ateles geoffroyi ornatus 52 CostaRica Ateles geoffroyi ornatus 50 CostaRica Ateles geoffroyi ornatus 23 CostaRica 96 Ateles geoffroyi ornatus 19 CostaRica 82 Ateles geoffroyi ornatus 40 CostaRica Ateles geoffroyi ornatus 61 CostaRica 36 Ateles geoffroyi panamensis/azuarensis 56 CostaRica Ateles geoffroyi panamensis/azuarensis PAN 16 Ateles geoffroyi panamensis/azuarensis 53 CostaRica Ateles geoffroyi panamensis/azuarensis 45 CostaRica 24 Ateles geoffroyi panamensis/azuarensis 43 CostaRica Ateles geoffroyi panamensis/azuarensis 20 CostaRica 44 Ateles geoffroyi panamensis/azuarensis 11 Boquete PAN 100 46 Ateles geoffroyi panamensis/azuarensis 11B Boquete PAN Ateles geoffroyi panamensis/azuarensis 13 Boquete PAN Ateles geoffroyi geoffroyi 3 CostaRica Ateles geoffroyi geoffroyi 8 CostaRica Ateles geoffroyi geoffroyi 9 CostaRica Ateles geoffroyi geoffroyi 10 CostaRica Ateles geoffroyi geoffroyi 11 CostaRica Ateles geoffroyi geoffroyi 13 CostaRica Ateles geoffroyi geoffroyi 14 CostaRica Ateles geoffroyi geoffroyi 17 CostaRica 72 Ateles geoffroyi geoffroyi 18 CostaRica Ateles geoffroyi geoffroyi 22 CostaRica Ateles geoffroyi geoffroyi 35 CostaRica Ateles geoffroyi geoffroyi 38 CostaRica 26 Ateles geoffroyi geoffroyi 48 CostaRica Ateles geoffroyi geoffroyi 49 Nicaragua Ateles geoffroyi geoffroyi 58 Nicaragua Ateles geoffroyi geoffroyi 63 Nicaragua 60 Ateles geoffroyi vellerosus 14 HONDURAS Ateles geoffroyi yucatensis Yucatan MEX 36 Ateles geoffroyi vellerosus 18 Peten GUAT Ateles geoffroyi vellerosus 75 Peten GUAT Ateles geoffroyi pan 74 Peten GUAT Ateles geoffroyi vellerosus 73 Peten GUAT 46 Ateles geoffroyi vellerosus 72 Peten GUAT Ateles geoffroyi vellerosus 71 Peten GUAT Ateles geoffroyi vellerosus 70 Peten GUAT Ateles geoffroyi yucatensis 67 Yucatan MEX Ateles geoffroyi yucatensis 66 Yucatan MEX

Historical Genetic Demography... Ateles geoffroyi frontatus 12 CostaRica Ateles geoffroyi frontatus 42 CostaRica 52 Ateles geoffroyi frontatus 33 CostaRica Ateles geoffroyi frontatus 65 CostaRica Ateles geoffroyi frontatus 59 CostaRica Ateles fusciceps rufiventris 89 Sucre COL 62 Ateles geoffroyi frontatus 55 CostaRica Ateles geoffroyi frontatus 47 CostaRica 26 Ateles geoffroyi frontatus 21 CostaRica Ateles geoffroyi frontatus 4 CostaRica Ateles geoffroyi frontatus 7 CostaRica Ateles geoffroyi frontatus 15 CostaRica Ateles geoffroyi frontatus 16 CostaRica 26 Ateles geoffroyi frontatus 28 CostaRica Ateles geoffroyi frontatus 29 CostaRica Ateles geoffroyi frontatus 32 CostaRica Ateles geoffroyi frontatus 41 CostaRica Ateles geoffroyi frontatus 57 CostaRica Ateles geoffroyi frontatus 60 CostaRica 90 Ateles fusciceps rufiventris 28 Atlantico COL Ateles fusciceps rufiventris 33 Atlantico COL Ateles belzebuth 14 Mocogua PER Ateles hybridus 7 BarcelonaZOO SPA Ateles hybridus 10 Santander COL 68 Ateles hybridus 13 ErfurtZOO GER Ateles hybridus 12 TwycrossZOO UK Ateles hybridus 11 RomagneZOO FRA 60 Ateles hybridus 10 RomagneZOO FRA Ateles hybridus 9 BarcelonaZOO SPA 66 Ateles hybridus 8 BarcelonaZOO SPA Ateles hybridus 6 RomagneZOO FRA 60 Ateles hybridus 2 Magdalena COL Ateles hybridus 25 Arauca COL Ateles hybridus 17 Maracaibo VEN Ateles hybridus 4 RomagneZOO FRA 18 68 Ateles hybridus 24 PTOWilches Santander COL 36 44 Ateles hybridus 9 PTOWilches Santader COL Ateles fusciceps fusciceps 59 ECU Ateles fusciceps rufiventris 69 Antioquia COL Ateles hybridus 11 Norte Santader COL 94 Ateles hybridus 68 Norte Santader COL 26 Ateles fusciceps rufiventris 70 Antioquia COL Ateles fusciceps rufiventris 71 Antioquia COL 24 Ateles fusciceps rufiventris 72 Antioquia COL Ateles fusciceps rufiventris 78 Antioquia COL Ateles fusciceps rufiventris 79 Antioquia COL Ateles fusciceps rufiventris 80 Antioquia COL 20 Ateles fusciceps rufiventris 97 Cordoba COL Ateles hybridus 2 Antioquia COL Ateles hybridus 4 Antioquia COL Ateles hybridus 1 Magdalena COL Ateles hybridus 3 Magdalena COL Ateles hybridus 5 RomagneZOO FRA Ateles hybridus 3 Antioquia COL 6 Ateles hybridus 14 ErfurtZOO GER Ateles chamek 60 PER Ateles marginatus 5 Santarem Pará BRA 38 Ateles marginatus 8 Santarem Pará BRA 60 Ateles marginatus 4 Santarem Pará BRA 20 Ateles marginatus 2 Santarem Pará BRA Ateles marginatus 2B Santarem Pará BRA 38 42 Ateles marginatus 1 Santarem Pará BRA Ateles marginatus 7 Santarem Pará BRA Ateles marginatus 1B Santarem Pará BRA 58 44 Ateles marginatus 3 Santarem Pará BRA 72 Ateles marginatus 6 Santarem Pará BRA 56

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Figure 3. (Continued).

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. Ateles chamek 3 STACruz BOL Ateles chamek 4 STACruz BOL Ateles chamek 26 Javari River BRA Ateles chamek 3 BOL 52 Ateles chamek 15 STACruz BOL 74 Ateles chamek 24 Branco River Acre BRA 70 Ateles chamek 6 STACruz BOL Ateles chamek 8 STACruz BOL Ateles belzebuth 69 Pastaza ECU Ateles belzebuth 52 Pastaza ECU Ateles belzebuth 9 Napo ECU Ateles belzebuth 50 Pastaza ECU 48 Ateles belzebuth 54 Morona Santiago ECU Ateles belzebuth 11 Nanay River PER 2 Ateles fusciceps rufiventris 73 Antioquia COL 16 48 Ateles belzebuth 5 Negro River BRA 54 Ateles belzebuth 15 Huila COL Ateles belzebuth 46 Amazon River PER Ateles belzebuth 62 Orellana ECU 18 10 Ateles belzebuth 39 SanMartin PER Ateles belzebuth 45 Amazon River PER 18 Ateles belzebuth 38 SanMartin PER 42 42 Ateles belzebuth 37 SanMartin PER Ateles belzebuth 42 Negro River BRA Ateles belzebuth 65 Pastaza ECU 42 34 Ateles belzebuth 51 Pastaza ECU 26 10 Ateles belzebuth 48 Amazon River PER 76 Ateles belzebuth 47 Amazon River PER 0 76 Ateles belzebuth 66 Pastaza ECU Ateles belzebuth 64 Napo ECU Ateles belzebuth 61 PER 86 Ateles belzebuth 43 Negro River BRA 8 Ateles belzebuth 8 Orellana ECU Ateles belzebuth 7 Negro River BRA Ateles belzebuth 16 Negro River BRA Ateles belzebuth 40 SanMartin PER Ateles belzebuth 53 Pastaza ECU Ateles belzebuth 57 Napo ECU 8 24 88 Ateles belzebuth 56 Morona Santiago ECU Ateles fusciceps 67 Montelibano Cordoba COL Ateles belzebuth 13 Huallaga River PER Ateles belzebuth 41 Huallaga River PER 26 58 50 Ateles belzebuth 12 SanMartin PER Ateles chamek 41 Mocagua PER Ateles chamek 32 Chazuta PER 88 Ateles chamek 79 BOL 68 Ateles chamek 42 20 Ateles chamek 51 Ucayali River PER Ateles chamek 49 Ucayali River PER 36 Ateles chamek 53 Ucayali River PER 48 42 Ateles chamek 47 Ucayali River PER Ateles chamek 45 Madre de dios PER

56

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Ateles chamek 64 Santa Rosa BOL Ateles chamek 78 BOL Ateles chamek 11 Beni River BOL Ateles chamek 55 Nanay River PER 54 Ateles chamek 58 Ballivian BOL Ateles geoffroyi grisescens 68 Choco COL 56 Ateles chamek 30 In. Leticia PER Ateles chamek 43 Loreto PER Ateles chamek 59 STA Ana Yacuma BOL Ateles chamek 81 STA Ana Yacuma BOL Ateles chamek 5 STA Cruz BOL Ateles chamek 63 Madidi BOL Ateles chamek 25 Maderia River BRA Ateles chamek 38 Nanay River PER 70 Ateles chamek 40 Nanay River PER Ateles chamek 123 Nanay River PER 10 Ateles belzebuth 44 Nanay River PER 86 Ateles chamek 56 Nanay River PER Ateles chamek 37 Nanay River PER Ateles chamek 26 In. Leticia PER 32 Ateles chamek 7 STA Cruz BOL Ateles chamek 35 Nanay River PER Ateles chamek 48 Ucayali River PER 32 Ateles chamek 50 Ucayali River PER Ateles chamek 52 Ucayali River PER 70 Ateles chamek 77 BOL Ateles chamek 46 Ucayali River PER 0 8 Ateles chamek 80 BOL Ateles paniscus 24 Surinam 46 Ateles paniscus 25 Trombetas River BRA Ateles paniscus 22 Trombetas River BRA Ateles paniscus 23 Trombetas River BRA Ateles paniscus 1 French Guiana Ateles paniscus 2 French Guiana Ateles paniscus 3 French Guiana Ateles paniscus 4 French Guiana Ateles paniscus 6 Uatama River BRA Ateles paniscus 8 Uatama River BRA Ateles paniscus 10 French Guiana Ateles paniscus 9 Uatama River BRA Ateles paniscus 11 French Guiana 60

48 100

26

24

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Lagothrix lagotricha 2 Lagothrix lagotricha 1 Lagothrix lagotricha 5 Lagothrix lagotricha 3 100 Lagothrix lagotricha 4

88

Figure 3. Neighbor-Joining tree with the Kimura (1980) 2P genetic distance with the 283 spider monkeys (Ateles) studied for three concatenated mitochondrial genes (COI, COII and Cyt-b). The number in the nodes are bootstrap percentages.

The second large differentiated clade mainly contained Ateles individuals from the Northern Caribbean in Colombia and Venezuela. It also had trans-Andean Ateles individuals (hybridus, fusciceps rufiventris, fusciceps fusciceps and different geoffroyi taxa). Many individuals of these taxa were intermixed within this large clade.

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There were six clusters within this large clade. 1- One cluster contained fusciceps fusciceps-fusciceps rufiventris-hybridus and had 16 individuals from Ecuador and Colombia and two hybridus individuals from one French zoo and Germany zoo respectively (94%); 2There was a small cluster of fusciceps rufiventris composed of two individuals from the Atlantic Department (Colombia) (90%); 3- One large cluster contained 55 geoffroyi individuals. They belonged to seven described morphological taxa of the Central American Ateles (yucatanensis, vellerosus, pan, frontatus, geoffroyi, panamensis, and azuarensis). There was also one fusciceps rufiventris from the Sucre Department (Colombia) (72%); 4One geoffroyi cluster only had individuals from Costa Rica (ornatus) (96%); 5- Another little cluster of fusciceps rufiventris had two individuals from Turbo (Antioquia) and from the Choco Department, both in Colombia (92%). 6- Finally, there was another large cluster containing fusciceps rufiventris from all the Colombian Departments where this species lives as well as all the fusciceps fusciceps individuals from Ecuador (except one). Similar to the large Amazonian clade, there were also some individuals of Amazon origins in the big Northern Caribbean Colombia and Venezuela/trans-Andean clade. One individual was classified as belzebuth from Caballococha (Amazon River, Peru), and related to haplotypes of hybridus, fusciceps fusciceps and fusciceps rufiventris. Also, one individual was classified as chamek from Yarinacocha, Ucayali River (Peru). It was within the main fusciceps rufiventrisfusciceps fusciceps haplotype clade.

DISCUSSION Current IUCN Classifications and the Evolutionary Demographics of Ateles IUCN classified paniscus as being in the least dangerous situation relative to all other Ateles taxa. It was listed at low risk in 1996, of least concern in 2000 and 2003, and in Nucleus II and vulnerable in 2008. However, our mitochondrial data showed this taxon to have the lowest gene diversity levels and no clear evidence of population expansions throughout its evolutionary history. Therefore, its evolutionary potential could be one of the lowest of the Ateles taxa, and this is not recorded in the IUCN classifications. A. chamek was classified by IUCN as in low risk (1996), least concern (2000, 2003), in Nucleus II and endangered (2008). However, the mitochondrial data showed that this taxon had a relatively high gene diversity levels and clear evidence of significant population expansions across its evolutionary history. Additionally, Ruiz-García et al., (2006) showed this taxon to have the highest gene diversity levels (expected heterozygosity and average number of alleles) for a set of nuclear DNA microsatellites. The estimated size of the wild population for this species was greater than 10,000 animals and was considered to be in a safe status according to Stevenson et al., (1992). Ruiz-García et al., (2006) used nuclear microsatellites to estimate the historically effective population size to be 8,000-61,000 and 6,000-47,600 based on coalescence and maximum likelihood procedures, respectively. If IUCN has changed the status of this taxon in the last two decades it is probably due to intense hunting pressure and habitat deforestation, even though this taxon inhabits the Western Amazon (Bolivia, Peru and western Brazilian Amazon), where the Amazon is still fairly preserved. However, the current genetic findings do not consider these circumstances and

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IUCN’s classification does not take into consideration that this is an Ateles taxon with great evolutionary potential. A similar situation is found for belzebuth. IUCN had classified this taxon as vulnerable but now it is considered endangered. However, both mitochondrial and nuclear DNA microsatellite markers showed elevated gene diversities. They also showed strong population expansions. The effective population size ranged from 4,000 to 36,000 and from 8,300 to 66,400 for the two procedures (Ruiz-García et al., 2006). Additionally, chamek and belzebuth, although they have very different coat colors, belonged to the same species. This complicates IUCN’s classifications. Thus, the evolutionary potential of belzebuth could be as important as that of chamek. IUCN has always classified marginatus as endangered. It is not affected by current human pressure but perhaps by climatological changes during the last 50,000 YA. About 80,000-70,000 YA, it was extremely cold during the early Pleni-glacial, which was the first extreme cold period in the fourth glaciation. Absy et al., (1991), Van der Hammen (1992) and Van der Hammen and Absy (1994) analyzed the vegetation in Carajas (Eastern Amazon) covering a time period starting approximately 65,000-51,000 YA. They determined that this current area of the Amazon forest was a savannah that probably formed during an extension of the early Pleni-glacial period. Liu and Colinvaux (1985) and Colinvaux and Liu (1987) analyzed Ecuadorian Andean valleys and the Amazon and concluded that the elevation limit of vegetation descended 27,000-34,000 YA. The temperature also descended 4.5°C during the middle and the upper Pleni-glacial periods. For example, one of the dryer and colder periods of the middle Pleni-glacial period occurred 30,000 YA. Bogota lagoon was dried at that period (Van der Hammem, 1992). Afterwards, another period of extreme cold occurred around 23,000 to 20,000 YA (Dryas I) (MacNeish, 1979). The Last Glacial Maximum (LGM) occurred around 19,000-16,500 YA. It was the moment where the extension of the snow reached the maximum in the Central Andes. Finally, around 14,500 to 12,000 YA (Dryas II), the cold was extreme at times. The glaciers reached their maximum surface area in the Manachaque Valley (Cordillera Blanca), in the Upismayo-Jalacocha at the Vilcanota Cordillera in Peru, in the Nevado of ChoqueYapu in Bolivia and in the Chimborazo in Ecuador. Rodbell and Seltzer (2000) showed that the glaciers in the Cordillera Blanca (San Martin Department in Peru) reached their maximum elevation (3,170-3,827 masl) approximately 12,000 YA. Today its elevations is 4,600 masl. Maslin and Burns (2000) showed that the Amazon River reached its maximum dry peak around 16,000-15,000 YA. This dry period continued until about 12,000 YA. The analysis with O18 isotopes revealed that the mouth of the Amazon River had only 40% of the water that it has today. In the tropical forest, the temperature decreased by 2°C to 6°C and in some areas as the Fuquene Lagoon near Bogota, the precipitation was only half of what it is today (Van der Hammen, 1992). The temperature of the Atlantic Ocean near the Brazilian coasts descended 6°C (Clark, 2002). These climatological changes could be more responsible for the limited geographic distribution of marginatus and its population decrease compared to current factors. The IUCN classification is probably more useful to hybridus because of its critically endangered status in 2008. Interestingly, the mitochondrial data showed that hybridus is not particularly impoverished in its gene diversity. It also has elevated amounts of mitochondrial gene diversity. However, Ruiz-García et al., (2006) found that A. hybridus showed the lowest levels of expected heterozygosity and average number of alleles for a set of microsatellites compared to other taxa of Ateles. Stevenson et al., (1992) claimed that only 100 to 1,000

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individuals of this taxon still exist in the wild. Ruiz-García et al., (2006) estimated the effective historical population size (based on the two procedures) to be 3,000-26,000 and 2,900-23,300 respectively. Ruiz-García (2003b), using other theoretical models, determined possible effective numbers for hybridus. These estimates from the average heterozygosity with the infinite allele and the step-wise mutation models showed values from 997 to 5,334. The fact that the mitochondrial gene diversity is not especially low for this taxon but the nuclear DNA microsatellite diversity is very low should agree quite well with a very recent genetic bottleneck in hybridus caused by human activity. Note, mitochondrial diversity can show ancestral diversity in a taxon and microsatellite diversity can show more recent diversity. However, the beginning of this decrease in genetic variability along with low effective numbers, could have begun around 15,000-20,000 YA as the BSP analysis registered. As we previously explained Dryas I, LGM and Dryas II could have affected hybridus because many forest of current Northern Colombia and Venezuela were highly affected. Correlated with this, Ruiz-García et al., (2015) detected a strong population decrease 14,000 YA for the lowland tapir population of Northern Colombia and Northwestern Venezuela (Tapirus terrestris). This is same area where hybridus inhabits, agreeing quite well with the situation of this population, which was considered critically threatened in 2004 by the IUCN (Constantino et al., 2006). This parallel population decrease of hybridus and T. terrestris in the same geographical area is related to the massive extinction of mammals across the Earth. This corresponds with the Younger Dryas (Dryas III), typical of Northern Europe and Scandinavia (Clapperton, 1993). The Younger Dryas was more drastic in the northern part than the southern part of South America. Thus, the situation of hybridus as one of the 25 most endangered primates in the world (2008-2010) (Mittermeier et al., 2009) seems justified. The case of fusciceps is more complex. IUCN has always classified fusciceps fusciceps as critically endangered and its populations has dropped by 90% in the last 45 years. Ateles fusciceps rufiventris was classified as vulnerable (1996, 2000, 2003), and then was recategorized as critically endangered in 2008. The geographic distribution of fusciceps fusciceps is restricted to some very small areas of the Pacific coast of Ecuador and this could correlate with its classification as critically endangered. However, the current study is the first to analyze this taxon at the molecular scale and we demonstrate that fusciceps fusciceps is undifferentiated from fusciceps rufiventris. A. fusciceps showed elevated mitochondrial gene diversity as well as elevated nuclear microsatellite diversity. Stevenson et al., (1992) estimated the wild population of this taxon to be about 1,000-3,000 individuals, which is extremely low compared to the effective number obtained by Ruiz-García et al., (2006) (12,000-95,000 and 12,300-99,000 respectively). As we will discuss in brief, both mitochondrial and microsatellites results detected two different groups of fusciceps rufiventris individuals (all had black coats), one more related to geoffroyi and other the more related to hybridus. The second group yielded the highest levels of microsatellite diversity. This was probably due to gene admixture involving hybridus which in turn overestimate the real effective numbers of fusciceps rufiventris. Nevertheless, the IUCN classification did not reflect the internal diversity within fusciceps or the high evolutionary potential of this Ateles taxon. Finally, geoffroyi (taken as a whole) was classified by the IUCN as a taxon of least concern in 2003, but as endangered in 2008. Both the mitochondrial gene and nuclear microsatellite diversities of goeffroyi were low. It was the second lowest after paniscus in the

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mitochondrial case and after hybridus in the microsatellite case. Also, after hybridus, it yielded the second lowest effective size numbers: 3,000-28,000 and 2,800-22,600. Its classification as endangered could reflect its census situation as well as its evolutionary potential. We can appreciate that the IUCN’s classification of a taxon helps to determine its potential endangered status. Sometimes the classification agrees with gene diversity levels and predictions of evolutionary potential (for instance, hybridus and geoffroyi). Other times it does not (for instance, chamek, belzebuth, fusciceps, paniscus).

