Jawless Fishes of the World : Volume 1 [1 ed.] 9781443889643, 9781443885829

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Jawless Fishes of the World

Jawless Fishes of the World: Volume 1 Edited by

Alexei Orlov and Richard Beamish

Jawless Fishes of the World: Volume 1 Edited by Alexei Orlov and Richard Beamish This book first published 2016 Cambridge Scholars Publishing Lady Stephenson Library, Newcastle upon Tyne, NE6 2PA, UK British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Copyright © 2016 by Alexei Orlov, Richard Beamish and contributors All rights for this book reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. ISBN (10): 1-4438-8582-7 ISBN (13): 978-1-4438-8582-9

TABLE OF CONTENTS

Volume 1 Preface ........................................................................................................ ix M. Docker Part 1: Evolution, Phylogeny, Diversity, and Taxonomy Chapter One ................................................................................................. 2 Molecular Evolution in the Lamprey Genomes and Its Relevance to the Timing of Whole Genome Duplications T. Manousaki, H. Qiu, M. Noro, F. Hildebrand, A. Meyer and S. Kuraku Chapter Two .............................................................................................. 17 Molecular Phylogeny and Speciation of East Asian Lampreys (genus Lethenteron) with reference to their Life-History Diversification Y. Yamazaki and A. Goto Chapter Three ............................................................................................ 58 Ukrainian Brook Lamprey Eudontomyzon mariae (Berg): Phylogenetic Position, Genetic Diversity, Distribution, and Some Data on Biology B. Levin, A. Ermakov, O. Ermakov, M. Levina, O. Sarycheva and V. Sarychev Chapter Four .............................................................................................. 83 Diversity and Distribution of the Hagfishes and Lampreys from Chilean Waters G. Pequeño and S. Sáez Chapter Five .............................................................................................. 94 Hagfishes of Mexico and Central America: Annotated Catalog and Identification Key A. Angulo and L. Del Moral-Flores

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Chapter Six .............................................................................................. 126 The Structure and Taxonomy of the Gill Pore in Lampreys of the Genus Entosphenus R. Beamish Chapter Seven.......................................................................................... 154 Review of Western Transcaucasian Brook Lamprey, Lethenteron ninae Naseka, Tuniyev & Renaud, 2009 (Petromyzontidae) S. Tuniyev, A. Naseka and C. Renaud Chapter Eight ........................................................................................... 191 A Nonparasitic Lamprey Produces a Parasitic Life History Type: The Morrison Creek Lamprey Enigma R. Beamish, R. Withler, J. Wade and T. Beacham Chapter Nine............................................................................................ 231 Description of the Larval Stage of the Alaskan Brook Lamprey, Lethenteron alaskense Vladykov and Kott 1978 C. Renaud, A. Naseka and N. Alfonso Chapter Ten ............................................................................................. 251 The Need for a New Taxonomy for Lampreys A. Kucheryavyy, I. Tsimbalov, E. Kirillova, D. Nazarov and D. Pavlov Part 2: Ecology and Life History Chapter Eleven ........................................................................................ 280 Distribution and Habitat Types of the Lamprey Larvae in Rivers across Eurasia D. Nazarov, A. Kucheryavyy and D. Pavlov Chapter Twelve ....................................................................................... 299 The Potential Roles of River Environments in Selecting for Streamand Ocean-Maturing Pacific Lamprey, Entosphenus tridentatus (Gairdner, 1836) B. Clemens, C. Schreck, S. Sower and S. van de Wetering Chapter Thirteen ...................................................................................... 323 The Formation of Fecundity in Ontogeny of Lampreys Yu. Kuznetsov, M. Mosyagina and O. Zelennikov

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Chapter Fourteen ..................................................................................... 346 Advances in the Study of Sea Lamprey Petromyzon marinus Linnaeus, 1758 in the NW of the Iberian Peninsula S. Silva, S. Barca and F. Cobo