New Insights on the Systematics of Ateles Our molecular results, the most complete to date for mitochondrial analyses, suggest the existence of only two (more conservative perspective) or three Ateles species, both valid for the biological and phylogenetic species concepts. If we take into consideration only the existence of monophyletic clades in a tree for defining species, then, only two Ateles species are recognized: A. paniscus (Linnaeus, 1758) and A. belzebuth (Geoffroy, 1806). A. paniscus (Linnaeus, 1758) was the first derived taxon from the hypothetical ancestor of the current spider monkeys. All the other haplotypes of different geographical origins and morphotypes were intermixed to some degree and the bootstrap percentages of the main clades within this large cluster were relatively low. As we showed, one of the main sub-clades contained mostly Amazonian individuals and one geoffroyi grisescens (Colombian Choco) and two fusciceps rufiventris individuals (Colombia). A second main sub-clade consisted of Northern South American and trans-Andean individuals. Additionally, there was one belzebuth from the Amazon River (Peru) and one chamek from Ucayali River (Peru). These are cases of genetic introgression more than recent hybridization (Ruiz-García et al., 2014) because there are large geographical distances among the specimens. This means that the split among these taxa is very recent. The elapsed time is probably short and no post and pre-zygote isolation mechanisms have emerged, constituting a unique species (Barton and Bengtsson, 1986; Coyne et al., 1994; Antonovics, 2006). This view agrees absolutely with the result of Pieczarka et al., (1989) with A. paniscus having 32 chromosomes, while all the other Ateles taxa had 34 chromosomes. Following this perspective within A. belzebuth some subspecies could be proposed. Within the Amazonian area four subspecies could be distinguished and are described here. 1. A. b. belzebuth (Geoffroy, 1806), includes the traditional belzebuth and chamek morphotypes (they are color variations of the same taxa; belzebuth [Geoffroy, 1806], has priority over chamek, [Humboldt, 1812]). From a coat pelage perspective, Elliot (1913) identified specimens with pelage characteristics of belzebuth in the territory of chamek (Chamicuros in the Huallaga River, Peru). We support their observations of hybrids between A. chamek and belzebuth at the Loreto Region in the Peruvian Amazon. Indeed, one of these specimens was enclosed in this study (phenotype of belzebuth with mitochondrial DNA of chamek). Belzebuth and chamek did not show monophyletic clades within the Amazonian clade and they were intermixed. Chamek presented the highest levels of gene sequence diversity at the aldolase A intron gene of all the taxa (Collins and Dubach, 2001). This aligns with the fact that chamek was

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. the taxon with the highest microsatellite gene diversity (Ruiz-García et al., 2006), thus suggesting chamek as the ancestral spider monkey clade. Medeiros et al., (1997) also concluded that chamek could represent the ancestral karyotype for Ateles. The finding of the 6b chromosome in A. chamek provided an important element to the identification of this taxon as the ancestor within this genus. Dutrillaux et al., (1986) found that 6b corresponded to 2 chromosomes within Lagothrix and Brachyteles. Therefore, since the 6b form only occurs in chamek, Lagothrix and Brachyteles it was interpreted as the ancestral form of Ateles. However, our mitochondrial gene diversity level for chamek is similar to other Ateles taxa. But, chamek individuals are the first to diverge within the Amazon clade. Thus, chamek (or better, the black morphotype of A. b. belzebuth) could be more related to the origins of the Amazon Ateles clade, but it is clear that the ancestor of A. paniscus was the first of the current Ateles taxa. In addition, Ruiz-García et al., (2006) revealed a strong connection between chamek and belzebuth by means of nuclear microsatellites. Therefore, chamek could also be the origin of belzebuth studied in Peru, Ecuador and Colombia. In time, the ancestor of belzebuth originated hybridus and probably crossed the Eastern Andes Cordillera. It originated the second fusciceps rufiventris population (Antioquia, Sucre, Bolivar, Córdoba, Atlantico). We have evidence that belzebuth crossed the Eastern Andes Cordillera. Brother Apolimar Maria (1913) recorded a specimen from the Tolima Department in the upper Magdalena Valley which was practically identical to A. belzebuth individuals that inhabited the eastern piedmont of the Eastern Andes. We have even located Ateles bones from the Huila Department in various Colombian museums on the other side of the Eastern Andes Cordillera. Indeed, we sampled and enclosed in this analysis one belzebuth individual from the Huila Department. This exemplar was molecularly undifferentiated from northern Brazilian and Ecuadorian belzebuth individuals. Hernández-Camacho and Cooper (1976) mentioned the existence of a zoological passage east of the Eastern Andes Cordillera into the upper Magdalena Valley. Several species of Primates such as Cebus apella, Lagothrix lagotricha lugens and Saimiri cassiquiarensis albigena, as well as other vertebrate species, have crossed this passage. It’s also possible that belzebuth originated fusciceps, and later, as our mitochondrial results support, the ancestor of Atlantic fusciceps originating hybridus. 2. A. b. marginatus (Geoffroy, 1809), which formed a monophyletic clade within the Amazonian group. 3. A. b. hybridus (Geoffroy, 1829) is the first derived taxon from the original ancestor of Northern South American and trans-Andean spider monkeys. One belzebuth haplotype was related to hybridus, which could mean that A. b. belzebuth originated to A. b. hybridus, although it could also be generated from fusciceps. This taxon showed an intense recent hybridization with the black spider monkeys (traditionally named fusciceps) that live on the other side of the Magdalena River. Probably, the black coat of fusciceps is dominant to the clear coat of hybridus and these hybrids are black (and thus morphologically classified as fusciceps) but have the mitochondrial DNA of A. b. hybridus. These frequent cases of recent hybridization (gene flow) were between fusciceps males x hybridus females. The reverse crossing of hybridus males with fusciceps females, cannot be detected by only studying mitochondrial DNA because the individuals have a black coat and contain mitochondrial fusciceps

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DNA. The gene flow between both morphotypes is still probably higher than we detected. Ruiz-García et al., (2006), by means of nuclear microsatellites, also detected a strong hybridization between hybridus and fusciceps. They showed that the AP74 microsatellite yielded a wide range of allele sizes. Hybridus basically only presented small sized alleles (130-132 base pairs), whereas belzebuth, chamek and the Atlantic fusciceps rufiventris population yielded these same alleles in addition to some larger sized alleles. However, other Ateles taxa only showed large sized alleles (fusciceps fusciceps, paniscus, geoffroyi and the Choco Pacific fusciceps rufiventris population). This supports that hybridus intensively hybridizes with the nearest Atlantic fusciceps populations. By using the nuclear microsatellite results, Ruiz-García et al., (2006) speculated on the origins of hybridus. Two hypotheses were described. It could be derived from belzebuth or from the Atlantic fusciceps rufiventris population by founder effect, although founder effect affects microsatellites less than other markers such as isoenzymes or plasma proteins. The possibility that hybridus arose because of founder effect and genetic drift could provide an explanation of why Collins and Dubach (2000a) found different relationships among the hybridus and the other Ateles clades. The parsimony analysis of the control region did not relate hybridus to any other clade, whereas the distance-based analysis clustered hybridus with A. geoffroyi/A. f. rufiventris with a bootstrap support of 65%. In contrast, the combined mitochondrial gene neighbor-joining analysis placed hybridus next to the clades of A. b. chamek/A. b. marginatus and A. geoffroyi/A. f. rufiventris. Now, we know that the differential results of Collins and Dubach (2000a) were influenced by a reduced number of hybridus specimens. Therefore, they did not detect the “gradient” effect which closely relates hybridus with fusciceps. Now, the microsatellite results, together with the new mitochondrial data, show that Amazonian haplotypes (like we found in belzebuth) could have originated hybridus. It could also be derived from a fusciceps population, which in turn was generated by belzebuth. This agrees with some authors, who concluded that hybridus could have originated directly from belzebuth. Or, it could be from the Magdalena River Valley and Atlantic fusciceps rufiventris population. From a chromosomal view, both origins are possible because hybridus has 6a, 6c, 7b and 14b chromosomes, while 7b is found in fusciceps rufiventris and 6c is found in belzebuth. On the other hand, these nuclear and mitochondrial results do not support the link between paniscus and hybridus as it was proposed by Medeiros et al., (1997) and based on chromosomal analysis (both taxa shared the 7b chromosome). The similarities of chromosome pair 7 between paniscus and other trans-Andean Ateles forms are not compatible with the molecular results. However, this chromosome could be polymorphic in A. belzebuth and in other Ateles individuals not yet studied and the apparent relationship between A. paniscus and the trans-Andean Ateles could be spurious. In fact, Collins and Dubach (2000a) showed that the haplotypes of hybridus did not exhibit the 25-base pair control region deletion that occurs in all the haplotypes of A. paniscus. On the other hand, Kunkel et al., (1980) detected the same 6c and 7b chromosomes in geoffroyi and hybridus in the same way as Froehlich et al., (1991) had revealed with morphometrics. This last study supported that hybridus and fusciceps were conspecifics just as our mitochondrial results supported.

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. Thus, our results showed that the western cordillera of the Andes and the Cauca River were not barriers to gene flow between fusciceps rufiventris and hybridus, such as was claimed by Collins and Dubach (2000a). Kunkel et al., (1980) and Medeiros et al., (1997) claimed that hybridus and fusciceps could be two different taxa such as karyotype analyses have revealed. These authors speculated that fusciceps rufiventris could be reproductively isolated from geoffroyi and hybridus because of the differences in chromosome pairs (5 and 6) but we have clearly showed high gene flow between fusciceps and hybridus. Also, Rossan and Baerg (1977) determined hybridization between fusciceps rufiventris and geoffroyi panamensis and they noted the location of a hybridization zone. Also, recall that no river has formed a barrier among Ateles species with the exception, may be, of some black-water rivers draining the Guianan highlands for A. paniscus (Collins and Dubach, 2000b). Indeed, Collins (2008) suggested, following Brown (1987) and Colinvaux (1993, 1996), that at the beginning of the Pliocene the gene flow across the Amazon Basin among Ateles populations should have been high. The river draining of this basin was not as massive as it is today and later the black-water drainage and associated unsuitable habitat of the Guianan shield (Froehlich et al., 1991; Ayres and Clutton-Brock, 1992; Norconk et al., 1996) split the ancestor of A. paniscus from the ancestor of the remaining Ateles (3.3-3.6 MYA). Probably, the split of A. paniscus should be more related to the existence of a relatively recent Amazon Lake in the Late Pliocene until the middle Pleistocene (by eustatic marine changes). This event connected the Orinoco and Amazon basins after its separation during the Miocene (Hoorn et al., 2010) and it could isolate the Guianan ancestor of the current A. paniscus. This agrees quite well with the views of Campbell (1990), Frailey et al., (1998), Klammer (1984), Marroig and Cerqueira (1997), Nores (1999, 2004) and Rossetti et al., (2005). A point supporting this large Amazon lake is the existence of a Pliocene (5 MYA) sea rise of 100 m for a duration of 0.8 MY (Haq et al., 1987).Thus, marine transgressions could have had a great influence on biotic diversification within the Amazon and it could also have affected the diversification in Ateles in the beginning. However, Medeiros et al., (1997) did not discount the possibility that animals with different karyotypes produce fertile offspring. This integrative evidence suggests that hybridus, fusciceps and geoffroyi constituted a unique species if we agree with our three Ateles hypothesis. In Table 6, we show the results of karyotypes found in different Ateles taxa. In some cases, the possible karyotype evolution correlated well with the molecular results. Nevertheless, in other cases, there was no agreement between karyotypes and molecules. Contrarily, for the Alouatta genus both karyotype and molecular results were very similar and this allows us to reconstruct the phylogeny and systematics of the howler monkeys with a very high precision (see Ruiz-García et al., 2016b in this book). This was not the case for Ateles, probably because the polymorphisms of several chromosomes in this genus is higher than the currently detected due to the small sample studied. Defler (2003) distinguished two possible subspecies of hybridus, A. h. hybridus, from the major part of the geographical range of this species, and A. h. brunneus from along the lower Cauca and Magdalena Rivers in the departments of Bolivar, Antioquia and Caldas. Animals of the second subspecies have different coat colors from those of the first subspecies. However, in the current study we did not find evidence of molecular differences

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between individuals of A. hybridus from both geographical areas for either microsatellites or mitochondrial DNA. Therefore, at a molecular level we could not differentiate between these two possible hybridus taxa. It’s likely that brunneus shows some pelage differences from the other hybridus population because its degree of mixture with fusciceps rufiventris is also higher. 4. A. b. geoffroyi (Kuhl, 1820) consisted of all Meso-American spider monkey individuals plus the fusciceps individuals where no hybridization with hybridus was detected. Therefore, two “fusciceps” groups were detected, one strongly mixed with hybridus and other more related to the Meso-American spider monkeys (the traditional geoffroyi). No mitochondrial differentiation was observed between the Colombian-Panamanian fusciceps rufiventris (= robustus) and the endemic Ecuadorian fusciceps fusciceps. The black or brown head pelage and the dorsum glossy black and the dorsum brownish black of both morphotypes probably do not have any phylogenetic signal. Under this molecular perspective and due to the limitations of the Linnaeus typological systematic nomenclature, the continuous transition of the fusciceps taxon between A. b. hybridus and A. b. geoffroyi is lost because hybridus (Geoffroy, 1829) and geoffroyi (Kuhl, 1820) precede fusciceps (Gray, 1866). Moreover, this classification questions all the subspecies based on pelage by Kellog and Goldman (1944). Indeed, this questioning was made by SilvaLopez et al., (1996) who showed that the typical white forehead patch of A. belzebuth is also present in many individuals of A. g. vellerosus. Furthermore, in most cases it is impossible to differentiate the pelage of A. g. vellerosus from that of A. g. yucatanensis. Maybe under a less strict point of view three Ateles species could be sustained agreeing absolutely with the morphological classificatory scheme of Froehlich et al., (1991): 1. A. paniscus. Our results ratify the molecular results of Collins and Dubach (2000a,b) and the isozyme analysis of Sampaio et al., (1993), who determined a genetic distance of 0.149 between paniscus and chamek, which is incompatible with the fact that both forms were subspecies of A. paniscus. Thus, A. paniscus is a monotypic species. 2. A. belzebuth (which should include the previous A. b. belzebuth, which in turn includes chamek, and A. b. marginatus). It’s interesting to remark that all the Amazonian Ateles forms could be a unique species, despite their different coat colors. Collins and Dubach (2000b) estimated that the initial diversification within A. belzebuth began around 2.4 MYA (beginning in the Pleistocene). Maybe the different color patterns could be related with some Pleistocene refugia (Napo Refugia, or island, for the belzebuth coat pattern, Inambari Refugia, or island, for the chamek coat pattern and the Pará Refugia, or island, for the marginatus coat pattern) if we follow the Pleistocene Refugia hypothesis (Haffer, 1969, 1982, 1987, 1997, 2008) or even if we follow the Recent Amazon Lake hypothesis (Nores, 1999, 2004). However, the Ateles populations, which survived in these refugia, should be of large size because the spider monkeys need specific habitat requirements (for instance, primary, evergreen and never flooded forests) and large territories (frugivore specialist). Thus, these populations could contain elevated levels of gene diversity.

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. This could prevent some modes of speciation (Mayr, 1954, 1963) and when refugia were again reconnected the different morphotypes colonized other refugia and it helping to mix the different Ateles populations and thus speciation did not occur. The chromosomal analysis of Nieves et al., (2005) showed a trichotomy among belzebuth, chamek and marginatus, which is also interpreted as a unique species. Collins and Dubach (2000b) and Collins (2008) claimed that the Pleistocene refugia hypothesis provides little support for the speciation events in Ateles, because some important splits in the spider monkeys were during the Pliocene. Still, Haffer (1997, 2008) showed that the dry-wet cycles in the Amazon (and other areas) were also present during Miocene and Pliocene. Thus, the importance of these dry-wet refugia could not be discarded for the beginning of the speciation processes in Ateles. 3. A. geoffroyi. In this case, this species contained three sub-species, A. g. hybridus, A. g. fusciceps and A. g. geoffroyi, with intense gene flow occurring among the three subspecies. This fact was also revealed through morphological observations. Indeed, Kellog and Goldman (1944) detected variable pelage characteristics within populations and intergraded where population distributions overlapped. This intergradation occurs among geoffroyi panamensis, fusciceps rufiventris and geoffroyi grisescens at the interface of Colombia and Panama. Rossan and Baerg (1977) located sympatric populations of geoffroyi and fusciceps in Eastern Panama, where both populations had hybridized to some extent along a contact zone. In addition, an Ateles specimen collected inside the territory of fusciceps robustus, (Catival, San Jorge River in Colombia) had a strong admixture of light-colored hairs on the back, similar to the hybridus phenotype (Hernández-Camacho and Cooper, 1976). Collins and Dubach (2000a) placed the two former species A. geoffroyi (Central America) and A. fusciceps, (Pacific Colombia and Ecuador and Northern Colombia) in the same group. The aldolase A gene also clustered two A. geoffroyi yucatanensis haplotypes with two A. f. robustus haplotypes but with a very low bootstrap support of 48%. Therefore, the aldolase A gene provided limited support compared to the mitochondrial genes for the inclusion of both the Central America A. geoffroyi and the South American A. fusciceps as one species (Collins and Dubach, 2001). Additionally, Froehlich et al., (1991) concluded in their morphological variation study that A. geoffroyi and A. fusciceps belonged to the same species. The coat colors of the two taxa are distinct overall, with the general trend of the production of darker pelages at the extremes of the Central American Isthmus (Konstant et al., 1985).However, although microsatellite results supported statistical significant differences among fusciceps rufiventris, geoffroyi and hybridus (RuizGarcía et al., 2006), our mitochondrial sequence analysis revealed a strong connection among these three taxa—considering them as a possible unique species. The Choco Pacific fusciceps rufiventris population diverged clearly from the Atlantic fusciceps rufiventris population for both microsatellites and mitochondrial DNA. However, the first population was practically identical to fusciceps fusciceps individuals studied from Ecuador for microsatellites. Our mitochondrial DNA that we previously discussed, also supported this. On the other hand, either of the two Colombian fusciceps rufiventris populations could have originated geoffroyi.

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In whichever case, our molecular evolutionary and population point of view (Darwin, 1859; Dobzhansky, 1937; Mayr, 1942, 1963; Simpson, 1944) applied to the systematics of Ateles is not compatible with the use of hybridus brunneus, fusciceps fusciceps, fusciceps rufiventris, and all the traditional subspecies within A. geoffroyi. This problem stems from the fact that the Linnaeus classificatory scheme is Aristotelian, scholastic and the scheme was created to classify static organisms that people thought would never evolve because they were created by God. Thus, in many cases there would be conflict between this typological philosophy and the evolutionary process which is continuous. As Wendt (1958) explained, there was intense criticism from French philosophers regarding reductive typological philosophy of Linnaeus (Montaigne, Gassendi, La Mettrie, but especially Buffon, 1749-1788: “Nature knows no systems”). However, the Linnaeus´s view was rapidly adopted by the scientists of his time because it was easy to understand and follow. Interestingly, some years later Linnaeus was aware that species evolved (case of Peloria monstruosa and the hybrid research of Réaumur). Later, he deleted comments about the invariability of species from Systema Naturae (1759). For instance, if we molecularly study three or four persons, with very marked physical characteristics from different continents (Africans, Europeans, Asians, Australians and Amerindians), we will probably generate a tree where each monophyletic clade is integrated by the individuals of each continent. In this case, we will probably claim that each continent contains a discrete race, or if we are working with other organisms, subspecies. This is the situation we find in many molecular systematic works. But what happens if we sample thousands and thousands of individuals, and not in just a few very discrete populations, but in many locations that span the greater geographical part of each continent? The perfect monophyletic clades for each core area will disappear. It’s probably that some populations share some characteristics (maybe morphological or biochemical, and, probably, for some characters but not for others) but the differences among distant populations should be more continuous or clinal than discrete ones. This is especially true if these distant populations correspond to individuals of the same species and the gene flow is not interrupted. This does not mean that some geographical races do not exist. Only some morphological characters (affected by positive natural selection or by gene drift) showed differences but not discretely, but rather as a gradient and the degree of pre-speciation was very limited. For this last reason, it may be better to use the term, geographic races, which doesn’t imply pre-speciation processes, as does the subspecies term. This is the situation of the human species (see Lewontin, 1972), and probably also of Ateles. It is very difficult to explain gradient differences with a typological discrete classification scheme such as the Linnaeus system. In this work (283 Ateles individuals), as well as that by Ruiz-García et al., (2016c this volume) (452 capuchin individuals), the number of individuals and the number of localities (distribution ranges) were greatly increased. With these additions we can begin to detect the “gradient” effect versus the “discrete” effect. In the case of Ateles, this is especially evident for hybridus. Collins and Dubach, (2000a,b, 2001), Collins, (2008) (molecular results) and Nieves et al., (2005) (chromosomal results) concluded that hybridus is a full species because this taxon conformed a monophyletic clade differentiated from fusciceps and geoffroyi. But they only analyzed three hybridus and four fusciceps specimens, in the molecular analysis. When we sequenced 94 fusciceps and 24 hybridus, we easily detected numerous hybrids.

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Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al. Table 6. Karyomorphs of Ateles taxa

Ateles taxa A. paniscus A. chamek A. belzebuth A. marginatus A. fusciceps rufiventris A. hybridus A. geoffroyi

Chromosome 5 Form c Form a Form a Form a Form b

Chromosome 6 Forms a, e Forms b, e Forms c, d Form d Form d

Chromosome 7 Form b Form a Form a Form a Form b

Chromosome 13 Form b Form a Form a Form a Form a

Chromosome 14 Form b Form a Form a Form a Form a

Form a Forms a, b

Form a, c Form c

Form b Form b

Form a Form a

Form b Form a

The most relevant conclusion of this work is that the real number of Ateles species (two or three) is considerably lower that the seven species currently accepted by the vast majority of the systematics and primatologists (Groves, 2001). What is a reasonable explanation of this difference? We suggest that earlier authors may have adopted a reductive view of the phylogenetic species concept without first securing enough samples and localities studied from a neutral molecular perspective. Indeed, the highest genetic distances among Ateles taxa is around 4% and many taxa pair comparisons are around 2-3%. For the three genes studied, other authors determined genetic distance values for different species within a genus of around 11% (for Cyt-b and COI; Bradley and Baker, 2001; Kartavtsev, 2011) and 6% (for COII; Ascunce et al., 2003). Clearly this shows the strong genetic relationships among the Ateles taxa. Maybe, the most important systematic contribution of this work is to show that hybridus is not a full and independent species as suggested by Collins and Dubach (2000a,b) and Nieves et al., (2005) because multiple individuals of hybridus and fusciceps shared similar or identical mitochondrial haplotypes.

ACKNOWLEDGMENTS Thanks to Dr. Diana Alvarez, Armando Castellanos, Andrés Laguna, Fernando Nassar, Luz Mercedes Botero, Marcela Ramírez, Luis Carrillo, Dr. B de Thoisy and Andrés Eloy Bracho for their respective help in obtaining spider monkey samples during the last 20 years. Thanks to Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Muñoz), to the Ministerio del Ambiente (permission HJK-9788) in Coca (Ecuador), to the Peruvian Ministry of Environment, PRODUCE (Dirección Nacional de Extracción y Procesamiento Pesquero), Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales from Peru, and to the Colección Boliviana de Fauna (Dr. Julieta Vargas) and to CITES Bolivia for their role in facilitating the obtainment of the collection permits in Colombia, Peru and Bolivia. The Costa Rican spider monkeys were sampled with the collection permits approved by the Costa Rican government to Dr. Gustavo Gutiérrez-Espeleta. Likely, All animal sampling in French Guiana was carried out in accordance with French animal care regulations and laws. Thanks also go to ARCAS (Guatemala) for providing hair samples of Ateles. Thanks also to the many people of diverse Indian tribes in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua,

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Angoteros, Orejón, Cocama, Kishuarana and Alamas), Bolivia (Sirionó, Canichana, Cayubaba and Chacobo), Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri and Yucuna) and Ecuador (Kichwa, Huaorani, Shuar and Achuar) who helped us to obtain samples of spider monkeys.

REFERENCES Absy, M. L., Cleef, A., Fournier, M., Martin, L., Servant, M., Siffedine, A., Ferreira da Silva, M., Soubies, F., Suguio, K., Turcq, B. and Van Der Hammen, T. (1991). Mise en évidence de quatre phases d’ouverture de la foret dense dans le sud-est de l’Amazonie au cours des 60 000 dernières années. Première comparaison avec d’autres régions tropicales. C. R. Acad. Sci. Paris., 312, 673 - 678. Akaike, H. (1974). A new look at the statistical model identification. IEEE Transactions on Automatic Control, AC-, 19, 716–723. Antonovics, J. (2006). Evolution in closely adjacent populations X: long term persistence of pre-reproductive isolation at a mine boundary. Heredity, 97, 33-37. Ascunce, M. S., Hasson, E. and Mudry, M. D. (2003). COII: a useful tool for inferring phylogenetic relationships among New World monkeys (Primates, Platyrrhini). Zoologica Scripta, 32, 397-406. Ashley, M. V. and Vaughn, T. A. (1995). Owl monkeys (Aotus) are highly divergent in mitochondrial cytochrome c oxidase (COII) sequences. International Journal of Primatology, 5, 793–807. Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, J. E., Reeb, C. A. and Saunders, N. C. (1987). Intraspecific phylogeographic: the mitochondrial DNA bridge between population genetics and systematics. Annual Review of Ecology, Evolution, and Systematics, 18, 489-522. Ayres, J. M. C. and Clutton-Brock, T. H. (1992). River boundaries and species range size in Amazonian primates. American Naturalist, 140, 531-537. Barton, N. and Bengtsson, B. O. (1986). The barrier to genetic exchange between hybridizing populations. Heredity, 57, 573-576. Bradley, R. D. and Baker, R. J. (2001). A test of the genetic species concept: cytochrome-b sequences and mammals. Journal of Mammalogy, 82, 960–973. Brown, K. S. (1987). Conclusions, synthesis, and alternative hypotheses. In Whitmore, T. C., Prance, G. T. (Eds). Biogeography and Quaternary history in Tropical America. (pp. 175210). Claredon Press, Oxford. Buffon, G. L. L. (1749-1788). Histoire naturelle, générale et particulière, avec la description du Cabinet du Roy., 36, volumes. Campbell, K. E. (1990). The geologic basis of biogeographic patterns in Amazonia. In Peters, G. and Hutterer, R. (Eds.). Vertebrates in the Tropics. (pp. 33-32). Bonn: Alexander Koenig Zoological Research Institute. Clapperton, C. (1993). Quaternary Geology and Geomorphology of South America. Ed. Elsevier. Amsterdam, The Netherlands. Clark, P. U. (2002). Early deglaciation in the Tropical Andes. Science, 298, 7a.

470

Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al.

Colinvaux, P. (1993). Pleistocene biogeography and diversity in tropical forests of South America. In Goldblatt, P., (Ed.). Biological Relationships between Africa and South America. (pp. 473-499). New Haven: Yale University Press. Colinvaux, P. (1996). Quaternary environmental history and forest diversity in the Neotropics. In Jackson, J. B. C., Budd, A. F., and Coates, A. G. (Eds.). Evolution and environment in Tropical America. (pp. 359-406). University of Chicago Press, Chicago. Colinvaux, P. A. and Liu, K. (1987). The late Quaternary climate of the western Amazon basin. In Berger, W., and Labegrie, L. (Eds.). Abrupt Climatic Change. (pp. 113-122). Riedel Dordrech. Collins, A. C. (1999). Species status of the Colombian spider monkey, Ateles belzebuth hybridus. Neotropical Primates, 7, 39-41. Collins, A. C. (2008). The taxonomic status of spider monkeys in the twenty-first century. In Campbell, C. J. (Ed). Spider monkeys. Behavior, ecology and evolution of the genus Ateles. (pp. 50-78). Cambridge University Press. Collins, A. C. and Dubach, J. M. (2000a). Phylogenetic relationships of spider monkeys (Ateles) based on mitochondrial DNA variation. International Journal of Primatology, 21, 381–420. Collins, A. C. and Dubach, J. M. (2000b). Biogeographic and Ecological forces responsible for speciation in Ateles. International Journal of Primatology, 21, 421-444. Collins, A. C. and Dubach, J. M. (2001). Nuclear DNA variation in spider monkeys (Ateles). Molecular Phylogenetics and Evolution, 19, 67–75. Constantino, E., Lizcano, D., Montenegro, O. and Solano, C. (2006). Danta Común. In Libro Rojo de los Mamíferos de Colombia. (pp. 1-433). Conservación Internacional Colombia, Ministerio de Ambiente, Vivienda y Desarrollo Territorial. Bogotá, Colombia. Coyne, J., Mah, K. and Cristianshen, A. (1994). Genetics of a pheromonal difference contributing to reproductive isolation in Drosophila. Science, 265, 1461-1464. Cummings, M. P., Otto, S. P. and Wakeley, J. (1995). Sampling properties of DNA sequence data in phylogenetic analysis. Molecular Biology and Evolution, 12, 814–822. Darwin, C. (1859). On the Origin of Species by Means of Natural Selection or the Preservation of Favoured Races in the Struggle for Life. (first edition), John Murray, London. deBoer, L. E. M. and deBruijn, M. (1990). Chromosomal distinction between the red-faced and black-faced black spider monkeys (Ateles paniscus paniscus and A. p. chamek). Zoo Biology, 9, 307-316. Defler, T. R. (2003). Primates de Colombia. Colombia, Bogotá: Conservación Internacional. Dobzhansky, Th. (1937). Genetics and the origin of species. Columbia University Press, New York. Dutrillaux, B., Couturier, J. and Viegas-Pequinot, E. (1986). Evolution chromosomique des Plathyrriniens. Mammalia, 50, 56-81. Elliot, D. G. (1913). A review of the primates. Vols. I and II. American Museum of Natural History, New York. Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294-299.

Historical Genetic Demography...

471

Frailey, C. D., Lavina, E. L., Rancy, A. and Pereira de Souza, J. (1988). A proposed Pleistocene/Holocene lake in the Amazon Basin and its significance to Amazonian geology and biogeography. Acta Amazonica, 18, 119-143. Froehlich, J. W., Supriatna, J. and Froehlich, P. H. (1991). Morphometric analysis of Ateles: Systematics and biogeographic implications. American Journal of Primatology, 25, 1-22. Fu, Y-X. (1997). Statistical tests of neutrality against population growth, hitchhiking and background selection. Genetics, 147, 915-925. Fu, Y. and Li, W. (1993). Statistical Tests of Neutrality of Mutations. Genetics, 133, 693-709. García, M. M., Caballín, M. R., Aragones, J., Goday, C. and Egozcue, J. (1975). Banding patterns of the chromosomes of Ateles geoffroyi with description of two cases of pericentric inversion. Journal of Medical Primatology, 4, 108-113. Groves, C. P. (1989). A theory of human and primate evolution. Claredon Press, Oxford. Groves, C. P. (2001). Primate taxonomy. Washington, DC: Smithsonian Institution Press. Haffer, J. (1969). Speciation in Amazonian forest birds. Science, 165, 131-137. Haffer, J. (1982). General aspects of the refuge theory. In Prance, G. T., (Ed.). Biological diversification in the tropics. New York: Columbia University. pp. 6-24. Haffer, J. (1987). Quaternary history of tropical America. In Whitmore, T.C., and Prance, G.T. (Eds.). Biogeography and Quaternary history in tropical America. Oxford: Clarendon and Oxford University Press. pp. 1-18. Haffer, J. (1997). Alternative models of vertebrate speciation in Amazonia: an overview. Biodiversity Conservation, 6, 451-476. Haffer, J. (2008). Hypotheses to explain the origin of species in Amazonia. Brazilian Journal of Biology 68, 917-947. Haq, B. U., Hardenbol, J. and Vail, P. R. (1987). Chronology of fluctuating sea levels since the Triassic. Science, 235, 1156–1167. Harpending, H. C. (1994). Signature and ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Human Biology, 66, 591-600. Harpending, H. C., Sherry, S. T., Rogers, A. R. and Stoneking, M. (1993). Genetic structure of ancient human populations. Current Anthropology, 34, 483-496. Hebert, P. D. N., Ratnasingham, S. and de Waard, J. R. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London B, 270 (Suppl.), S96-9. Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. and Francis, C. M. (2004). Identification of birds through DNA barcodes. PLoS Biology, 2, 1657-63. Hernández-Camacho, J. and Cooper, R. W. (1976). The nonhuman primates of Colombia. In Thorington Jr RW, and Heltne PG (Eds.), Neotrop. Primates: Field Studies and Conservation (pp. 35-69). Washington D.C.: National Academy of Sciences. Hershkovitz, P. (1968). Metachromisms or the principle of evolutionary change in mammalian tegumentary colours. Evolution, 22, 556-575. Hershkovitz, P. (1969). The evolution of mammals in southern continents. VI. The recent mammals of the neotropical region: a zoogeographic and ecological review. Quarterly Review of Biology, 44, 1-70. Hershkovitz, P. (1972). The recent mammals of the Neotropical region: A zoogeographic and ecological review. In Keast, A.F., Erk, C., and Glass, B. (Eds.), Evolution, mammals and southern continents. (pp. 311-431). Albany, State University of New York.

472

Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al.

Hill, W. C. O. (1962). Primates Comparative Anatomy and Taxonomy. V. Cebidae. Part. B. Edinburgh University Press, Edinburgh. Hoorn, C. and Wesselingh, F. P. (2010). Amazonia: landscape and species evolution. A look into the past. Wiley-Blackwell Publishing Ltd. pp. 1-446. Hudson, R. R., Boss, D. D. and Kaplan, N. L. (1992). A statistical test for detecting population subdivision. Molecular Biology and Evolution, 9, 138-151. Jacobs, S. C., Larson, A. and Cheverud, J. M. (1995). Phylogenetics relationships and orthogenetic evolution of coat color among tamarins (genus Saguinus). Systematic Biology, 44, 512-532. Kartavtsev, Y. (2011). Divergence at Cyt-b and Co-1 mtDNA genes on different taxonomic levels and genetics of speciation in animals. Mitochondrial DNA, 22, 55-65. Kellog, R. and Goldman, E. A. (1944). Review of the spider monkeys. Proceedings of the United States Natural Museum, 96, 1-45. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111-120. Klammer, G. (1984). The relief of the extra-Andean Amazon basin. In Sioli, H. (Ed.). The Amazon. Limnology and landscape ecology of a mighty tropical river and its basin. (pp. 47-83). Dordrecht: Junk Publishers. Konstant, W., Mittermeier, R. A. and Nash, S. D. (1985). Spider monkeys in captivity and in the wild. Primate Conservation, 5, 82-109. Kunkel, L. M., Heltne, P. G. and Borgaonkar, D. S. (1980). Chromosomal variation and zoogeography in Ateles. International Journal of Primatology, 1, 223-232. Lavergne, A., Ruiz-García, M., Catzeflis, F., Lacote, S., Contamin, H., Mercereau-Puijalon, O., Lacaste, A. and Thoisy, B. (2010). Taxonomy and phylogeny of squirrel monkey (genus Saimiri) using cytochrome b genetic analysis. American Journal of Primatology, 72, 242-253. Lewontin, R. (1972). The apportionment of human diversity. Evolutionary Biology, 6, 391398. Librado, P. and Rozas, J. (2009). DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-1452 | doi: 10.1093/bioinformatics/btp187. Liu, K. B. and Colinvaux, P. A. (1985). Forest changes in the Amazon basin during the last glacial maximum. Nature, 318, 556-557. MacNeish, R. S. (1979). The early man remains from Pikimachay Cave, Ayacucho Basin, Highland Peru. In Humprey, R. L., Standford, D. (Eds.). Pre-Llano cultures of the Americas: Paradoxes and possibilities. Washington D.C.: Anthropological Society of Washington. pp. 1-47. María Brother Appolinar, (1913): Catálogo del Museo del Instituto de La Salle. Bol. Soc. Cienc. Nat. Inst. La Salle, 1. Bogotá. Marroig, G. and Cerqueira, R. (1997). Plio-Pleistocene South American history and the Amazon Lagoon hypothesis: a piece in the puzzle of Amazonian diversification. Journal of Computational Biology, 2, 103-119. Maslin, M. A. and Burns, S. J. (2000). Reconstruction of the Amazon Basin effective moisture availability over the past 14,000 years. Science, 290, 2285-2287. Mayr, E. (1942). Systematics and the Origin of Species. New York, Columbia University Press.

Historical Genetic Demography...