Volume 2 Part 3: Demography, Stock Assessment, Fisheries and Conservation Chapter Fifteen ............................................................................................ 2 Lampreys in Central Europe: History and Present State L. Hanel and J. Andreska Chapter Sixteen ......................................................................................... 32 Distribution of Arctic and Pacific Lampreys in the North Pacific A. Orlov and A. Baitaliuk Chapter Seventeen ..................................................................................... 57 Trends in the Catches of River and Pacific Lampreys in the Strait of Georgia J. Wade and R. Beamish Chapter Eighteen ....................................................................................... 73 Trends of Pacific Lamprey Populations across a Broad Geographic Range in the North Pacific Ocean, 1939-2014 J. Murauskas, L. Schultz and A. Orlov Chapter Nineteen ....................................................................................... 97 Observations on the Catch and Biology of Hagfish (Eptatretus stoutii) from an Exploratory Fishery in Northwest Mexico F. Márquez–Farías, R. Lara–Mendoza, O. Zamora–García and E. Ramírez–Félix Chapter Twenty ....................................................................................... 115 Sea Lamprey Fisheries in the Iberian Peninsula M. Araújo, S. Silva, Y. Stratoudakis, M. Gonçalves, R. Lopez, M. Carneiro, R. Martins, F. Cobo and C. Antunes

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Chapter Twenty One................................................................................ 149 Insights Gained through Recent Technological Advancements for Conservation Genetics of Pacific Lamprey Entosphenus tridentatus J. Hess Chapter Twenty Two ............................................................................... 160 Developing Techniques for Artificial Propagation and Early Rearing of Pacific Lamprey Entosphenus tridentatus for Species Recovery and Restoration R. Lampman, M. Moser, A. Jackson, R. Rose, A. Gannam and J. Barron Part 4: Species Interactions Chapter Twenty Three ............................................................................. 196 Common Behavioral Adaptations in Lamprey and Salmonids E. Kirillova, P. Kirillov, A. Kucheryavyy and D. Pavlov Chapter Twenty Four ............................................................................... 214 The Silver Lamprey and the Paddlefish P. Cochran and J. Lyons Chapter Twenty Five ............................................................................... 234 Relationships between Pacific Lamprey and Their Prey A. Orlov In Memoriam: Dr. Philip A. Cochran (1955-2015) ................................. 286 J. Lyons In Memoriam: Dr. Sako B. Tuniyev (1983-2015) ................................... 304 B. Tuniyev In Memoriam: Dr. Yuriy K. Kuznetsov (1937-2015) .............................. 318 N. Bogutskaya, A. Naseka, K. Fedorov and O. Zelennikov List of Reviewers..................................................................................... 323 List of Contributors ................................................................................. 325

PREFACE

Hagfishes and lampreys, the extant jawless fishes of the world, are rare gems. With approximately 110–120 described species (roughly 70–80 hagfish and at least 40 lamprey species), they represent less than 0.2% of all extant vertebrates. However, as sole survivors at the base of the vertebrate family tree (surviving at least four of the five mass extinction events since the Cambrian explosion), these two lineages—dating back some 400–500 million years—can tell us much about the evolution of early vertebrates. The origin of vertebrates represents one of the major jumps in animal evolution, with the active, sentient vertebrates being distinguished from the largely sessile non-vertebrate chordates (the lancelets and tunicates) by a suite of major innovations (e.g., a cranium and pronounced cephalization; paired sense organs; an axial skeleton and muscle segmentation; more complex circulatory, respiratory, digestive, and endocrine systems; and a glomerular kidney). Given the poor fossil record, hagfishes and lampreys—so-called living fossils—are helping us understand the events that occurred at the dawn of vertebrate evolution. Whether the extant jawless fishes are monophyletic (with hagfishes and lampreys as each other’s closest living relative) or paraphyletic (with hagfishes representing an earlier offshoot from the vertebrate family tree and lampreys sharing an ancestor more recently with the jawed vertebrates) has been the subject of much debate. Although recent molecular evidence strongly supports hagfishes and lampreys as a monophyletic group, it is nevertheless important to recognize the long independent evolutionary histories of each lineage and their significant morphological, physiological, and ecological differences. Hagfishes are exclusively marine (and, unique among vertebrates, are isomotic with the environment), lack image-forming eyes and even remnant vertebral arches, and show direct development. Lampreys, in contrast, were the first extant vertebrates to invade fresh water (and the anadromous species are able to osmoregulate in both fresh and salt water), have well-developed eyes as adults and rudimentary vertebral arches, and show indirect development (undergoing a dramatic metamorphosis after the prolonged larval stage). Hagfishes and lampreys are also being used increasingly in biomedical and biomimetic research. For example, the slime of hagfishes—one of the traits that make them so reviled by fishermen—is a source of inspiration