473

Mayr, E. (1954). Changes in genetic environment and evolution. In Huxley, J., Hardy, A.C., Ford, E. B., (Eds). Evolution as a Process. Allen and Unwin. London. Pp. 157-180. Mayr, E. (1963). Animal Species and Evolution. Harvard University Press: Cambridge, Massachusetts. Medeiros, M. A., Barros, R. M. S., Pieczarka, J. C., Nagamachi, C. Y., Ponsa, M., Garcia, M., Garcia, F. and Egozcue, J. (1997). Radiation and speciation of spider monkeys, genus Ateles, from cytogenetic viewpoint. American Journal of Primatology, 42, 167-178. Mittermeier, R. A. and Coimbra-Filho, A. F. (1977). Primate conservation in Brazilian Amazonia. In Prince Rainier III of Monaco, and Bourne, G. H. (Eds.). Primate Conservation. Academic Press, New York. Mittermeier, R. A., Kinzey, W. G. and Mast, R. B. (1989). Neotropical Primate Conservation. Journal of Human Evolution, 18, 597-610. Mittermeier, R. A., Wallis, J., Rylands, A. B. and Ganzhorn, J. U. et al., (2009). Primates in peril: The world’s 25 most endangered primates 2008-2010. Primate Conservation, 24, 157. Montgelard, C., Catzeflis, F. M. and Douzery E. (1997). Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Molecular Biology and Evolution, 14, 550–559. Moore, W. (1995). Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution, 49, 718–726. Nieves, M., Ascunce, M. S., Rahn, M. I. and Mudry, M. D. (2005). Phylogenetic relationships among some Ateles species: the use of chromosomic and molecular characters. Primates, 46, 155-164. Norconk, M. A., Sussman, R. W. and Phillips-Conroy, J. P. (1996). Primates of Guyana shields. In Norconk, M. A., Rosenberger, A. L., and Garber, P. A. (Eds.). Adaptative radiations of Neotropical Primates. Plenum Press, New York. pp. 69-83. Nores, M. (1999). An alternative hypothesis for the origin of Amazonian bird diversity. Journal of Biogeography, 26, 475-485. Nores, M. (2004). The implications of Tertiary and Quaternary sea level rise events for avian distribution patterns in the lowlands of northern South America. Global Ecology and Biogeography, 13, 149-162. Pieczarka, J. C., Nagamachi, C. Y. and Barros, R. M. S. (1989). The karyotype of Ateles paniscus paniscus (Cebidae, Primates): 2n = 32. Revista Brasileira de Genética, 12, 543551. Plautz, H. L., Goncalves, E. C., Ferrari, S. F., Schneider, M. P. C. and Silva, A. (2009). Evolutionary inferences on the diversity of the genus Aotus (Platyrrhini, Cebidae) from mitochondrial cytochrome c oxidase subunit II gene sequences. Molecular Phylogenetics and Evolution, 51, 382–387. Posada, D. and Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817-818. Ramos-Onsins, S. E. and Rozas, J. (2002). Statistical properties of new neutrality tests against Population growth. Molecular Biology and Evolution, 19, 2092–2100. Rodbell, D. T. and Seltzer, G. O. (2000). Rapid ice margin fluctuations during the Younger Dryas in the tropical Andes. Quaternary Research, 54, 328-338.

474

Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al.

Rogers, A. R. and Harpending, H. C. (1992). Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution, 9, 552569. Rogers, A. R., Fraley, A. E., Bamshad, M. J., Watkins, W. S. and Jorde, L. B. (1996). Mitochondrial mismatch analysis is insensitive to the mutational process. Molecular Biology and Evolution, 13, 895-902. Rosenberger, A. L. and Strier, W. G. (1989). Adaptative radiation of the atelinae primates. Journal of Human Evolution, 18, 717-750. Rossan, P. N. and Baerg, D. C. (1977). Laboratory and feral hybridization of Ateles geoffroyi panamensis Kellogg and Goldman, 1944, in Panama. Primates, 18, 235-237. Rossetti, D. F., Toledo, P. M. and Goes, A. M. (2005). New geological framework for Western Amazonia (Brazil) and implications for biogeography and evolution. Quaternary Research, 63, 78–89. Ruiz-García, M. (2003a). Molecular population genetic analysis of the spectacled bear (Tremarctos ornatus) in the Northern Andean Area. Hereditas, 138, 81-93. Ruiz-García, M. (2003b). Análisis genético conservacionista de los géneros Lagothrix y Ateles (Atelidae, Primates) mediante loci microsatélites: Evidencia de un reciente cuello de botella en Lagothrix lagotricha y en Ateles paniscus chamek. In: Nassar, F., Pereira, V., Savage, A. (Eds.). Primatología del Nuevo Mundo: Biología, Medicina, Manejo, Conservación. Chapter 11. Fundación Araguatos, Bogotá DC, 272-285. Ruiz-García, M. (2007). Genética de Poblaciones: Teoría y aplicación a la conservación de mamíferos neotropicales (Oso andino y delfín rosado). Boletín de la Sociedad Española de Historia Natural, 102, 99-126. Ruiz-García, M. (2013). The genetic demography history and phylogeography of the Andean bear (Tremarctos ornatus) by means of microsatellites and mtDNA markers. In Molecular Population Genetics, Evolutionary Biology and Conservation of Neotropical Carnivores. Ruiz-García, M., Shostell, J. (Eds.). Nova Science Publishers. New York. pp. 129-158. Ruiz-García, M., Orozco-terWengel, P., Payán, E. and Castellanos, A. (2003). Genética de Poblaciones molecular aplicada al estudio de dos grandes carnívoros (Tremarctos ornatus – Oso andino, Panthera onca- jaguar): lecciones de conservación. Boletín de la Sociedad Española de Historia Natural, 98, 135-158. Ruiz-García, M., Orozco-terWengel, P. Castellanos, A. and Arias, L. (2005). Microsatellite analysis of the spectacled bear (Tremarctos ornatus) across its range distribution. Genes and Genetics Systems, 80, 57-69. Ruiz-García, M., Parra, A., Romero-Aleán, N., Escobar-Armel, P. and Shostell, J. M. (2006). Genetic characterization and phylogenetic relationships between the Ateles species (Atelidae, Primates) by means of DNA microsatellite markers and craniometric data. Primate Report, 73, 3–47. Ruiz-García, M., Castillo, M. I., Vásquez, C., Rodriguez, K. and Pinedo-Castro, M. et al. (2010). Molecular phylogenetics and phylogeography of the white-fronted capuchin (Cebus albifrons; Cebidae, Primates) by means of mtCOII gene sequences. Molecular Phylogenetics and Evolution, 57, 1049-1061. Ruiz-García, M., Vásquez, C., Camargo, E., Leguizamon, N., Castellanos-Mora, L. F., Vallejo, A., Gálvez, H., Shostell, J. and Alvarez, D. (2011). The molecular phylogeny of

Historical Genetic Demography...

475

the Aotus genus (Cebidae, Primates). International Journal of Primatology, 32, 1218– 1241. Ruiz-García, M., Castillo, M. I., Lichilin, N. and Pinedo-Castro, M. (2012a). Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, C. xanthosternos and C. nigritus, Cebidae), by means of mitochondrial COII gene sequences. Folia Primatologica, 83, 100-125. Ruiz-García, M., Castillo, M. I., Ledezma, A., Leguizamon, N. and Sánchez, R. et al. (2012b). Molecular systematics and phylogeography of Cebus capucinus (Cebidae, Primates) in Colombia and Costa Rica by means of the mitochondrial COII gene. American Journal of Primatology, 74, 366-380. Ruiz-García, M., Pinedo-Castro, M. and Shostell, J. M. (2014). How many genera and species of wolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular analysis of Lagothrix flavicauda, an endemic Peruvian primate species. Molecular Phylogenetics and Evolution, 79, 179–198. Ruiz-García, M., Castellanos, A., Bernal, L. A., Navas, D., Pinedo-Castro, M. and Shostell, J. M. (2015). Mitochondrial gene diversity of the mega-herbivorous of the genus Tapirus (Tapiridae, Perissodactyla) in South America and some insights on their genetic conservation, systematics and the Pleistocene influence on their genetic characteristics. Advances in Genetics Research, 14, 1-51. Ruiz-García, M., Lichilin, N., Gutierrez-Espeleta, G., Castillo, M. I., Wallace, R. and Escobar-Armel, P. (2016a). Molecular phylogeny of all the Ateles taxa (Atelidae, Primates) by means of mitochondrial genes and microsatellites. Molecular Phylogenetics and Evolution, (submitted). Ruiz-García, M., Cerón, A., Pinedo-Castro, M. and Gutierrez-Espeleta, G. (2016b). Which howler monkey (Alouatta, Atelidae, Primates) taxa is living in the Peruvian Madre de Dios River Basin (Southern Peru)? Results from mitochondrial gene analyses and some insights in the phylogeny of Alouatta. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Ruiz-García, M., Castillo, M. I. and Luengas, K. (2016c). It is misleading to use Sapajus (robust capuchins) as a genus? A review of the evolution of the capuchins and suggestions on their systematics. In Ruiz-García, M., Shostell, J. M. (Eds.). Phylogeny, Molecular Population Genetics, Evolutionary Biology and Conservation of the Neotropical Primates. Nova Science Publishers, Inc. New York, USA. Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 405–425. Sambrock, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Laboratory manual. 2nd edition. V1. Cold Spring Harbor Laboratory Press. New York. Sampaio, M. I., Schneider, M. P. C. and Schneider, H. (1993). Contribution of genetic distances studies to the taxonomy of Ateles, particularly Ateles paniscus paniscus and Ateles paniscus chamek. International Journal of Primatology, 14, 895-903. Schwarz, G. E. (1978). Estimating the dimension of a model. Annals of Statistics, 6, 461-464. Sheed, D. H. and Macedonia, J. M. (1991). Metachromism and its phylogenetic implications for the genus Eulemur. Folia Primatologica, 57, 221-231. Silva-López, G., Motta-Gill, J. and Hernández, A. I. (1996). Taxonomic notes on Ateles geoffroyi. Neotropical Primates, 4, 41-44.

476

Manuel Ruiz-García, Nicolás Lichilín, Pablo Escobar-Armel et al.

Simonsen, K., Churchill, G. and Aquadro, C. (1995). Properties of Statistical Tests of Neutrality for DNA Polymorphism Data. Genetics, 141, 413-429. Simpson, G. G. (1944). Tempo and Mode in Evolution. Columbia University Press, New York. Stevenson, M. F., Foose, T. J. and Baker, A. (1992). Primate conservation assessment and management plan. IUCN/SSC Captive Breeding Specialist Group (CBSG), Appley Valey, Minnesota. Strier, K. B. (1992). Atelinae adaptations: Behavioral strategies and Ecological constraints. American Journal of Physical Anthropology, 88, 515-524. Strobeck, C. (1987). Average number of nucleotide differences in a sample from a single subpopulation: a test for population subdivision. Genetics, 117, 149-153. Tamura K., Stecher G., Peterson D., Filipski A. and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution, 30, 27252729. Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585-595. Van der Hammen, T. (1992). La paleoecología de Suramérica Tropical. Cuarenta años de investigación de la historia del medio ambiente y de la vegetación. In Historia, Ecología y Vegetación (pp. 1-411). Bogotá, Colombia: Corporación Colombiana para la Amazonía-Araracuara. Van der Hammen, T. and Absy, M. L. (1994). Amazonia during the last glacial. Palaeogeography, Palaeoclimatology, Palaeoecology, 109, 247-261. Van Roosmalen, M. G. M. (1980). Habitat Preferences, diet, feeding strategy, and social organization of the black spider monkey (Ateles paniscus paniscus) in Surinam. PhD. Thesis, University of Wageningen, Netherlands. Walsh, P. S., Metzger, D. A. and Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques, 10, 506513. Weir, B. S. and Hill, W. G. (2002). Estimating F-statistics. Annual Review of Genetics, 36, 721–750. Wendt, H. (1958). Tras las huellas de Adán. Editorial Noguer, Barcelona. Wolfheim, J. H. (1983). Primates of the New World: Distribution, Abundance and Conservation. Seattle, University of Washington Press.

CONSERVATION BIOLOGY

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 14

CAPUCHIN MONKEYS IN AMAZONIAN MANGROVE AREAS Ricardo R. Santos1, R. G. Ferreira2 and A. Araujo2 1

Center for Agrarian and Environmental Sciences, Federal University of Maranhão, Brazil 2 Department of Physiology, Federal University of Rio Grande do Norte, Brazil

ABSTRACT Mangroves have been suggested as refuges for primates that may experience habitat loss and illegal hunting in terra firme forests. The northern coast of Brazil is considered one of the largest mangrove extensions in the world and may have many primate species yet to be reported in this habitat. In this chapter we focus on surveys of capuchin monkeys in mangrove areas of the northern coast of Brazil to determine if their distribution and pattern of occupation are associated with the refuge hypothesis. We surveyed thirty-three areas in the main estuaries of the east coast of the Brazilian Amazon, in Pará and Maranhão States for the presence of two species of capuchin monkeys in mangrove forests. Sapajus apella extensively occupied mangroves and also occupied terra firme forests. In addition, there was a limited number of areas in which they lived as resident groups in mangroves. S. libidinosus is present in three estuaries at the eastern end of the study area and they use the mangrove only as residents. There are no records of groups using both habitats. The two species occupy mangroves in different areas, following their geographic distributions on terra firme forests. The presence of successful groups in the mangrove (resident groups) are possibly associated with historical changes in the terra firme forests and not to human factors such as habitat loss and illegal hunting.

Keywords: sapajus, distribution, flooded forest, habitat use

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INTRODUCTION The geographical distribution of primate species may be associated with ecological or historical factors (Lehman and Fleagle 2006) and the influence of these variables is what sets the limits of distribution. In capuchin monkeys, climatic and vegetational factors have been described as distribution boundaries between different species (Vilanova et al. 2005). However, the factors associated with the dispersion of these animals to mangrove areas remain unknown. Capuchin monkeys are widely distributed in Brazil (Silva Jr. 2001; Rylands et al. 2004, 2005), but records of flooded mangrove forests are scarce. However, the northern coast of Brazil is strongly influenced by the Amazon River, has one of the largest mangrove forests in the world (Souza-Filho 2005), and may provide wide range of primates (Santos and Bridgeman 2016). Based on the geographical distribution of primates four species are likely to have colonized the mangroves of the northern coast of Brazil: Cebus kaapori (Ka’apor Capuchin), Cebus olivaceus (wedge-capped capuchin) and Sapajus apella (tufted capuchin) from the Amazon forest and Sapajus libidinosus (bearded capuchin) from Cerrado and Caatinga (Silva Jr. 2001; Alfaro et al. 2012). Although these last two species are widely distributed, our knowledge on the occurrence of capuchin monkeys in the mangroves of Brazil’s northern coast is limited to only a few recorded locations for S. apella and C. olivaceus (Fernandes 1991; Fernandes and Aguiar 1993; Silva Jr. 2001). Also, our knowledge of how primates live in mangroves and how they differ from their upland neighbors is limited to a few studies (Santos et al. 2016a,b). A lack of information may simply be due of the constraints imposed by the environment for conducting observations (Barnett et al. 2016). A postulated hypothesis is that mangroves can act as refuges for species that are subjected to loss of habitat and hunting pressure (Galat-Luong and Galat 2005; Nowak 2012; Gonedélé Bi et al. 2013; Beltrão-Mendes and Ferrari 2016; Nowak and Coles 2016). However, on the northern coast of Brazil, mangroves have been suggested as habitats frequently used by terra firme groups (Fernandes and Aguiar 1993), but depending on the localization and extension of these flooded forests, we suggest that they can also shelter resident groups. Thus, through the distribution of two species of capuchin monkeys in mangroves, we attempt to identify how these primates use the mangroves and checked which factors may explain their distribution patterns. Additionally, we analyze if the occupation by potential resident groups is linked to the mangrove being used as a refuge.

METHODS Study Area This study was carried out on the northern coast of Brazil, east of the Amazon River’s mouth, in the mangroves of the main estuaries. We collected data at 33 locations in northeastern Pará and throughout the Maranhão. These mangroves are presented largely as a continuous forest whose landscape is intersected by tidal canals, rivers and formations of islands of different sizes (Souza-Filho 2005).

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The study area presents four large bays, the Gurupi, Turiaçu, Cumã and Tubarão associated with the Amazon, and another located under the influence of arid climate of the Brazilian Cerrado called Delta do Parnaíba. The areas have average annual rainfall of 2,300 mm (Fisch et al. 1998) to 1600 mm (IBAMA 2003) respectively and tides ranging from 4 m to 7.5 m (Souza-Filho 2005) providing high productivity (Menezes et al. 2008).

Survey Mangroves areas were found through satellite imagery and accessed by boat, canoe or walking. Neighboring mangrove areas were also inspected in order to identify if there were indications of potential corridors for the animals helping them to move from non-flooded forests to mangroves. In this case, the type of vegetation (mangrove or terra firme) and the presence/absence of the capuchin monkeys were recorded. The presence of capuchin monkeys was recorded through sightings and interviews, records of captive animals from local residents, and indirect records such as the presence of feeding sites used by animals for consumption of shellfish (see description of feeding sites on Santos 2010 and Santos et al. 2016a).

RESULTS We confirmed the presence of capuchin monkeys in mangrove areas within all 33 sites surveyed (Figure 1). Only S. apella and S. libidinosus were recorded in surveyed areas. S. apella is present in 77% of the bays and its distribution in Brazilian Amazon mangroves seems to coincide with the northeast limit of the species’ distribution in Amazonian upland forests. S. libidinosus has limited distribution in the mangrove and its occurrence was confirmed only in three estuaries in the eastern portion of the north shore (Table 1).

1

4

2

5

8

3 67 9

14 10,11,12

Pará

Atlantic Ocean Atlântico

13 15 16 17 18

2º S

Amazon

19 20

22

Maranhão

27

28 29 30

Sand dunes

25 23

31 32

■ ■ ■

26 21

33

24

km

Baixada Maranhense

Cerrado 44º W

Figure 1. Surveyed areas of the northern coast of Brazil (northeastern Pará and Maranhão States). Sapajus apella ( ), Areas: 1-30; Sapajus libidinosus ( ), Areas: 31-33. See Table 1 for details. Larger circles are based on Fernandes 1991; Fernandes and Aguiar 1993, and Silva Jr 2001.

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Table 1. Localities surveyed, cities and methods used to record the presence of Sapajus apella (1 - 30) and Sapajus libidinosus (31 - 33) Area 1

Locality

City Salinópolis

2

Praia da Corvina, Baía do Maracanã Fazenda Salina, Baía do Caeté

3

Rio Arapiranga, Baía do Gurupi

Carutapera

4

Ilha de Tucundiua, Baía do Tromaí “Ilha” de Fora, Baía do Tromaí

Luis Domingues Luís Domingues Luís Domingues Luis Domingues Godofredo Viana Godofredo Viana Godofredo Viana Godofredo Viana Godofredo Viana Cândido Mendes Bacuri

5 6 7

Povoado Livramento, Baía do Tromaí Porto, Baía do Tromaí

8

Rio Aurizona, Baía do Tromaí

9

Campo, Baía do Tromaí

10

Porto, Baía do Tromaí

11

Canal Barão, Baía do Tromaí

12

Boca do Alemão, Baía do Tromaí Rio Maracaçumé, Baía do Maracaçumé Povoado Assu, Baía do Turiaçu

13 14 15

Bragança

Bacuri

17

Povoado Portugal, Baía do Turiaçu Povoado Trajano, Baía do Turiaçu Porto, Baía do Turiaçu

18

Porto, Baía do Cabelo da Velha

Cururupu

19

Rio Pericumã, Baía de Cumã

Guimarães

20

Reserva do CLA (Centro de Lançamento de Alcântara), Baía de São Marcos Povoado Cujupe, Baía de São Marcos

Alcântara

16

21

Bacuri Bacuri

Alcântara

Geographic coordination 0º36’14.93”S/ 47º22’25.42”W 0°55’21.30”S/ 46º40’11.50”W 1º11’58.53”S/ 46º01’26.42”W 1°05'54.03"S/ 45°51'05.41"W 1°14'46.31"S/ 45°55'15.88"W 1º19’09.14”S/ 45º55’44.75”W 1º19’13.02”S/ 45º53’38.81”W 1º16’13.28”S/ 45º46’27.36”W 1º22’41.17”S/ 45º47’10.26”W 1º25’21.17”S/ 45º46’25.06”W 1º24’03.04”S/ 45º49’06.07”W 1º23’11.44”S / 45º44’42.74”W 1º27’22.23”S/ 45º43’41.18”W 1º38’37.36”S/ 45º15’53.07”W 1º37’57.41”S/ 45º14’30.34”W 1º41’03.72”S/ 45º09’49.77”W 1º43’34.95”S/ 45º08’27.60”W 1º49’52.21”S/ 44º52’14.66”W 2°07’51.28"S/ 44°35’45.91"W 2°20'01.24"S/ 44°21'48.73"W 2°30’13.87”S/ 44°31’11.20”W

Method 3 1,3 3 1*,3 3 3 1,2,3 3 3 3 3 3 3 3,4 3,4 3,4 2,3 3 2,3 1,3

3

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Capuchin Monkeys in Amazonian Mangrove Areas Area

Locality

22

City

Porto do Baltazar, Baía de São Bacurituba Marcos 23 Praia de Itapeúna (rio Mearim), Cajapió Baía de São Marcos 24 Porto (rio Mearim), Baía de São Cajapió Marcos 25 Perizes - Estreito dos Mosquitos Bacabeira (rio Mearim), Baía de São Marcos 26 Praia do Araçagy, Baía de São São José de Marcos Ribamar 27 Ilha de Curupu, between Baía de Raposa São Marcos and Baía de São José de Ribamar 28 Povoado Iguaíba, Baía de São Paço do José de Ribamar Lumiar 29 Povoado Mojó, Baía de São José Paço do de Ribamar Lumiar 30 Sítio Aguahy, Baía de São José São José de de Ribamar Ribamar 31 Rio Preguiças, Área de Proteção Barreirinhas Ambiental do Rio Preguiças e Pequenos Lençóis 32 Rio Novo, Área de Proteção Paulino Neves Ambiental do Rio Preguiças e Pequenos Lençóis 33 Delta do rio Parnaíba, Baía de Tutóia Tutóia 1. Sighting; 2. Captive animals; 3. Interview; 4. indirect records. * Cranial skeleton. 1-2. Pará State, Brazil; 3-33. Maranhão State, Brazil.