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for the production of extra strong fabrics and petroleum-free plastics. The protein threads of hagfish slime are finer than spider silk (and 100 times thinner than human hair) but just as strong. Hagfish slime (or a synthetic gel containing its fibers) is also being explored for treating accident victims or use during surgery; since it expands when it contacts blood (i.e., saline water), it could potentially be used to stop blood flow. In New Zealand, hagfish slime is used by the Maori as a cleaning agent. The lamprey central nervous system has long been used as a model in neurological studies, and lampreys—since they are unique among known vertebrates in their ability to recover nearly full function after complete spinal cord transection—are being used extensively in research into spinal cord regeneration. The relatively simple and well-studied neural networks of lampreys have also led to the development of lamprey-inspired biorobotic systems for studying high level motor tasks. In addition, the buccal gland secretions of parasitic lampreys are being investigated as a potential source of novel anti-coagulants (comparable to hirudin from medicinal leeches), local anaesthetics, and immunosuppressants, and the ability of lampreys to survive the programmed loss of the gall bladder and biliary tree during metamorphosis and to store and tolerate high concentrations of iron in various body tissues make them excellent model organisms for research into treatment for cholestasis and hemochromatosis in humans. Despite their incredible scientific value, however, hagfishes and lampreys are generally underappreciated (or actively loathed) by the general public. Sometimes referred to as “scavengers of the deep” and “vampires of the deep,” respectively, hagfishes and lampreys are often considered pests of commercial food fisheries—hagfishes given their tendency to feed on commercially-valuable fish caught on lines and in pots; lampreys due largely to the decimation of the commercial and recreational fisheries in the Great Lakes following the invasion and spread of the sea lamprey. More respect is warranted, of course, particularly given the important ecosystem services that hagfishes and lampreys perform. The burrowing and scavenging behavior of hagfishes helps in the turnover of substrate and recycling of organic material in deep-sea environments (so that “earthworms of the deep” might be a better name for them), and they are important food items for pinnipeds and other marine mammals. Similarly, larval lampreys are important in nutrient cycling in rivers and streams, the carcasses of spent anadromous lampreys are a significant source of marine-derived nutrients in freshwater systems, and lampreys— during all stages—are important food sources for other animals. Lampreys are also ecosystem engineers; the burrowing and feeding activities of

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larval lampreys significantly increase substrate oxygen levels and the nestbuilding activity of spawning lampreys increases streambed complexity in ways that appear to benefit other organisms. When hagfishes and lampreys are appreciated, they are often overexploited. Traditionally, hagfishes were fished in Asia for their meat—for consumption by humans and domestic animals and for use as bait. These fisheries expanded in Japan in the 1940s, when other fish stocks were being depleted, and in Korea in the 1980s—particularly following increased demand for hagfish skin, used for “eelskin” leather products. After local Asian stocks were exhausted, hagfish fisheries expanded into North America and New Zealand, followed, unfortunately, by serial depletion of most of these stocks. Lampreys (despite the bad reputation now given to them by sea lamprey in the Great Lakes) have also been long appreciated as food by many cultures (e.g., the Maori, Native Americans in the Columbia River Basin, and throughout much of Europe and Asia). Traditional and commercial fisheries continue throughout many of these areas, although lamprey numbers have declined in recent decades, due largely to the effects of industrialization and urbanization, and some fisheries (e.g., for Caspian lamprey in Russia and Azerbaijan) are no longer viable. Many hagfish and lamprey species are now of conservation concern. On the IUCN (International Union for the Conservation of Nature) Red List of Threatened Species (as of July 2015), 24 hagfish and 32 lamprey species have been evaluated (approximately 32 and 78% of described hagfish and lamprey species, respectively). Of the 24 hagfish species evaluated, three are considered at some risk of extinction (Critically Endangered 1, Vulnerable 1, Near Threatened 1), 15 are listed as Least Concern, and six are considered Data Deficient (i.e., with insufficient information to assess risk of extinction). Of the 32 generally recognized lamprey species evaluated, eight are considered at some risk of extinction (Critically Endangered 2, Endangered 1, Vulnerable 3, Near Threatened 2), 20 are listed as Least Concern, and four are considered Data Deficient. There is a rapidly growing body of research, therefore, being conducted in support of conservation and management. As outlined below, many of the chapters in this book relate to the conservation needs of hagfishes and lampreys. Beyond their value in evolutionary and biomedical research, their ecosystem services, and their economic value, hagfishes and lampreys have always garnered respect and study from at least a small dedicated group of scientists interested in these fascinating creatures for their own sake. Many of the biomedical applications, for example, are offshoots of