Geographic coordination 2º43’05.60”S/ 44º41’35.57”W 2º50’56.98”S/ 44º38’02.90”W 2º53’10.29”S/ 44º40’25.70”W 2°45'58.31"S/ 44°22'56.93"W 2°26’40.31”S/ 44°8’39.92”W 2º25’15.00”S/ 44º04’21.29”W 2º28’05.89”S/ 44º05’47.83”W 2º30’15.08”S/ 44º03’58.22”W 2°41'21.09"S/ 44°10'2.89"W 2º39’17.57”S/ 42º41’07.80”W

Method 3 3 3 3

2,3 1,2,3

3 2,3 1,3 1,2,3,4

2º42’43.09”S/ 42º31’05.98”W

1,2,3,4

2º45’55.02”S/ 42º16’28.82”W

3

Mangrove Use by Capuchin Monkeys The use of mangroves by S. apella and S. libidinosus occurs in two different ways: 



Groups use both mangrove and terra firme forest (Figure 2A): this use of both habitats was observed only in S. apella, prevailing in 77% of the survey areas. (Areas: 1-21, 29-30, Figure 1); Groups living in mangrove: the animals use the mangroves as sleeping and feeding sites but do not move to the upland forest (Figure 2B). These groups are termed residents groups. They correspond to the overall geographic distribution of S. libidinosus (Areas: 31-33) and make up 23% of the S. apella surveyed in mangroves (Areas: 22-28).

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Ricardo R. Santos, R. G. Ferreira and A. Araujo A

B

Figure 2. Mangrove use by capuchin monkeys. (A) Terra firme forests (background) are largely connected to mangroves (foreground) enabling the movement of animals between habitats (Photo from Area 21, povoado Cujupe, Baía de São Marcos, Alcântara, Maranhão). (B) Bearded capuchins (Sapajus libidinosus) survive in mangroves throughout their life (Photo from Area 31, Rio Preguiças, Barreirinhas, Maranhão). Photo credits: Ricardo Rodrigues dos Santos.

Due the extensive distribution range of S. apella in the mangrove, it is possible that the records for this species are underestimated. Since the presence of resident groups is considered a key factor to successful colonization of capuchin monkeys in the mangrove, here we provide a description of the mangrove areas colonized in a successful manner by both species. Some localities need further inquiries because they are considered to be potential areas of resident groups not confirmed during this study (Figure 1).

Localities in Mangrove Suggested for Shelter Residents Groups of Sapajus apella Information from interviews suggest that large estuaries interspersed with canals and mangrove islands (between sites 3-19) may be potential occupation areas for S. apella. Furthermore, these areas may provide isolation from populations in terra firme forests. These estuaries have not been thoroughly surveyed and records in mangroves were restricted to more accessible areas close to the terra firme forests.

Mangrove Areas with Resident Groups Sapajus apella In the region formed by large seasonal lakes termed Baixada Maranhense (Maranhão), between sites 22 and 25, the presence of large natural grasslands form an ecosystem around a mangrove (Figure 3A). This seems to limit the connectivity of primates with the terra firme forest. These non-flooded areas are formed by Amazonian tree species with many palm trees, mainly Orbignya phalerata (Arecaceae), named Matas de Cocais. Seasonal flooded fields bordering mangroves were also observed at site 2, in Northeastern Pará. However, at this site we also noted the use of terra firme forests by tufted monkeys coming from continuous mangrove areas.

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C

D

Figure 3. Examples of where resident groups of capuchin monkeys were recorded: (A) in mangroves surrounded by a seasonally flooded field. The photo (Area 22: Porto do Baltazar, Baía de São Marcos, Bacurituba, Maranhão) was taken during the dry season and a mangrove can be seen in the background; (B) living on mangrove islands (Photo from Area 32: Rio Novo, Área de Proteção Ambiental do Rio Preguiças e Pequenos Lençóis, Paulino Neves, Maranhão); (C) surrounded by sand dunes (Photo from Area 31: Rio Preguiças, Área de Proteção Ambiental do Rio Preguiças e Pequenos Lençóis, Barreirinhas, Maranhão); and (D) surrounded by restinga vegetation (Photo from Area 31). Photo credits: Ricardo Rodrigues dos Santos.

The occupation of S. apella in island areas (26 to 28) where restinga vegetation and sand dunes surround mangroves, seems to limit S. apella to an isolation pattern similar to that observed in the occupied estuaries for S. libidinosus (Figure 3B).

Sapajus libidinosus Although the presence of the Amazon supports a continuous ranges of mangroves, mangroves can also have naturally fragmented distributions. We observed S. libidinosus most often in these fragmented areas such as in 31, 32, and 33. This result is due to the presence of estuaries and extensive sand dune fields encroaching on the mangroves (Figure 3C). The restinga vegetation surrounding these mangroves seems to limit the distribution of these animals to the mangrove as well. It is not clear if any connection exists between primates from mangroves and non-flooded areas (Figure 3D).

DISCUSSION Brazilian mangroves are among the most productive mangrove ecosystems around the world (Schaeffer-Novelli et al. 1990, 2000) and even in the Amazon mangroves, the diversity

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of mangrove primates is low (Santos and Bridgeman 2016). However, this may be due to limited research in this habitat. In this study we surveyed the occurrence, distribution and mangrove use by S. apella and S. libidinosus on the northern coast of Brazil. We tested the hypothesis of resident groups using mangroves as refuges. As opposed to what is observed in the distribution of capuchin species from the Amazon or Cerrado of the Brazil (Silva Jr. 2001; Alfaro et al. 2012), mangroves do not seem to be a limiting factor for the expansion of either species. In the areas surveyed, the dispersion into the mangrove seems to have been successful in both species in proportion to the geographic distribution encountered. However, a large part of the mangrove used by S. apella appears to reflect different patterns in comparison to S. libidinosus. The occupation of mangroves by S. apella predominantly occurs where mangroves have continuity with the remaining forests of Amazonian terra firme. Groups that successfully occupy mangroves use them as sites for sleeping and foraging. We observed groups essentially in areas where mangrove forests were associated with seasonal flooded fields. This represents an insignificant proportion of their distribution in this habitat. As noted by Fernandes and Aguiar (1993), in areas where mangroves have close association with terra firme forests, such as in the occurrence of S. apella, these flooded forests appear to be used by primates as alternative habitats. Conversely, in estuaries used by S. libidinosus, successful occupation of mangroves seems to be associated with isolation from the populations of Caatinga and Cerrado in northern Brazil. The restricted distribution of S. libidinosus in mangrove areas in the eastern portion of Brazil’s northern coast may be associated with a lower number of estuaries in this region. The colonization of estuaries may be due to general dispersion followed by isolation. Resident groups of primates would then use the mangroves as sleeping and feeding sites. In this region, the mangroves are not used as secondary habitats and restinga vegetation close to mangroves seems to work as a barrier for these mangrove populations from the dry areas of the Cerrado and Caatinga. In this condition, S. libidinosus individuals develop and spend their lives in mangroves. The condition recorded in S. libidinosus is observed in a few mangroves used by S. apella. Bearded capuchins use the mangroves as feeding and sleeping sites, not returning to the terra firme. Obviously, that does not remove the possibility of movement of primates between mangrove and neighboring land in narrow ranges along different generations. But there is no evidence of it for the bearded capuchin. We recognize habitat loss as a possible cause for primate colonization of mangroves (Nowak 2012). However, our study provides no data supporting this as a causative factor for the dispersal of both species of capuchin monkeys to mangroves and the presence of resident groups. Thus, the successful colonization of mangroves seems to be associated with biogeography factors. With the natural decrease of upland forests and advancement of restinga or flooded fields, mangrove forests may have acted as remaining habitats. This may be connected to an increase in the number of animals isolated from terra firme populations. In the studied areas, the occupation of the mangrove may be dated back to the beginning of the Holocene, when the mangrove forests in Maranhão were more towards the continent’s interior (Behling and Costa 1997). Historical changes on cover of coastal land in Maranhão are reported to affect different taxonomic groups (Oren 1988). This may have influenced the use of mangroves by resident

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groups of capuchin monkeys. Currently, it is possible that the successful occupation of mangroves by resident groups of capuchin monkeys in Brazil's northern coast is limited by physical and vegetation barriers. This may have helped to isolate capuchin populations from their original distribution in terra firme forests. Thus, we propose that occupation of mangroves by capuchin monkeys on the northern coast of Brazil cannot be directly associated with anthropic factors such as habitat loss. The mangrove use by capuchin monkeys might at first be associated with exploratory behavior or be related to seasonal food resources in terra firme forests along coastal areas. This may be seen in most of the distribution of S. apella. The primates, in this case, would behave as mangrove visitors, a peripheric habitat of their distribution. The natural landscape change over time in large areas of vegetation of restinga and sand dunes or seasonal flooded fields, and estuarine dynamics with the formation of islands surrounded by tidal channels, may have functioned as the main factors that led to colonization and success of these animals in estuaries. This made the resulting residents dependent on available resources in the mangrove forests. The successful occupation, mainly by resident groups of S. libidinosus shows that mangroves are not impenetrable frontiers and reveals the adaptability of these primates.

ACKNOWLEDGMENTS We are grateful to Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) for financial support (no. APP-01038/08) and to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a scholarship granted to R.R.S. (# 142604/2005-4) and A.A. (# 302012/2006-0).

REFERENCES Alfaro, J. W. L, Boubli, J. P., Olson, L. E., Di Fiori, A., Wilson, B., Gutiérrez-Espeleta, G. A., Chiou, K. L., Schulte, M., Neitzel, S., Ross, V., Schwochow, D., Nguyen, M. T. T, Farias, I, Janson, C. H. and Alfaro, M. E. (2012). Explosive Pleistocene range expansion leads to widespread Amazonian sympatry between robust and gracile capuchin monkeys. Journal of Biogeography, 39, 272–288p. Barnett, A. A, Hawes, J. E., Pontes, A. R. M., Layme, V. M. G., Chism, J., Wallace, R., Ferrari, S., Beltrão-Mendes, R., Wright, B., Haugaasen, T., Bezerra, B., Matsuda, I. and Santos, R. R. (2016). Survey and study methods for flooded habitats. In: Primates in flooded habitats: ecology and conservation. Adrian A. Barnett, Ikki Matsuda, and Katarzyna Nowak (Editors). Cambridge University Press, UK. In press. Behling, H. and Costa, M. L. (1997). Studies on Holocene tropical vegetation, mangrove and coast environments in the state of Maranhão, NE Brazil. Quaternary of South America and Antartic Peninsula, 10, 93-118. Beltrão-Mendes, R. and Ferrari, S. F. (2016). Mangrove forests as a key habitat for the conservation of the critically endangered yellow-breasted capuchin, Sapajus xanthosternos, in the Brazilian Northeast. In: Primates in flooded habitats: ecology and

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conservation. Adrian A. Barnett, Ikki Matsuda, and Katarzyna Nowak (Editors). Cambridge University Press, UK. In press. Fernandes, M. E. B. (1991). Tool use and predation of oysters (Crassostrea rizophorae) by the tufted capuchin, Cebus apella, in brackish water mangrove swamp. Primates, 32, 529-531. Fernandes, M. E. B. and Aguiar, N. O. (1993). Evidências sobre a adaptação de primatas neotropicais às áreas de mangue com ênfase no macaco-prego Cebus apella apella. A Primatologia no Brasil, 4, 67-80. Fisch, G., Marengo, J. A. and Nobre C. (1998). Uma revisão geral sobre o clima da Amazônia. Acta Amazônica, 28, 101-126. Galat-Luong, A. and Galat, G. (2005). Conservation and survival adaptations of Temminck’s red colobus (Procolobus badius temmincki), in Senegal. International Journal of Primatology, 26, 585-603. Gonedélé Bi, S., Béné, J. C. K., Bitty, E. A., Kassé, B. K., N’Guessan, A., Koffi, D. A., Akpatou, K. B. and Koné, I. (2013). Roloway guenons (Cercopithecus diana roloway) and white-naped mangabey (Cercocebus atys lunulatus) prefer mangrove habitats in Tanoé Forest, South-Eastern Ivory Coast. Journal of Ecosystem and Ecography, 3, 126. IBAMA. (2003). Plano de manejo do Parque Nacional dos Lençóis Maranhenses. Ministério do Meio Ambiente, Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis. São Luís, MA. 499p. Lehman, S. M. and Fleagle, J. G. (2006). Biogeography and primates: a review. In: Primate Biogeography. Shawn. M. Lehman; John. G. Fleagle (editors). Springer. 1-58p. Menezes, M. P. M., Berger, U. and Mehlig, U. (2008). Mangrove vegetation in Amazonia: a review of studies from the coast of Pará and Maranhão states, north Brazil. Acta Amazonica, 38, 403-420. Nowak, K. (2012). Mangrove and peat swamp forests: refuge habitats for primates and felids. Folia Primatologica, 83, 361-376. Nowak, K. and Coles, R. (2016). Worldwide patterns in the ecology of mangrove-living monkeys and apes. In Adrian A. Barnett, Ikki Matsuda, and Katarzyna Nowak (Eds.). Primates in flooded habitats: ecology and conservation.). Cambridge University Press, UK. In press. Oren, D. C. (1988). Uma reserva biológica para o Maranhão. Ciência Hoje, 44, 36-45. Rylands, A. B. (2004). Taxonomy, distribution and conservation. Where and what are they and how did they get there? In D. M. Fragaszy, E. Visalbergui, and L. Fedigan (Eds.). The complete capuchin. (pp. 13-35). Cambridge University Press. Rylands, A. B., Kierulff, M. C. M. and Mittermeier, R. A. (2005). Notes on the taxonomy and distributions of the capuchin monkeys (Cebus, Cebidae) of South America. Lundiana, 6, 97-110. Santos, R. R. (2010) Uso de ferramentas por macacos-prego em manguezais. PhD Thesis. Universidade Federal do Rio Grande do Norte, Natal, Brazil. Santos, R. R. and Bridgeman, L. L. (2016). Mangrove-living primates in the neotropics: an ecological review. In Adrian A. Barnett, Ikki Matsuda, and Katarzyna Nowak (Eds.). Primates in flooded habitats: ecology and conservation. Cambridge University Press, UK. In press. Santos, R. R., Araujo, A., Fragaszy, D. M. and Ferreira, R. G. (2016a). The role of tools in the feeding ecology of bearded capuchins living in mangroves. In Adrian A. Barnett, Ikki

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Matsuda, and Katarzyna Nowak (Eds.). Primates in flooded habitats: ecology and conservation. Cambridge University Press, UK. In press. Santos, R. R., Bridgeman, L. L., Supriatna, J., Siregar, R., Winarni, N. and Salmi, R. (2016b). Behavioural ecology of mangrove primates and their neighbors. In Primates in flooded habitats: ecology and conservation. Adrian A. Barnett, Ikki Matsuda, and Katarzyna Nowak (Editors). Cambridge University Press, UK. In press. Schaeffer-Novelli, Y., Cintrón-Molero, G., Adaime, R. R. and Camargo, T. M. (1990). Variability of mangrove ecossistems along the Brasilian coast. Estuaries, 13, 204-218. Schaeffer-Novelli, Y., Cintrón-Molero, G., Soares, M. L. G. and De-Rosa, T. (2000). Brazilian Mangroves. Aquatic Ecosystem Health and Management, 3, 561-570. Silva Jr, J. de S. (2001). Especiação nos macacos-pregos e caiararas, gênero Cebus Erxleben, 1777 (Primates, Cebidae). PhD Thesis. Universidade Federal do Rio de Janeiro, Rio de Janeiro. Souza-Filho, P. W. (2005). Costa de manguezais de macromaré da Amazônia: cenários morfológicos, mapeamento e quantificação de áreas usando dados de sensores remotos. Revista Brasileira de Geofísica, 23, 427-435. Vilanova, R., Silva Jr., J. S., Grelle, C. E. V, Marroig, G. and Cerqueira, R. (2005). Limites climáticos e vegetacionais das distribuições de Cebus nigritus e Cebus robustus (Cebinae, Platyrrhini). Neotropical Primates, 13, 14-19.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 15

HOW DOES THE COLOMBIAN SQUIRREL MONKEY COPE WITH HABITAT FRAGMENTATION? STRATEGIES TO SURVIVE IN SMALL FRAGMENTS Xyomara Carretero-Pinzón1,3,*, Thomas R Defler2 and Manuel Ruiz-Garcia1 1

Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética, Departamento de Biología, Facultad de Ciencias, Pontificia Universidad Javeriana. Bogotá DC., Colombia 2 Universidad Nacional de Colombia, Biology Department, Bogotá, Colombia 3 Proyecto Zocay, Colombia

ABSTRACT Habitat loss and fragmentation are the main threats to Neotropical primates. These processes affect the behavior of primate species, changing their feeding and movement patterns. We compared activity budget, daily range, home range and diet of two groups of Colombian squirrel monkeys (Saimiri cassiquiarensis albigena) living in continuous and fragmented areas of Colombia. Additionally, we compared group size, male/female and female/immature ratios from groups in both areas. We collected behavioral data from both groups of S. c. albigena using slow scan sampling every 5 minutes. Additionally, ad libitum group size and composition from other S. c. albigena groups were collected and used for the group size and composition comparison between sites. We found that diet composition varies although percentage of time consuming different food items remains similar for arthropods, fruits and flowers but not for leaves. Leaves were not observed being consumed in the fragmented area. Proportion of time spent consuming each food item was higher for all food items except for young leaves in the fragmented areas compared with the continuous area. Group composition and size were reduced in fragmented areas as well as home range. However, average daily range is higher in fragmented areas. On the other hand, stationary foraging is reduced in fragmented areas compared with the continuous area, while moving and foraging activity increased. *

Corresponding author: University of Queensland, School of Geography, Planning and Environmental Management, Brisbane, Australia.

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Xyomara Carretero-Pinzón, Thomas R Defler and Manuel Ruiz-Garcia Additionally, resting activity is reduced in fragmented areas, while moving increases for the same area when compared with the continuous area. In conclusion, the Colombian squirrel monkey’s strategy in fragmented landscapes is to spend more time feeding, increasing the proportion of pioneer species chosen for the diet that are found in forest edges and living fences. Living fences in their home ranges increases the space available for food resources.