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such basic research, and hagfishes and lampreys continue to astonish and amaze us. Studies using baited video cameras, for example, have captured images showing hagfishes actively hunting and preying on other fishes (suggesting that their ecological role in deep-sea ecosystems is more diverse than previously thought) and others showing the instantaneous effectiveness of hagfish slime at deterring predators; in less than half a second of being grasped by a shark or other fish, jets of slime filled the mouth and gill chamber of the predator, causing it to visibly choke and move away. A study looking at coiling behavior in Pacific hagfish demonstrated that individuals showed significant “handedness,” that is, individuals preferentially coiled in a clockwise or counterclockwise direction. Recent discoveries in lampreys include finding a chemical alarm cue in sea lamprey (reminiscent of the well-studied "Schreckstoff" found in cyprinids) that is released into the water when a conspecific is injured, eliciting avoidance behavior. Also interesting is the discovery of adipose tissue in the dorsal rope of sexually-mature male sea lamprey that produces heat when the male is in the presence of an ovulated female; this is the only known example of a thermogenic fat in a non-mammal. Interestingly, handedness in lampreys (in terms of both attachment to a host during parasitic feeding and mating behavior) has long been observed. Hagfishes and lampreys are also physiological wonders: Pacific hagfish are able to tolerate and recover from exposure to high concentrations of ammonia while burrowed inside their meals of decomposing carcasses, and anadromous sea lamprey have a high capacity for urea excretion that enables them to feed on basking sharks (the world’s second largest fish) and other ureosmotic elasmobranchs. The growing appreciation for hagfishes and lampreys is indicated by the number of special symposia and publications dedicated to these jawless fishes in recent years, including this book, which is the main output from a one-day symposium at the 2014 American Fisheries Society annual meeting. Jawless Fishes of the World is a truly international effort, including contributions from authors from four continents (Asia, Europe, North America, South America) and 13 countries (Brazil, Canada, Chile, Costa Rica, Czech Republic, Germany, Greece, Japan, Mexico, Portugal, Russia, Spain, and the United States of America). Consisting of 25 chapters organized into four sections, Jawless Fishes of the World provides a wealth of information on a broad range of topics, and includes chapters on some of the better-studied wide-ranging species (e.g., Pacific hagfish and anadromous sea, Arctic, and Pacific lampreys) and many of the less well-known species (e.g., lamprey species with more limited freshwater distributions). It is exciting to see such diversity included in a