Keywords: Saimiri cassiquiarensis albigena, fragmentation, diet, activity budgets, daily range, home range

INTRODUCTION Two of the main threats for primate species worldwide are habitat loss and fragmentation (Rylands et al., 2008, Marsh et al., 2013). These processes imply a set of changes in composition and spatial arrangement of the landscape, producing fragments of different sizes and landscapes with varying numbers of fragments and forest proportions (Forman and Godron, 1986; Fahrig 2003). This composition and spatial arrangement can determine the survivorship and persistence of primate species in the short, medium and long-term. It has been hypothesized that each primate species’ survivorship and persistence depends on their species traits, such as diet specialization, home range requirements, body size, and group size, making them more tolerant or susceptible to the effects of habitat loss and fragmentation (Onderdonk and Chapman, 2000; Boyle and Smith, 2010; Carretero-Pinzón et al., 2015). Therefore a small body size species will be expected to tolerate habitat loss and fragmentation processes more than larger species that require more space and more resources. However that is not the only trait that can influence the survivorship of a particular species. For example, its diet (especially the proportion of fruit) as well as its home range size have been found to be important traits that can explain the presence of some primate species in forest fragments in the Brazilian Amazon (Boyle and Smith, 2010). Primate species’ responses to habitat loss and fragmentation seem to be determined by the combinations of those traits. A recent review of primate species’ responses to habitat loss and fragmentation across traits did not find any consistent pattern in primate responses to these processes when analyzed across species traits (Carretero-Pinzón et al. 2015). Neotropical primates are species dependent on forest cover, and any changes in forest spatial arrangement and amount will affect their behavior and survivorship in the landscape (Arroyo-Rodriguez et al., 2013). Fragmentation and habitat loss produce changes in primate behavior (in terms of activity patterns, daily distance and home ranges) but, how different species respond to the effects and their magnitude remains unclear. Even less information is available about behavioral changes as consequences of these processes. Some studies have reported changes in diet composition (Ateles geoffroyi: Gonzalez-Zamora et al., 2011; Chaves et al., 2012; Alouatta caraya: Bicca-Marques et al., 2009; Alouatta palliata: Dunn et al., 2010; Alouatta pigra: Pozo-Montuy and Serio-Silva, 2007; Pozo-Montuy et al., 2013; Chiropotes satanas chiropotes: Boyle et al., 2012), daily ranges (Alouatta pigra: PozoMontuy and Serio-Silva, 2007; Chiropotes satanas chiropotes: Boyle et al., 2009; Boyle and Smith, 2010) and activity patterns (Ateles geoffroyi: Gonzalez-Zamora et al., 2011; Alouatta palliata: Asencio et al., 2007; Alouatta pigra: Pozo-Montuy et al., 2013; Cebuella pigmaea:

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 493 De la Torre et al., 2013) when compared with groups of the same species in continuous forests. Saimiri is a genus of small neotropical primates (869 to 1000 g, Defler, 2010), that live in large groups in different types of habitat, with large home ranges (Defler, 2010). Most of the studies of Saimiri have been done in continuous forests (Terborgh, 1983; Boinski 1987, 1989; Boinski and Mitchell, 1994; Mitchell 1990). Only one study has occurred in fragmented areas for one species of the Saimiri genus (Rodriguez-Vargas, 2003). This study assesses the genetic structure of Saimiri oerstedii populations in the fragmented distribution of this species finding that habitat loss and fragmentation have an effect on the metapopulation structure of this endemic Central American species. Saimiri cassiquiarensis albigena (= S. sciureus albigena; for several recent changes in the nomenclature and systematics of Saimiri, see Ruiz-García et al., 2015) is a vulnerable subspecies, endemic to the Colombian Llanos, with most of its current distribution consisting of forest fragments of different sizes (Carretero-Pinzon et al., 2009, 2013). Studies of S. c. albigena have been limited to some undergraduate theses of continuous forests (CarreteroPinzon 2000, Angulo 2001) and a master thesis of a fragmented area (Carretero-Pinzon 2008), from which data are presented here. This chapter aims to answer the following four questions. 1) How does the diet of a group of S. c. albigena change in a fragmented area when compared with a group in a continuous forest? 2) How much do the home and daily ranges of a group of S. c. albigena change in a fragmented area compared with one in a continuous area? 3) How much does the activity budget of a group of S. c. albigena change in a fragmented area compared with one in a continuous area? 4) How much do the group size and composition of S. c. albigena change in fragmented areas versus continuous areas?

Figure 1. Study site locations: a. San Martin Area (Fragmented area) and b. Tinigua National Park (Continuous area).

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MATERIAL AND METHODS Data for this chapter comes from two sets: one collected in a continuous area at Tinigua National Park (January to May 1999, Carretero-Pinzon, 2000) and the other collected in a fragmented area (San Martin area: August 2005 to January 2007, Carretero-Pinzon 2008) (Figure 1). Both data sets were collected with the same methodology (see below). Additionally, ad libitum group size and composition from other S.c.albigena groups were collected and were used for the group size and composition comparison between both sites (data from 1999, 2004-2014, Carretero-Pinzon, unpublished data).

STUDY SITES DESCRIPTION Tinigua National Park: Continuous Area This study site is located on the right bank of the Duda River in the Tinigua National Park (Centro de Estudios Ecológicos La Macarena (CIEM): 2º40’ N and 74º10’ W, Figure 1a). The area has two seasons: dry season (December – March: maximum 100 mm) and wet season (April – November: maximum 3000 mm) (Kimura et al., 1994). The Park’s forest types have been characterized by Hirabuki (1990) and Stevenson et al., (1994, 2004). Seven primate species are found at this area: red howler monkeys (Alouatta seniculus), white-bellied spider monkey (Ateles belzebuth), woolly monkeys (Lagothrix lagothricha), dusky titi monkeys (Callicebus ornatus), black-capped capuchins (Sapajus apella), Colombian squirrel monkeys (S. c. albigena) and Brumback’s night monkeys (Aotus brumbacki) (Yoneda, 1990).

San Martin Area: Fragmented Area This study area is composed of forest fragments of different sizes surrounded by pastures (range: 0.2 – 59.8 ha) and connected by living fences of different heights (natural hedgerows, Carretero-Pinzón et al., 2010). The area is located near the town of San Martín, Meta Department, in the Colombian Llanos, at Santa Rosa and Arrayanes farms (3°3’30”N and 73°35’40” W, elevation 350 meters; Figure 1b). There is a wet season from April-November (average 1777 mm), and a dry season from December-March (average 357 mm). The annual average temperature is 26°C (Carretero-Pinzón, 2008). The original land-cover was a mosaic of lowland forest, gallery forest and natural savannas. Five sympatric primates have been recorded in the area: red howler monkeys (A. seniculus), black-capped capuchins (S. apella), Colombian squirrel monkeys (S. c. albigena), dusky titi (C. ornatus) and Brumback’s night monkeys (A. brumbacki) (Carretero-Pinzón, 2013). The vegetation of these fragments shared species richness with differences in the importance of plant species in each fragment (Stevenson and Aldana, 2008; Carretero-Pinzón, unpublished data). Fragmentation is an ongoing process in this region due to palm oil plantations, petrol exploitation and livestock practices (Wagner et al., 2009; Carretero-Pinzón and Defler, in press).

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 495

PRIMATE DATA COLLECTION Tinigua National Park: Continuous Area One group of 53 S. c. albigena individuals was selected and habituated in this area. Continuous following was made from January to May of 1999 (total sampling, 369 hours; 15 days of habituation as this groups was previously habituated to human presence). Behavioral and ecological data were collected by scan sampling every 5 minutes (1 minute of continuous sampling followed by 4 minutes without data) (Altman, 1978). Samplings were made from 6:00 to 18:00. Every 10 minutes the group location was recorded and related to the existing trail system. Natural variation in pelage characters and body scars allowed individual identification of S. c. albigena (Mitchell, 1990). Data collected included: food type (arthropods, fruits, flowers and leaves), location every 10 minutes, and time spent in foraging, moving with foraging, moving, resting with foraging, resting and social activities (CarreteroPinzón, 2000). The classification of activities follows Terborgh (1983) and Bonski (1987).

San Martin Area: Fragmented Area We selected and habituated one group of 42 S. c. albigena individuals at this study area. Continuous following was made from August 2005 to January 2007 (total sampling, 1113 hours; six months of habituation was required for this group). The sampling protocol was the same as for the continuous area (see above).

Data Analysis For each group we used χ2 tests (Zar, 1996) to determine whether the frequency of the food items consumed in the continuous and fragmented area was significantly different from random. Also, we use χ2 tests (Zar, 1996) to test whether the frequency of the activity budget of each group was significantly different from random, at the continuous and fragmented areas. For the statistical analysis we used R software (www.r-project.org).

RESULTS Diet The study group in the continuous area consumed a total of 84 plant species in four months. This included fruits (57 spp.), flowers (4 spp.) and leaves (2 spp.) (Table 1). The study group in the fragmented area consumed 91 plant species in 13 months of study consisting of fruits (80 spp.) and flowers (10 spp.) (Table 2). No leaves were consumed by this study group during the entire 13 months of study. The main families consumed by the study group in the continuous area were: Fabaceae, followed by Burseraceae, Moraceae and Cecropiaceae. In the fragmented areas most of the consumed species belonged to the

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Melastomataceae family (especially pioneer species found on fragment edges and living fences), followed by the Annonaceae, Burseraceae, Fabacea and Moraceae families. The percentage of time consuming each food item was similar across groups (arthropods: Continuous 79.36%, Fragmented 79.67%; fruits: Continuous 19.67%, Fragmented: 20.27%; flowers: Continuous 0.22%, Fragmented 0.07% and young leaves: Continuous 0.75%, Fragmented 0%). However, the proportion of scan samplings per hour that each group spent consuming the same items was significantly different between sites (Figure 2; p < 0.05).

Home Range and Daily Distance Home range was reduced in the fragmented area, to less than half (100 ha, Figure 3) of that found in the continuous forest (240 ha, Figure 4). Low or no overlapping of the home range with neighboring groups was observed in the fragmented areas. Home range of the study group in the fragmented area included forest fragments, pastures and living fences (natural hedgerows). The area of forest fragments was 50 ha in total. However average daily range was higher in fragmented areas (Continuous: 1017 m; fragmented area: 2237 m). Table 1. Plant species consumed by the S. c. albigena group in the continuous area (Tinigua National Park)

Species Annonaceae Oxandra mediocris Duguetia odorata Ruizodendron ovale Burseraceae Crepidospermun rhoifolium Protium glabescens Protium sagotianum Protium sp Protium robustus Caesalpinaceae Bauhinia guianensis Cannabaceae Celtis schippi Cecropiaceae Pouroma bicolor Pouroma mollis Cecropia sp Pouroma petiolulata Combretaceae Combretum laxum Cyclanthaceae Carludovica palmata Fabaceae Inga alba Inga sp Inga marginata Inga pilosula Dalbergia sp. Inga edulis Inga brachyrachys Inga punctata Inga acrocephala Inga umbellifera Inga sp 50 Inga bonplondiana

Type of item consumed Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fl Fr Fr Fr Fr Fr HJ Fr Fr Fr Fr Fr Fr Fr

Species Flacourtiaceae Mayna odorata Melastomataceae Miconia sp. Miconia trinervia Meliaceae Trichilia mayrasiana Moraceae Pseudolmedia laevis Ficus cf americana Pseudolmedia laevigata Pseudolmedia obliqua Nyctaginaceae Neea verticillata Poaceae Guadua angustifolia Polygonaceae Coccoloba densifrons Putranjivaceae Drypetes amazonica Rubiaceae Duroia hirsuta Sapindaceae Paullinia obovata Sapotaceae Sarcaulus brasiliensis Solanaceae cf Solanum aturense Solanum lepidotum Sterculaceae Herrania nitida Violaceae Leonia glycycarpa Rinorea lindeniana

Type of item consumed Fr Fr Fr Fr Fr Fr Fr Fr Fr, Fl HJ Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr

Species Indeteminated #1 Indeterminated #2 Sp 39 Sp 37 Sp 11 Sp 40 Sp 43 Sp 45 Sp 49 Sp 47 Sp 33 Sp 55 Indeterminated #3 Indeterminated #4

Type of item consumed Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fl Fl

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 497 Table 2. Plant species consumed by the S.c. albigena group in the fragmented area (San Martin area)

*

Type of item Type of item Species consumed Species consumed Species Anacardiaceae Fabaceae Myristicaceae Tapirira cf. guianensis Fr Inga fastuosa Fr Virola sebifera Anonnaceae Inga sp1 Fr Virola sp1. Guatteria punctata Fr Inga bonplondiana Fr Virola sp2. cf. Rollinia edulis Fr Inga cf. alba Fr Irianthera laevis* Xylopia polyantha Fr Clitoria javitensis Fl Myrtaceae Xylopia aromatica Fr Flacourtiaceae Indeterminated XCP142 Annona sp. Fr Diospyros c.f pseudoxylopia Fr Ochnaceae Araliaceae Gnetaceae Ouratea sp.1 Schefflera morototoni Fr, Fl cf. Gnetum nodiflorum Fr Ouratea sp.2 Arecaceae Lauraceae Piperaceae Euterpe precatoria Fr Ocotea oblonga Fr Piper marginatum Mauritia flexuosa Fr, Fl Lecythidaceae Rubiaceae Oenocarpus bataua Fr, Fl Escheweilera bracteosa* Fr Genipa americana Attalea maripa* Fl Malpighiaceae Duroia hirsuta Bignonaceae Byrsonima crispa Fr cf. Psychotria sp. Arrabidaea sp2. Fl Marcgraviaceae Psychotria cf. deflexa Boraginaceae Norantea guianensis Fr Psychotria sp. 1 Cordia bicolor Fr Indeterminated XCP160 Fl Psychotria racemosa Cordia nodosa Fr Melastomataceae Salicaceae Burseraceae Bellucia grossularioides Fr Ryania spaciosa Protium sp Fr Miconia sp1 Fr Sapindaceae Protium glabrescens Fr Miconia trinervia Fr Cupania sp 1 (cf scrobiculata) Protium heptaphyllum Fr Henriettella cf. goudotiana Fr Sapotaceae Trattinnickia cf. aspera Fr Miconia sp2 Fr Sarcaulus brasiliensis Crepidospermum rhoifolium Fr Miconia sp3 Fr Cecropiaceae Miconia elata Fr Pouroma bicolor Fr, Fl Miconia sp4 Fr Cecropia membranaceae Fr, Fl Indeterminated XCP098 Fr Chrysoballanaceae Indeterminated XCP099 Fr Hirtella americana Fr Indeterminated XCP109 Fr Licania subarachnophylla Fr Indeterminated XCP110 Fr Clusiaceae Indeterminated XCP117 Fr Garcinia madruno Fr cf. Miconia ternatifolia Fr Combretaceae Meliaceae Combretum laxum Fr Guarea guidonia Fr Convolvulaceae Mendonciaceae Maripa peruviana Fr Mendoncia lindavii Fr, Fl Cucurbitaceae Moniminiaceae Cayaponia cf. capitata Fr Siparuna guianensis Fr Dilleniaceae Moraceae Davilla nitida Fr Ficus americana Fr Erythroxylaceae Ficus cf. obtusifolia Fr Erythroxylon sp. Fr Pseudolmedia obliqua Fr Euphorbiaceae Ficus trigona Fr Pera arborea Fr Perebea xanthochyma Fr Alchornea triplinervia Fr Pseudolmedia laevis Fr Hyeronima alchorneoides Fr *This species were consumed by Saimiri cassiquiarensis albigena during ad libitum observations of other groups

Type of item consumed Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr Fr

This species were consumed by Saimiri cassiquiarensis albigena during ad libitum observations of other groups

Figure 2. Proportion of scan samplings per hour of the study groups consuming arthropods, fruits, flowers and young leaves at both study sites.

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Figure 3. Home range of San Martin area group of Saimiri cassiquiarensis albigena.

Figure 4. Home range of Tinigua National Park group of Saimiri cassiquiarensis albigena.

Activity Budget Regardless of living in continuous or fragmented environments, S. c. albigena spent most of their time on either stationary foraging or moving/foraging of the five activities we measured. S. c. albigena in the continuous area spent more of their time on stationary foraging and resting compared to those in the fragmented area (Figure 5a). Whereas S. c. albigena in the fragmented area spent relatively more time on moving/foraging and moving relative to those living in the continuous area. The proportion of scan sampling per hour that each group spent on each activity was significantly different between the sites (Figure 5b; p < 0.05).

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 499

Group Size and Composition Group composition and size were reduced in fragmented areas. The study group in the fragmented area was composed of 43 individuals: 4 – 6 males, 8 females, 22 juveniles and 7 infants (born between December and January of 2006). The study group in the continuous area was composed of 53 individuals: 4 males, 14 females, 7 subadults of unknown sex, 14 juveniles and 14 infants born between February and April of 1999. At both sites solitary and bachelor groups of males were observed (range 1 to 7 males). At Tinigua National Park a fusion with a neighboring group was observed (5 times). Fission of the study group was also observed (one time, two subgroups at a ratio of 200 m). In the fragmented area, only fission and fusion of the study group was observed without neighboring groups involved. However, an adult male disappeared for a couple of months and returned with a female and a newborn infant, possibly from a neighboring group (Carretero-Pinzon, 2008).

Percentage of time spent in each activity

a. 60 50 40

30 20 10 0

F

Df

De

D

Voc

D

Voc

Activities Continuous area

Fragmented area

b. Proportion of scan samplings/hour

50 45 40 35

30 25 20 15 10

5 0 F

Df

De

Activities Continuous area

Fragmented area

Figure 5. Activity budget of the study groups of S. c. albigena in a continuous and fragmented area: a. Percentage of time spent in each activity for both groups, and b. Proportion of scan samplings per hour for each activity for both groups. Activities are labeled: stationary foraging (F), moving and foraging (DF), resting (De), moving (D), and vocalizations (Voc).

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Table 3. Group size, composition, male/female ratio, and female/immature ratio of study groups and other groups of Saimiri cassiquiarensis albigena in the study areas No. Group Group Size No. Males No. Females No. subadults No. Juveniles No. Infants Male/Female Ratio Female/Immature ratio Tinigua National Park: Continuous area 0.4 0.29 14 14 7 14 4 53 1* 0.48 0.375 6 24 3 16 6 55 2 0.39 0.36 36 14 5 55 3 0.42 0.34 Average San Martin Area: Fragmented area 0.28 0.75 7 22 0 8 6 43 1* 0.45 0.8 11 5 4 25 2 0.44 0.5 18 8 4 30 3 0.62 0.5 13 8 4 25 4 0.5 0.57 14 7 4 25 5 0.42 0.8 12 5 4 25 6 0.47 0.625 17 8 5 30 7 0.625 0.4 8 5 2 15 8 0.8 1.5 5 4 6 15 9 0.42 0.6 12 5 3 20 10 0.42 0.6 12 5 3 20 11 0.4 0.67 15 6 4 25 12 0.42 0.6 12 5 3 20 13 0.42 0.5 19 8 4 31 14 0.375 0.5 16 6 3 25 15 0.313 0.8 16 5 4 25 16 0.313 0.8 16 5 4 25 17 0.4 0.67 15 6 4 25 18 0.28 0.4 18 5 2 25 19 0.375 0.5 16 6 3 25 20 0.29 0.6 17 5 3 25 21 0.29 0.6 17 5 3 25 22 0.4 0.67 15 6 4 25 23 0.4 0.67 15 6 4 25 24 0.37 0.57 19 7 4 30 25 0.5 0.33 18 9 3 30 26 0.4 0.67 15 6 4 25 27 0.29 0.6 17 5 3 25 28 0.5 0.57 14 7 4 25 29 0.38 0.4 13 5 2 20 30 0.62 0.5 13 8 4 25 31 0.38 0.4 13 5 2 20 32 0.5 0.57 14 7 4 25 33 0.375 0.5 16 6 3 25 34 0.37 0.57 19 7 4 30 35 0.42 0.375 19 8 3 30 36 0.47 0.43 15 7 3 25 37 0.375 0.5 16 6 3 25 38 0.8 0.25 10 8 2 20 39 0.3 0.67 20 6 4 30 40 0.375 0.5 16 6 3 25 41 0.43 0.59 Average

*

*Study Groups for each area

Study Groups for area

Ad libitum observations of group size and composition in both areas revealed similar average female/immature ratios at both areas (continuous area: 0.42; fragmented area: 0.43), but almost double of the male/female ratio for the fragmented area when compared with the continuous area (continuous area: 0.34; fragmented area: 0.59). In the fragmented area group sizes ranged from 15 – 43 in forest fragments (range of forest fragments: 0.2 – 1080 ha; Table 3) and over 50 individuals in the continuous area.

DISCUSSION Habitat loss and fragmentation produced changes in Colombian squirrel monkey behavior (S. c. albigena), which adapts to these processes, changing activity budgets, diet, home range and daily ranges. These changes in behavior have been described for other species such as spider and howler monkeys in Mexico (Asencio et al., 2007; Pozo-Montuy and Serio-Silva 2007; Bicca-Marques et al., 2009; Dunn et al., 2010; Gonzalez-Zamora et al., 2011; Chaves et

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 501 al., 2012; Pozo-Montuy et al., 2012), northern bearded saki monkeys and pygmy marmosets in the Amazon (Boyle et al., 2009; Boyle and Smith 2010; Boyle et al., 2012; De la Torre et al., 2013). The range of changes in these behaviors is wide and varied, so no general pattern has been found, except for an increase in feeding activity in fragmented areas, as the ones observed in the present study (Carretero-Pinzon et al., 2015.). The use of new plant species as well as an incremental increase in consumption by species in continuous areas are less important than has been reported in other studies as a consequence of reduced access to fruits in forest fragments (Pozo-Montuy and Serio-Silva 2007; Bicca-Marques et al., 2009; Dunn et al., 2010; Boyle et al., 2009, 2012; Chaves et al., 2012; Pozo-Montuy et al., 2012). Reduction in space leads to a reduction in home range and the exploitation of isolated trees, living fences and other resources, such as pioneer plant species to complement their diet (Pozo-Montuy et al., 2012). The importance of pioneer plant species was evident in the number of species from the Melastomatacea family included in the group from the fragmented area, compared with the group in the continuous area. These species were found mainly in fragment edges and living fences that were used by this same group especially when fruit production in the fragments was reduced (Carretero-Pinzon and Defler, in press). Additionally, the reduction in resource availability leads to possible effects on fitness reduction as proposed for northern bearded sakis and mantled howlers (Boyle and Smith, 2010; Dunn et al., 2010). This fitness reduction is supported by a reduction or non-presence of immature individuals in groups living in fragment areas (Boyle and Smith, 2010), contrary to what was found in this study, which showed a similar female/immature ratio in both areas. However, low variation in group size through time (in San Martin group, Carretero-Pinzón unpublished data) could suggest a high mortality of juveniles that needs to be investigated. A higher male/female ratio observed in Colombian squirrel monkeys in the fragmented area could suggest dispersal problems for both sexes due to habitat loss and fragmentation. Although bachelor groups and solitary individuals were observed in the fragmented area, some of them were found in isolated fragments which reduces their reproductive potential (Carretero-Pinzon, unpublished data). Landscape configuration and the presence of living fences could be important to improve individual dispersion and solitary individuals probabilities of reproduction (Carretero-Pinzon et al., 2010). Additionally, a reduction in group size was observed in the fragmented area that could be related to high mortality, although no data is available to support this, but domestic dog predation has been observed (Carretero-Pinzón 2013). Group size variations, less individual dispersion and lower reproduction have been reported in fragmented areas for other primate species (Boyle and Smith, 2010; Boyle et al., 2012; Estrada et al., 2002; Srivastava et al., 2001; Umapathy and Kumar, 2000). Changes in the activity budgets of groups living in fragments have shown an increase in the time spent in foraging activities, while traveling alone is reduced as a strategy to adapt to resource limitations in fragmented habitats (Chaves et al., 2011; Gonzalez-Zamora et al., 2011). This same pattern was shown by the study group in the fragmented area, which spent more time in activities related to feeding (foraging and moving with foraging). A tendency to maximize energy through more moving and foraging activity (combined) seems to be part of the behavioral changes of Colombian squirrel monkeys in fragmented areas, at least for this study period. These changes in the activity patterns are also related to an increase in more dispersed resources, namely arthropods (Janson and Chapman, 1999), which is one of the main components of the squirrel monkey’s diet (Boinski, 1989; Boinsky and Fowler, 1989).

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The increased travelling time in the search for arthropods for the fragmented area group also increases its daily ranges. This seems to support the tendency to maximize their energy by increasing movements while searching for arthropods and fruit trees when they are both abundant (Rosenberger and Strier, 1989). We used the beginning of the wet season as the study period analyzed for both groups, a time when fruit production is high (Stevenson et al., 1994; Carretero-Pinzon, 2008). Additionally, the beginning of the wet season has been reported as a period of abundance of herbivorous arthropods due to an increase in new leaves (Boinski and Fowler, 1989). Therefore, the study period corresponds to the time when the main resources in the Colombian squirrel monkey diet are more abundant and the monkeys maximize the intake of energy by increasing their feeding time. In conclusion, the Colombian squirrel monkeys’ strategy in fragmented landscapes is to spend more time feeding and increasing the proportion of pioneer species found in forest edges and living fences. The animals also include live fences in their home ranges to increase the space available for food resources.

ACKNOWLEDGMENTS Fieldwork in both sites mentioned in this chapter would not have been possible without the help and permissions from various institutions, grants and individuals. At Tinigua National Park: J. Ahumada, K. Izawa, C. A. Mejía, A. Nishimura and Ministry of Environment and Unidad de Parques Nacionales, as well as field assistants Alvaro, Meyo and Ramiro. At San Martin Area: Sanchez-Rey, Novoa and Enciso families for their long-term support and permission for XCP to live on their farms. Funding for fieldwork (2004 – 2014) was provided by Becas IEA (2009, 2011; Conservation International Colombia, Fundación Omacha and Fondo para la Acción Ambiental y la Niñez), RHD Grant from School of Geography, Planning and Management, The University of Queensland (2013-2014), Idea Wild and private funding (farms support).

REFERENCES Altmann, J. (1974). Observational study of behavior: Sampling methods. Behaviour, 49, 227267. Angulo, M. A. (2001). Determinación del rango de hogar y patrones de actividad de una manada de mono ardilla (Saimiri sciureus) en el Parque Nacional Natural Tinigua. Undergraduate Thesis, Universidad de los Andes, Bogotá, Colombia. Arroyo-Rodriguez, V., González-Perez, I. M., Garmendia, A., Solà, M. and Estrada, A. (2013). The relative impact of forest patch and landscape attributes on black howler populations in the fragmented Lacandona rainforest, Mexico. Landscape Ecology, 28, 1717-1727. Asensio, N., Cristóbal-Azkarate, J., Días, P. A. D., Veà-Baro, J. J. and Rodríguez-Luna, E. (2007). Foraging habits of Alouatta palliata mexicana in three forest fragments. Folia Primatologica, 78, 141–153.

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 503 Bicca-Marques, J. C., Barboza-Muhle, C., Mattjie-Prates, H., Garcia de Oliveira, S. and Calegaro-Marques, C. (2009). Habitat impoverishment and egg predation by Alouatta caraya. International Journal of Primatology, 30, 743–748. Boinski, S. (1987). Habitat use in squirrel monkeys (Saimiri oerstedii) in Costa Rica. Folia Primatologica, 49, 151-167. Boinski, S. (1989). The positional behavior and substrate use of squirrel monkeys: ecological implications. Journal of Human Evolution, 18, 659-677. Boinski, S. and Fowler, N. L. (1989). Seasonal patterns in a tropical lowland forest. Biotropica, 21(3), 223-233. Boinski, S. and Mitchell, C. L. (1994). Male residence and association patterns in Costa Rican squirrel monkeys (Saimiri oerstedii). American Journal of Primatology, 34, 157169. Boyle, S. A., Lourenço, W. C., da Silva, L. R. and Smith, A. T. (2009). Travel and spatial patterns change when Chiropotes satanas chiropotes inhabit forest fragments. International Journal of Primatology, 30, 515–531. Boyle, S. and Smith, A. (2010). Behavioral modifications in northern bearded saki monkeys (Chiropotes satanas chiropotes) in forest fragments of central Amazonia. Primates, 51, 43–51. Boyle, S. A., Zartman, C. E., Spironello, W. R. and Smith, A. T. (2012). Implications of habitat fragmentation on the diet of bearded saki monkeys in central Amazonian forest. Journal of Mammalogy, 93(4), 959-976. Chaves, O. M., Stoner, K. E. and Arroyo-Rodriguez, V. (2011). Seasonal differences in activity patterns of geoffroyi’s spider monkeys (Ateles geoffroyi) living in continuous and fragmented forests in southern Mexico. International Journal of Primatology, 32, 960– 973. Chaves, O. M., Stoner, K. E. and Arroyo-Rodriguez, V. (2012). Differences in diet between spider monkey groups living in forest fragments and continuous forest in Mexico. Biotropica, 44(1), 105–113. Carretero-Pinzon, X. (2000). Un estudio ecológico de Saimiri sciureus y su asociación con Cebus apella, en la Macarena, Colombia. Undergraduate thesis. Pontificia Universidad Javeriana, Bogotá, Colombia. pp 88. Carretero-Pinzón, X. (2008). Efecto de la disponibilidad de recursos sobre la ecología y comportamiento de Saimiri sciureus albigena en fragmentos de bosque de galería, San Martín (Meta – Colombia). Masters thesis, Pontificia Universidad Javeriana, Bogotá, Colombia. pp. 132. Carretero-Pinzón, X. (2013). An eight year life history of a primate community in fragments at Colombian Llanos. In Primates in Fragments: Complexity and Resilience, Developments in Primatology: Progress and Prospects. Marsh, L.K., and Chapman, C.A. (Eds.). Springer Science+Business Media, New York, USA, pp. 159-182. Carretero-Pinzon, X., Defler, T. R., McAlpine, C. A. and Rhodes, J. R. (2015). What do we know about the effect of patch size on primate species across life history traits? Biodiversity and Conservation, 25 (1), 37-66. Carretero-Pinzon, X. and Defler, T. R. (in press). Primates and flooded forest in the Colombian Llanos. In: Primates in flooded habitats: ecology and conservation. Barnett A.A., Matsuda I. and Nowak K. (Eds.). Cambridge. Cambridge University Press.

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Carretero-Pinzón, X., Ruíz-García, M. and Defler, T. R. (2010). Uso de cercas vivas como corredores biológicos por primates en los llanos orientales. In Pereira, V., Stevenson, P. R., Bueno, M. L. and Nassar-Montoya, F. (Eds.). Libro del Primer Congreso Colombiano de Primatología. (pp. 91-98). Fundación Universitaria San Martin, Bogotá. De la Torre, S., Yepez, P., Nieto, D. and Payaguaje, H. (2013). Preliminary evaluation of the effects of habitat fragmentation on habitat use and genetic diversity of pygmy marmosets in Ecuador. In Marsh, L.K., and Chapman, C.A. (Eds.). Primates in Fragments: Complexity and Resilience, Developments in Primatology: Progress and Prospects. (pp. 437-446). Springer Science+Business Media, New York, US. Defler, T. R. (2010). Historia Natural de los Primates Colombianos. Universidad Nacional de Colombia, Bogotá, Colombia. pp. 612. Dunn, J. C., Cristobal-Azkarate, J. and Vea, J. J. (2010). Seasonal variations in the diet and feeding effort of two groups of howlers in different sized forest fragments. International Journal of Primatology, 31, 887-903. Estrada, A., Mensoza, A., Castellanos, L., Pacheco, R., Van Belle, S., Garcia, Y. and Muñoz, D. (2002). Population of the black howler monkey (Alouatta pigra) in a fragmented landscape in Palenque, Chiapas, Mexico. American Journal of Primatology, 58, 45-55. Fahrig, L., (2003). Effects of habitat fragmentation on biodiversity. Annual Review of Ecology and Systematics, 34, 487–515. Forman, R. T. T. and Godron, M. (1986). Landscape Ecology. John Wiley and Sons, Inc. USA, pp. 619. Gonzalez-Zamora, A., Arroyo-Rodriguez, V., Chaves, O. M., Sanchez-Lopez, S., Aureli, F. and Stoner, K. E. (2011). Influence of climatic variables, forest type and condition on activity patterns of Geoffroyi’s spider monkeys throughout Mesoamerica. American Journal of Primatology, 73, 1189-1198. Hirabuki, Y. (1990). Vegetation and landform structure in the study area of La Macarena: A physiognomic investigation. Field Studies of New World Monkeys, La Macarena, Colombia, 3, 35-48. Janson, C. H. and Chapman, C. A. (1999). Resource and primate community structure. In Primate Communities. Fleagle, J.G., Janson, C.H., and Reed, K.E. (Eds). (pp. 237-267). Cambridge University Press, UK. Kimura, K., Nishimura, A., Izawa, K. and Mejia, C. A. (1994). Annual changes of rainfall and temperature in the tropical seasonal forest at La Macarena, Field Station Colombia. Field Studies of New World Monkeys. La Macarena, Colombia, 9, 1-3. Marsh, L. K., Chapman, C. A., Arroyo-Rodriguez, V., Cobden, A. K., Dunn, J. C., Gabriel, D., Ghai, R., Nijman, V., Reyna-Hurtado, R., Serio-Silva, J. C. and Wasserman, M. D. (2013). Primates in fragments 10 years later: once and future goals. In Primates in Fragments: Complexity and resilience, Developments in Primatology: Progress and prospects. Marsh, L.K., Chapman, C.A. (Eds.). (pp. 503-523). Springer Science+Business Media, New York. Mitchell, C. L. (1990). The ecological basis for female social dominance: A behavior study of the squirrel monkey (Saimiri sciureus) in the wild. Ph.D. Dissertation, Princeton University, Princeton. pp 189. Onderdonk, D. A. and Chapman, C. A. (2000). Coping with forest fragmentation: the primates of Kibale National Park, Uganda. International Journal of Primatology, 21(4) 587–611.

How Does the Colombian Squirrel Monkey Cope with Habitat Fragmentation? 505 Pozo-Montuy, G. and Serio-Silva, J. C. (2007). Movement and resource use by a group of Alouatta pigra in a forest fragment in Balancan, Mexico. Primates, 48, 102–107. Pozo-Montuy, G., Serio-Silva, J. C., Chapman, C. A. and Bonilla-Sanchez, Y. M. (2013). Resource use in a landscape matrix by an arboreal primate: evidence of supplementation in black howlers (Alouatta pigra). International Journal of Primatology, 34, 714-731. Rodriguez-Vargas, A. R. (2003). Analysis of the hypothetical population structure of the squirrel monkey (Saimiri oerstedii) in Panama. In Primates in Fragments: Ecology and Conservation. Marsh, L.K. (Ed.) Kluwer Academic/Plenum Publisher, New York, pp. 5362. Rosenberger, A. L. and Strier, K. B. (1989). Adaptative radiation of the ateline primates. Journal of Human Evolution, 18, 717-750. Ruiz-García, M., Luengas-Villamil, K., Leguizamón, N., de Thoisy, B. and Gálvez, H. (2015). Molecular phylogenetics and phylogeography of all the Saimiri taxa (Cebidae, Primates) inferred from mt COI and COII gene sequences. Primates, 56, 145-161. Rylands, A. B., Williamson, E. A., Hoffmann, M. and Mittermeier, R. A. (2008). Primate surveys and conservation assessments. Oryx, 42(3), 313-314. Srivastava, A., Biswas, J. and Bujarbarua, P. (2001). Status and distribution of Golden langurs (Trachypithecus geei) in Assam, India. American Journal of Primatology, 55, 1523. Stevenson, P. R., Quiñones, M. J. and Ahumada, J. A. (1994). Ecological strategies of woolly monkeys (Lagothrix lagothricha) at Tinigua National Park, Colombia. American Journal of Primatology, 32, 123-140. Stevenson, P. R., Suescun, M. and Quiñones, M. J. (2004). Characterization of forest types at the CIEM, Tinigua Park, Colombia. Field Studies of Fauna and Flora. La Macarena, Colombia, 14, 1-20. Stevenson, P. R. and Aldana, A. M. (2008). Potential effects of Ateline extinction and forest fragmentation on plant diversity and composition in the western Orinoco Basin, Colombia. International Journal of Primatology, 29, 365–377. Terborgh, J. (1983). Five new world primates. A study in comparative ecology. Princeton University Press. pp 260. Umapathy, G. and Kumar, A. (2000). The demography of the lion-tailed macaque (Macaca silenus) in rain forest fragments in the Anamalai Hills, South India. Primates, 41(2), 119126. Wagner, M., Castro, F. and Stevenson, P. R. (2009). Habitat characterization and population status of the dusty titi monkey (Callicebus ornatus) in fragmented forest, Meta, Colombia. Neotropical Primates, 16(1), 18-24. Yoneda, M. (1990). The difference of tree size used by five cebid monkeys in Macarena, Colombia. Field Studies of New World Monkeys, La Macarena, Colombia, 3, 13-18. Zar, J. H. (1996). Biostatistical analysis. Prentice Hall International Editions. Third Edition. Upper Saddle River, New Jersey, 662.

In: Phylogeny, Molecular Population Genetics … Editors: M. Ruiz-Garcia and J. Mark Shostell

ISBN: 978-1-63485-165-7 © 2016 Nova Science Publishers, Inc.

Chapter 16

CALLICEBUS ORNATUS, AN ENDEMIC COLOMBIAN SPECIES: DEMOGRAPHY, BEHAVIOR AND CONSERVATION Xyomara Carretero-Pinzon1,3 and Thomas R. Defler2 1

University of Queensland, School of Geography, Planning and Environmental Management, Brisbane, Australia ²Universidad Nacional de Colombia, Biology Department, Bogotá, Colombia 3 Proyecto Zocay, Colombia

ABSTRACT Habitat loss and fragmentation are two of the main threats to primate species worldwide. Studies of species of Callicebus in forest fragments have highlighted the species´ ecological plasticity and adaptability of this genus. The aim of this chapter is to evaluate ecological and demographic aspects of dusky titi monkeys in fragmented areas and to elucidate possible features that make them tolerant to habitat loss and fragmentation. Using census data collected from two databases: 1) from a large fragment (1080 ha, from 2008-2013) and 2) from 46 fragments (range: 4.87-1080 ha, from 20132014), we found that Callicebus ornatus use of forest edge is significantly different from random (p < 0.05) for the largest fragment. All the other observations of C. ornatus groups made in this study were in forest fragment edges. These data support our hypothesis that this species prefers forest fragment edges to the interior of a forest. Vegetation analysis showed that forest near the edge of a large fragment was 1.4 times more diverse (D = 0.953) than forest 300-400 m from the forest edge (D = 0.92). We also found that group sizes and density indices are not significantly affected by fragment size (p > 0.05), although average group size and the average density indices for fragments of 1-50 ha present higher values compared to the other fragment size categories. Therefore, we did not find support for our hypothesis of fragment size as a determinant of group size and density for C. ornatus. A more detailed exploration of landscape, forest structure and resource availability variables is needed to understand how habitat loss and fragmentation is affecting this species. The findings in this chapter supports the ecological adaptability and plasticity of Callicebus ornatus through its ability to use the edge of forest fragments.