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single volume and such attention paid to traditionally “non-charismatic” species. Part 1 of Jawless Fishes of the World—which focuses on the evolution, phylogeny, diversity, and taxonomy of hagfishes and lampreys—shows that there has been no waning of interest in the evolution of these ancestral vertebrates. The first chapter, for example, takes advantage of the nowavailable sea lamprey and Arctic lamprey genomes to explore the timing of two large-scale genome duplication events that occurred sometime during the evolution of early vertebrates—and that likely permitted the large number of vertebrate-specific innovations mentioned above. Several other chapters in this section tackle hagfish and lamprey taxonomy; taxonomy is challenging because, compared to bony fishes (with their vertebrae, scales, fin rays, and many other meristic and morphometric characters), there are relatively few taxon-distinctive morphological characters in hagfishes and lampreys. Furthermore, given the morphological conservation seen in these lineages over long periods of evolutionary time (modern hagfishes and lampreys look eerily similar to those captured in the fossil record more than 300 million years ago), it is not surprising that extant species—that diverged from each other so comparatively recently— are difficult to distinguish. However, the chapter describing the use of gill pore papilla number and structure for resolving lamprey phylogeny sounds like the sort of thing that J. D. McPhail and C. C. Lindsey had in mind in 1970 when they suggested in their Freshwater Fishes of Northwestern Canada and Alaska that “an imaginative search for new taxonomic characters in lampreys might be fruitful.” Molecular characters, particularly mitochondrial DNA sequence, have also helped to resolve lamprey phylogenies, showing, for example, that some species (e.g., Lethenteron sp. S)—despite being morphologically indistinguishable from other species—represent divergent evolutionary lineages. Other chapters in this section cover the taxonomy and diversity of several less wellknown species or populations, including: hagfishes from Mexico, Central America, and Chile; the pouched and short-headed lampreys in Chile (representatives of the two Southern Hemisphere families which receive more attention in Australia and New Zealand); and the West Transcaucasian and Alaskan brook lampreys (which include the first descriptions of the adult and larval stages, respectively). There has also been no diminution of interest in lamprey “paired species,” a term first coined in 1959 by G. Zanandrea, although J. C. C. Loman made the observation more than 100 years ago that European river and brook lampreys are morphologically similar (particularly as larvae) but the nonparasitic brook lamprey delays metamorphosis and accelerates sexual

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maturation relative to the river lamprey (which “inserts” a parasitic feeding phase between metamorphosis and sexual maturation). Investigation of paired species has been reinvigorated in recent years with the advent of molecular DNA techniques, which have shown that many paired species are “barcode indistinguishable” and that they sometimes—but not always—show evidence of contemporary gene flow when sympatric. As explored in two chapters in this section, this has renewed the debate regarding whether paired species are “real” (i.e., distinct) species. The population of western brook lamprey in Morrison Creek on Vancouver Island that is able to produce some individuals capable of feeding parasitically adds to this fascinating debate. Part 2 deals with the ecology and life history of lampreys, the study of which has generally been more feasible than that of deep-sea hagfishes. In lampreys, habitat requirements of the prolonged freshwater larval stage have been relatively well-characterized, although the secretive nature of the larvae makes accurate population assessment difficult. Far less is known about the parasitic feeding phase, particularly in anadromous species; this is a theme also echoed throughout Parts 3 and 4. The complex and finely-orchestrated life history of migratory lampreys, which experience different habitat requirements at each stage and well-timed movements between them, makes them particularly vulnerable to environmental perturbation (e.g., climate change, dams that act as barriers to migration and alter natural stream flow regimes). Chapters in this section describe advances in electrofishing methods for assessing and monitoring larval lamprey populations, a mark-recapture study that helps fill gaps in our knowledge about the oceanic phase of the anadromous sea lamprey, and investigations into the potential effects of climate and anthropogenic changes on life history of the anadromous Pacific lamprey. Also of great interest is the report of landlocked sea lamprey in Spain, where some post-metamorphic feeding was observed in the Portodemouros Reservoir but no individuals were found to complete their life cycle in fresh water—presumably due to a limited prey base. Given the apparent ease with which sea lamprey colonized the Great Lakes, it is important to understand that it may not be osmoregulatory constraints that generally restrict sea lamprey populations to feeding at sea; predicted climate-driven increases in the productivity of fresh water relative to marine systems (as well as stocking of lakes and reservoirs with host fishes) could potentially permit anadromous lampreys to become invasive in fresh water. Part 3 focuses on research in support of hagfish and lamprey conservation and fisheries management. As mentioned above, knowledge