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Keywords: Callicebus ornatus, habitat loss, fragmentation, use of edges, birth season

INTRODUCTION Habitat loss and fragmentation are two of the main threats to primate species worldwide (Rylands et al., 2008; Marsh et al., 2013). These two processes occur at the landscape scale, affecting the amount of habitat available and how separate the fragments are in the landscape (Forman and Godron, 1986; Fahrig, 2003) and producing changes in species demography, behaviour and ecology (Boyle et al., 2009; Chapman et al., 2010; Gonzalez-Zamora et al., 2011; Boyle et al., 2012; Carretero-Pinzon, 2013a). Primate studies in fragments have been done mainly at the patch scale, evaluating the effect of fragment size and isolation on the primate species presence or absence and their densities (Harcourt and Doherty 2005; ArroyoRodriguez et al., 2013; Arroyo-Rodriguez and Fahrig 2014; Benchimol and Peres 2013). Primate responses to habitat loss and fragmentation have been found to be consistent across species, with increases of density, feeding behavior and parasitic prevalence and diversity, while genetic diversity and individual species´ presence decrease (Carretero-Pinzón et al., 2015). In Colombia, the main drivers of deforestation are population growth and migration, infrastructure projects, palm oil plantations, agriculture and cattle ranching (Etter et al., 2006; Fedepalma, 2014; Ecopetrol, 2015). These drivers are also some of the main factors affecting the loss of habitat for the dusky titi monkey (Carretero-Pinzon, 2013b). Studies with species of Callicebus in forest fragments have highlighted the ecological plasticity and adaptability of the species of this genus (Pyritz et al., 2010). Occupancy data for species such as C. moloch have not found evidence of fragment size effects (Michalski and Peres 2005). However, for species living in caatinga forest (C. coimbrai and C. barbarabrownae) fragment size and groundwater seem to be important for their presence (Ferrari et al., 2013). Densities reported for C. ornatus in fragments are higher than those reported for the species in continuous areas (Polanco-Ochoa and Cadena 1993; Wagner et al., 2009; Carretero-Pinzon, 2013a). However, these two cited studies have small sample sizes for fragments and a larger sample is needed to understand if fragment size is affecting density and if so, in what way it is affecting it. The dusky titi monkey (Callicebus ornatus) is a small Neotropical primate that lives only in Colombia. Studies of this species have been limited to undergraduate theses of short duration (6 months or less) focused on the ecology of this species in a continuous area (Polanco-Ochoa and Cadena, 1993) and ecology, behavior and densities in fragments of different sizes (Sanchez, 1998; Ospina, 2006; Wagner et al., 2009). Although dusky titi monkeys inhabit secondary forests and seem to adapt well to this habitat (Defler, 2010), we still don’t know how much habitat loss and fragmentation are affecting their demography, ecology and behavior. Additionally, it seems this species prefers areas near fragment edges which can give them some advantages in fragmented landscapes where small and degraded fragments are available in which edge effects predominate. Studies of the effects of edge variables on forest dynamics in Amazonia have found that edge variables can affect forest dynamics up to 300 m from the edge (Laurence et al., 1998). Such forest dynamics affect forest-dependant animal populations such as primates. Primate studies have found that some

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species can be tolerant to edge effects and this tolerance can provide advantages for survival in forest fragments (Lehman et al., 2006; Quemere et al., 2010). The aim of this chapter is to evaluate ecological and demographic aspects of dusky titi monkeys in fragmented areas, in order to elucidate possible features that make them tolerant to habitat loss and fragmentation. We hypothesized that the dusky titi monkeys is a species that prefers forest fragment edges to forest interior and this is one of the main features that allows them to survive in fragmented areas. Additionally, we hypothesized that forest edges might have a superior offering of foods compared to the interior in a large forest fragment. We also hypothesized that group size and density of this species is determined by fragment size. In order to test these hypotheses we ask the following questions: 1) Are dusky titi monkeys more common in fragment forest edges compared with fragment forest interior in a large fragment where edge and interior can be defined (1080 ha)? 2) Is the offering of foods somehow superior near the edge of large fragments? 3) Is there an effect of fragment size on dusky titi monkey group size and density? In addition, in this chapter we present data defining a birth season for Callicebus ornatus in fragmented areas and a list of plant species consumed by this species in the area.

MATERIALS AND METHODS Study Area This study was conducted near the town of San Martín de Los Llanos and neighboring towns (3°45’05.67”N 73°44’23.09”W to 3°25’10.57”N 73°26’20.00”W), Department of Meta, in the Colombian Llanos. This area has fragments of different sizes (range 4.61-1080 ha, Figure 1) surrounded by pastures, natural savannas, palm oil plantations and small scale agriculture. The presence of small forest fragments (less than 1 ha), isolated trees and live fences is common in some parts of the study area (Figure 2). Elevation varies from 300-400 meters. Five Neotropical primates are found in this area: Sapajus apella fatuellus, Alouatta seniculus, Saimiri cassiquiarensis albigena, Callicebus ornatus, and Aotus brumbacki (Carretero-Pinzón, 2013a). The weather in this area is characterized by a wet season (AprilNovember, average 1777 mm) and a dry season (December-March, average 357 mm), with an annual average temperature of 26°C (Carretero-Pinzón, 2008). The vegetation of these fragments shared species richness with differences in the importance of plant species in each fragment (Stevenson and Aldana, 2008; Carretero-Pinzón, unpublished data). Fragmentation is an ongoing process in this region due to livestock practices, palm oil plantations and petrol exploitation (Wagner et al., 2009; Carretero-Pinzón 2013b; Carretero-Pinzón and Defler, in press).

Primate Surveys The data presented in this chapter come from two databases. One database is of the largest fragment in the area (1080 ha) in which census surveys have been done from 2008 to 2013, every two to four months. These surveys were done in transects of 400-2600 m, in

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which edge effects were considered to penetrate the fragment up to 300 m from the edge (sensu Laurence et al., 1998, 2011). The other database includes survey data from 46 fragments conducted during 2013-2014. Surveys from both data sets were conducted from 0530 to 1100 and again from 1330 to 1630. Transects were walked at approximately 1.5 km/h. When a primate group was observed, a minimum of 15 minutes was taken to count the group members and to determine group composition (number of males, females, juveniles and infants). The coordinates of each group observation were registered using a GPS. Surveys were not conducted during heavy rain. Ad libitum observations of group composition and independent infants of the same groups in the largest (1080 ha) forest and three groups of a small fragment (23 ha) were taken. Plant species consumed during the census and during ad libitum observations were also recorded (data 2008-2014).

Data Analysis Data surveys from the largest fragment were pooled and we used χ2 tests (Zar, 1996) to test whether the frequency of observation of Callicebus ornatus groups in the fragment interior and fragment edge were significantly different from random. Also, we used a linear regression (Zar, 1996) to test if fragment size had an effect on group size and density index of Callicebus ornatus. Density indices were calculated as the total of individuals observed in a fragment divided by the number of surveys conducted in that fragment. For the statistical analysis we used R software (www.r-project.org).

Vegetation Analysis A system of measured trails was developed outlining two hectares of forest. The first hectare was measured in a grid of four squares 50 m X 50 m (oriented linearly) under the home ranges of two Callicebus ornatus close to the forest edge (10-20 m). The second hectare was located in a large square (100 m X 100 m), divided into four parts (50 m X 50 m) by trails and located 300-400 m from the forest edge. All trees with DBH of 10 cm and above were marked in each hectare square with flagging tape and each individual tree was assigned a number. All DBH and tree heights were registered. Species determinations were made with the collaboration of Francisco Castro, a local botanic who is an expert on the local llanos vegetation. A Simpson Diversity Index was carried out for each 1 ha plot (Simpson, 1949).

RESULTS Forest Fragment Edge Versus Interior Use by Groups of Callicebus ornatus We conducted a total of 641 census hours (634.4 km) in the largest fragment (1080 ha), in which 92 vocal (66%) and visual (34%) contacts with Callicebus ornatus groups were registered. The proportion of observations of C. ornatus groups at the forest edge was significantly different from observations in the forest interior for both types of observations (Figure 3, p > 0.05).

Figure 1. Locations of fragments sampled in the San Martin area, Colombia (total area of 1932 km2).

Figure 2. Detail of the study area showing living fences and isolated trees (detailed area: 114 km2).

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Figure 3. Proportion of Callicebus ornatus groups observed at the forest fragment edge versus the forest fragment interior, for auditive and visual types of observations.

Fragment Size Effects on Group Size and Density Index We observed a total of 351 groups; this includes all groups observed and differentiated from the largest fragment and all groups observed in the other 46 fragments during 20132014. Although fragment size did not explain the variation in group size of C. ornatus, a tendency to reduce the group size, especially in fragments over 100 ha was observed (Figure 4; R2 = 0.001435, df = 149, p = 0.6442).

Figure 4. Variation of the average group size of Callicebus ornatus with fragment size.

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Similarly, fragment size did not explain the density index found in the study area (Figure 5, R2 = 0.001435, df = 149, p = 0.6442), however a tendency to reduce the density index in fragments over 50 ha was observed.

Figure 5. Density index of Callicebus ornatus in fragments of different size.

Diversity of Hectare Plots The hectare plot near the edge of the forest (Plot 1) had 1.4 times more tree diversity (D = 0.953) as compared to the forest interior (Plot 2) (D = 0.92). Plot 1 had 452 trees of 10 cm and above, as compared to Plot 2 with 320 trees of 10 cm and above. The trees in Plot 1 were represented by 32 families and 75 species as compared to Plot 2, which had 24 families and 54 species of trees.

Additional Observations A birth season has been observed in the study area with dependent infants (infants carried solely by the males) from December to March (73 observations): smallest infants have been observed in December and January. Data from repeated observations of the same groups in small fragments have shown independent movements of infants starting after three months of age and total independent movement after six months (repeated ad libitum observations of three groups). A list of plant species consumed by the C. ornatus groups observed during the census is shown in Table 1. All the plant species consumed by the groups observed during the censuses were consumed for their fruits and the main plant families were Burseracea, Annonaceae, Fabaceae and Melastomataceae.

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Table 1. List of plant species consumed by Callicebus ornatus groups observed during census (data from 2008-2013) Type of Type of item item Species consumed Species consumed Annonaceae Fabaceae Rollinia cf edulis Fr Inga fastuosa Fr Xylopia sp. Fr Inga bonplondiana Fr Xylopia polyantha Fr Inga cf. alba Fr Guatteria punctata Fr Malpighiaceae Arecaceae Byrsonima crispa Fr Euterpe precatoria Fr Marcgraviaceae Mauritia flexuosa Fr Norantea guianensis Fr Burseraceae Melastomataceae Protium glabrescens Fr Miconia elata Fr Trattinickia aspera Fr Bellucia grossularoides Fr Protium cf. robustus Fr Miconia trinervia Fr Crepidospermum rhoifolium Fr Henriettella cf. goudotiana Fr Protium heptaphyllum Fr Myristicaceae Cecropiaceae Virola sebifera Fr Pouroma bicolor Fr Virola sp1. Fr Clusiaceae Virola sp2. Fr Garcinia madruno Fr Irianthera laevis* Fr Euphorbiaceae Myrtaceae Hyeronima alchorneoides Fr Psidium guajaba Fr

Species Moraceae Pseudolmedia laevis Ficus americana Salicaceae Ryania spaciosa

Type of item consumed Fr Fr Fr

DISCUSSION The preference for forest fragment edges of Callicebus ornatus demonstrates that the species is ecologically adaptable enough to cope with habitat loss and fragmentation. These features make them more tolerant to habitat loss and fragmentation processes. However, this needs to be investigated in more detail as habitat loss and fragmentation are landscape processes. The high frequency of observations (visual and auditory) of C. ornatus in forest fragment edges can be explained by several aspects of their ecology. First, C. ornatus is a frugivorous species that complements its diet with arthropods (Fruit: 60%, Arthropods: 25.5%, Sanchez, 1998; Ospina, 2006). In addition, the ability of C. ornatus to exploit pioneer species such as fruits from Bellucia spp., Miconia spp., that are common in forest fragment edges (Sanchez, 1993; Ospina, 2006), give them an advantage for its frugivorous diet. Second, analysis of the vegetation diversity in the largest study fragment showed clearly that tree diversity was higher close to the edge and that there were considerably more trees available at a diameter at breast height (DBH) of 10 cm and above. So the fruit offerings were considerably more numerous in this part of the forest, compared to the forest 300-400 m towards the forest interior. Additionally, arthropod consumption could be increased due to an increase of arthropods in forest edges influenced by the amount of light in these areas (Marsh, 2003; Richards and Coley, 2007). Lastly, C. ornatus prefers to use habitats in which the proportion of vines is high (Polanco-Ochoa and Cadena, 1993), characteristic of forest fragment edges (Silver and Marsh, 2003).

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Although we did not find significant effects of fragment size on C. ornatus group sizes and densities, a trend towards higher values in average group size and density indices for fragment forests under 50 ha was observed. Small fragment sizes are usually found in highly transformed landscapes (175 species of fruit in approximately 10 months), yet they fed from some species for multiple months, ingesting both young and mature seeds. The same pattern was noted for Chiropotes albinasus (Santos et al., 2013) and Cacajao hosomi in Brazil (Boubli and Tokuda 2008). These behaviors supersede characteristics of specific habitats. Thus Cacajao and Chiropotes appear to have a built-in strategy to deal with resource rarity. They can open mechanically protected fruits, make use of very young to mature seeds, and travel great distances daily in very large home ranges to locate resources. Their highly derived dentition, a morphological specialization, has expanded their dietary niche (Norconk and Veres, 2011), although their diet each month is usually dominated by seeds. Feeding studies suggest that P. pithecia is more generalized in their choice of food types than Chiropotes and Cacajao (Setz et al., 2013). The former ingest more leaves (often, but in low quantities) and insects, as well as seeds, arils, and ripe fruit pulp (Kinzey and Norconk, 1993; Norconk, 1996; Norconk, 2006; Norconk and Setz, 2013). We noted above that Inga spp. (mesocarp) is the top-ranked annual resource for Pithecia spp.

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c. Ecological Effects of Sympatry among Pitheciines Whereas Chiropotes and Cacajao are essentially allopatric throughout most of their ranges, Pithecia populations potentially overlap widely with Chiropotes and to a lesser degree with Cacajao (see Figure 4). However, the only studies of sympatric Chiropotes and Pithecia have taken place in the Guianas (Gregory and Norconk, 2013). Pithecia spp. north of the Amazon (P. pithecia and P. chrysocephala) seem to be ecologically very similar, making extensive use of the understory (Setz et al., 2013). Walker (1996), observed Pithecia pithecia and Chiropotes satanas on separate islands in Lago Guri, and found that Pithecia fed in smaller, shorter trees, with smaller crown diameters and traveled in the understory (mean height, 6.8 m) compared to Chiropotes (13.5 m). Free-ranging Pithecia and Chiropotes differ in home range size (68-148 ha vs. 200-559 ha, respectively) (Gregory and Norconk, 2013: 287). Thus, even though they both have a diet that is high in seeds, they rarely interact. We are not aware of any studies of larger-bodied Pithecia (e.g., P. irrorata) that are apparently sympatric with Chiropotes, although Barnett et al. (2013) referred to Pithecia and Cacajao as having different habitat preferences particularly with regard to flooded habitats. Larger-bodied Pithecia in the central and western Amazon were found higher in the forest. Peres’ (1993) study of P. albicans in a small area outside of the range of either Chiropotes or Cacajao presents an interesting contrast to Guianan sakis. P. albicans is the largest bodied Pithecia (see Table 1), thus seems largely convergent on Chiropotes in body size and habitat use with a much larger home range. Finally, it should be noted that population densities, particularly in Pithecia populations south of the Amazon, are very low compared to those north of the Amazon in the Guianas (Norconk and Setz, 2013).

PITHECIINE CONSERVATION ISSUES Globally, primates are threatened by habitat loss and fragmentation, disease, hunting, and climate change (Chapman and Gogarten, 2012; Oates, 2013), and approximately 48% of primate taxa are listed by the IUCN as Critically Endangered, Endangered, or Vulnerable (Mittermeier et al., 2009; IUCN, 2015). The pitheciines follow similar patterns: 75% of the Chiropotes species assessed by IUCN1 are listed as Critically Endangered or Endangered, and 75% of Cacajao species and 20% of Pithecia species are listed as Vulnerable (IUCN, 2015; Boyle, 2014). Of the species assessed by the IUCN as of 2014, the population trends were listed as decreasing for 75% of Cacajao and Chiropotes species, and for 40% of Pithecia species (Boyle, 2014). The forest habitat of some pitheciine species has been heavily fragmented, and some areas have undergone recent forest loss (see Figure 1). Overall, the geographic range of the pitheciines has been heavily modified in parts of the Brazilian states of Amazonas, Maranhão, Mato Grosso, Pará, Rondônia, Roraima, and Tocantins; the Colombian departments of Caqueta, Meta, and Putumayo; the Ecuadorian provinces of Napo, Orellana, and Sucumbíos; and the Venezuelan state of Bolívar (see Figure 1A). In addition, recent forest loss has also 1

The IUCN Red List of Threatened Species does not include the recent taxonomic revisions made to Pithecia by Marsh (2014). The most recent assessment of Chiropotes in 2008 included four, not five, species of Chiropotes due to taxonomic inconsistencies between Silva Jr. and Figueiredo (2002) and Bonvicino et al. (2003).

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occurred on a smaller scale in French Guiana, Guyana, Peru, and Suriname. In examining the geographic range of Cacajao, Chiropotes, and Pithecia, Boyle (2014) found that the area occupied by Chiropotes had the greatest amount of forest loss from 2000 to 2010 (11% loss of forest), the greatest amount of modified land cover (8% of geographic range), and the smallest mean size of forest fragments (240 ha; see Figure 1C). Pithecia and Cacajao have fared better in regard to forest loss (1% for both genera), modified land cover (3% and 1%, respectively), and mean size of forest fragments (4059 ha and 1642 ha, respectively), but both genera have experienced forest loss and fragmentation (Boyle, 2014; Figures 1B and 1D). The connection between diet and conservation is summarized well by Chapman et al. (2012:161): “... an understanding of primate foraging strategies and their diversity is necessary to construct informed conservation management plans.” A better understanding of pitheciine diet will include additional information on preferred dietary items, fallback foods, fruit size, fruit hardness, fruit mechanical and chemical protection, the nutritional composition of the foods, nutrient requirements for the primates, and foraging decisions in response to variables such as group size, group composition, habitat area, habitat quality, and spatiotemporal location of food resources. The heavy use of unripe fruits and their seeds, and ability to eat seeds from young to mature fruit, is likely to be an advantage to pitheciines that live in seasonal habitats, and habitats that have been modified. Understanding how the variables mentioned earlier in this paragraph impact pitheciine diet could be very helpful in predicting how pitheciine populations can respond to changes in resource availability due to climate change and/or due to increased habitat loss and fragmentation.

Figure 9. Net primary productivity (from -1.0 to 6.5 gC/m2/day) in the geographic range of (A) all pitheciines; (B) Cacajao; (C) Chiropotes; and (D) Pithecia. Productivity data are from NASA Earth Observations (NEO), hydrological data are from USGS HydroSHEDS (Lehner et al., 2006), geographic range data are from the IUCN Red List (IUCN, 2014), and site data represent a compilation of studies and surveys (Barnett et al., 2012; Boyle, 2015; Boyle et al., 2015; Boyle et al., in press; Defler, 2013; Hirsch et al., 2002; Marsh, 2014).

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a. Forest Loss and Fragmentation One of the main conservation concerns for pitheciines is forest loss and fragmentation (Boyle, 2014; Ferrari et al., 2013a, b, c; Lehman et al., 2013; Porter et al., 2013). Globally, human activities have modified more than 75% of the Earth’s land that is not covered by ice (Ellis and Ramkutty, 2006). The extent to which the geographic ranges of Cacajao, Chiropotes, and Pithecia species have been impacted can be great: modified land cover represents 27% and 19%, respectively, of the geographic ranges for Chiropotes satanas and Chiropotes utahickae, and these two species experienced forest loss between 2000 and 2012 of 14% and 21%, respectively (Boyle, 2014). Much of the forest loss and fragmentation within the pitheciines’ geographic ranges is due to agriculture and cattle ranching (Lehman et al., 2013), flooding of forests for the construction of hydroelectric dams (Benchimol and Peres, 2015; Ferrari et al., 2013a; Finer and Jenkins, 2012), mining (Lehman et al., 2013), and logging (Lehman et al., 2013). It does not appear as though forest loss and fragmentation will cease in the near future as there are plans for the construction of additional dams (Fearnside, 2015). To date, there have been studies of Chiropotes and Pithecia in forest fragments in Brazil or on islands created by flooding for hydroelectric dams in Venezuela and Brazil (see Boyle, 2014 for a compilation; Benchimol and Peres, 2015), but no published studies of Cacajao in fragmented habitats. In general, these forest fragments or forested islands can exhibit local extinctions of primate populations, particularly in the smaller fragments or on the smaller islands (Benchimol and Peres, 2015; Boyle et al., 2013). Cacajao and Chiropotes typically have large home ranges (up to 1200 ha and 1000 ha, respectively) in continuous forest (Bowler and Bodmer, 2011; Shaffer, 2013b). Although there does not appear to be any published studies of Cacajao in forest fragments, there have been studies of Chiropotes in living in forest fragments (or forested islands)