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of the basic biology and demography of deep-sea hagfishes and the oceanic feeding phase of anadromous lampreys is generally lacking— dangerously so in some species. Chapters in this section review the conservation status of lampreys in central Europe; present key baseline information gathered from an exploratory Pacific hagfish fishery off the coast of Baja California in Mexico; and shed more light on the enigmatic marine phase of Arctic, Pacific, and western river lampreys, that is, on their distribution (both geographically and in the water column), population trends, and their potential effect on (and dependence on) host fish populations. Two other chapters outline recent advances in the artificial propagation and conservation genetics and genomics of Pacific lamprey in the Columbia River basin. As is being done with European river lamprey (e.g., in Finland, Estonia, and Latvia), methods are being developed to rear Pacific lamprey larvae in hatcheries as part of a supplementation program to reintroduce or augment locally or functionally extinct populations. Rearing strategies to help get more early-stage larvae through the “survival bottleneck” would also be highly valuable to researchers interested in heritability of feeding type in paired species and, among other things, the relative contribution of genetic versus environmental factors in lamprey sex determination. The recent technological advancements in conservation genomics is helping to identify management units (given that lampreys do not home to their natal streams and thus do not form populations that can be easily delineated based on geographic location), and predict and monitor the success of adult translocations. Part 4 explores the interactions between parasitic lampreys and their hosts. This section includes fascinating insights (from the late Philip Cochran—a rare gem himself—and his long-time collaborator John Lyons) into the ancient predator-prey relationship between silver lamprey and American paddlefish in the Mississippi River drainage. The silver lamprey is not a recent invader to fresh water, and it and the paddlefish appear to have shared a long evolutionary history living in “peaceful coexistence.” The paddlefish is unlikely to be much affected by one or a few parasitic silver lamprey (given its large body size and its breaching behavior); the silver lamprey, in turn, has a few tricks of its own “up its sleeve” (e.g., a relatively larger oral disc where its co-occurs with paddlefish and attachment sites within the paddlefish’s branchial cavity that presumably provide some protection from dislodgement when the paddlefish breaches). The silver lamprey-paddlefish “story” serves as a good counterpoint to the negative image of blood-sucking sea lamprey in the Great Lakes.

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Hagfishes and lampreys have survived—as important components of their respective (and dynamic) ecosystems—for hundreds of millions of years. Over evolutionary time, hagfishes—living in predominantly deepsea habitats—were likely protected from the periodic upheaval experienced in shallow sea and terrestrial ecosystems. Lampreys, as generalists, also managed to persist when other—perhaps more specialized—lineages perished. For example, since lampreys appear not to be philopatric (locating spawning tributaries by attraction to larval pheromones, a good indicator of contemporary rather than historical larval rearing habitat quality), they are likely less finely tuned to local environmental conditions than species that exhibit strong site fidelity. That parasitic species lack strong host specificity (more than 50 host species, for example, have been reported for anadromous sea lamprey) means that they are less likely to be impacted from changes in the distribution and abundance of individual host species; “facultative ureotelism” even allows them to feed on basking and other large sharks! “Facultative parasitism” or at least the apparent ease with which lamprey feeding and migratory type can evolve (e.g., allowing populations to “abandon” the parasitic feeding phase and associated migrations when the substantial mortality incurred during this stage is no longer compensated for by the opportunity to attain a larger size and higher fecundity) may also allow lampreys “to keep their options open” under changing conditions. The “staying power” exhibited by hagfishes and lampreys to date, however, should not allow us to be complacent; although these two lineages have survived through four mass extinction events, individual species did not. Research in support of the management and conservation of the extant jawless fishes of the world— those species that are commercially valuable and those that are not— therefore needs to continue.

Margaret F. Docker Department of Biological Sciences University of Manitoba Winnipeg, Manitoba, Canada

PART 1: EVOLUTION, PHYLOGENY, DIVERSITY, AND TAXONOMY

CHAPTER ONE MOLECULAR EVOLUTION IN THE LAMPREY GENOMES AND ITS RELEVANCE TO THE TIMING OF WHOLE GENOME DUPLICATIONS TEREZA MANOUSAKI, HUAN QIU, MIYUKI NORO, FALK HILDEBRAND, AXEL MEYER AND SHIGEHIRO KURAKU

Background The genomes of two lamprey species have been sequenced, and this has provided the basis for genome-wide comparison of molecular evolution between jawless fishes and the rest of vertebrates. Molecular phylogenetic analyses of jawless fish genes increased our knowledge of the evolutionary time scale of diversification of hagfishes and lampreys, as well as of gene redundancy in their genomes. It was shown that the ancestor of jawed vertebrates experienced two rounds of whole genome duplications (Dehal & Boore, 2005). However, it has been controversial whether this event occurred before or after the ancestors of extant jawless fishes diverged from the lineage which gave rise to jawed vertebrates (e.g., Escriva et al., 2002; reviewed in Kuraku, 2013). Recent molecular phylogenetic studies showed that the whole genome duplications occurred before the radiation of all extant vertebrates including hagfishes and lampreys (Kuraku et al., 2009a), and this scenario has been confirmed by later studies including the genomic analysis of Petromyzon marinus (sea lamprey) (Hoffmann et al., 2010; Smith et al., 2013). In this chapter, we analyze peculiar characteristics of the lamprey genomes, focusing mainly on protein-coding regions, to propose potential factors that act as barriers to the understanding of the timing of whole genome duplications.

Molecular Evolution in the Lamprey Genomes

3

Jawless fish in molecular phylogenetics Extant jawless fishes, also called cyclostomes, are comprised of hagfishes and lampreys and diverged from the stem lineage that gave rise to jawed vertebrates (gnathostomes) about 600-500 million years ago (Kuraku et al., 2009b; Figure 1-1). They have been proven to be a monophyletic group, based on molecular phylogenies of both mitochondrial and nuclear genes (reviewed in Kuraku, 2008; Figure 1-1). So far, the so-called ‘phylogenomics’ approach, involving more than hundreds of genes on nuclear genomes (Kumar et al., 2012), has not been applied to elucidate the relationships between hagfish, lamprey and jawed vertebrates in such a high resolution as demonstrated for other long-standing questions in animal phylogeny (e.g. Misof et al., 2014). This is mainly due to the lack of large-scale sequence information for hagfish (see Delsuc et al., 2006).

Figure 1-1. Overview of the lamprey phylogeny based on molecular data. Evolutionary time scale within the cyclostome lineage is based on previous literature (Kuraku & Kuratani, 2006; Kuraku et al., 2009b). Branch lengths in the other lineages roughly correspond to evolutionary times inferred from molecular data (Hedges et al., 2006). The phylogenetic relationships among the Mordaciinae, Geotriinae and Petromyzontinae remain to be carefully analyzed with multiple genes (reviewed in Kuraku, 2008).

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Lamprey genome sequencing The first jawless fish species for which whole genome sequencing was started was the sea lamprey Petromyzon marinus. Its genome was sequenced with the so-called Sanger method using DNA extracted from the adult liver. An early version of the genome assembly was made public in 2007 at the UCSC Genome Browser (http://genome.ucsc.edu/), which is still available there as version 3 (petMar1; http://hgdownload.soe.ucsc. edu/goldenPath/petMar1/bigZips/). Later, an improved genome assembly, designated version 7 (petMar2), was generated and used as the final product in the genome-wide analysis by the genome consortium (Smith et al., 2013). In the meantime, it was reported that the sea lamprey experiences programmed genomic rearrangement (PGR) in somatic cell lineages (Smith et al., 2009; Smith et al., 2012). It is thus likely that DNA extracted from the source material for the whole genome sequencing was incomplete and heterogeneous, derived from a mixture of somatic cells with differentially rearranged genomes. In 2013, the genome assembly of another northern hemisphere species, the Arctic lamprey Lethenteron camtschaticum (formerly known as L. japonicum), based on Roche 454 sequencing platform, was also released (Mehta et al., 2013; http://jlampreygenome.imcb.a-star.edu.sg/). This L. camtschaticum project employed genomic DNA extracted from the mature testis, which could have contributed to a larger size and higher continuity of the genome assembly because germline cells possess the intact genome (Table 1-1). This project focused on the evolutionary history of Hox gene clusters – a long-standing theme regarding cyclostome gene phylogeny (reviewed in Kuraku, 2011; Kuraku and Meyer, 2009). To provide a comparison of assembly statistics between these two genomes, we recomputed basic metrics (see Bradnam et al., 2013) using the latest genome scaffold sequences downloaded from Ensembl (for P. marinus) and NCBI (for L. camtschaticum) (Table 1-1).

LetJap1

Lethenteron camtschaticum 1.031

0.886

Total # of bases (Gbp)

86,125

25,006

# of scaffolds

923.6

79.7

Scaffold N50 (Kbp)

17.2

26.8

%N

11,640

3,631

Max scaffold length (Kbp)

0.867

0.201

Min scaffold length (Kbp)

5

The statistics in this table are based on computations using all publicly available scaffolds and are partly different from those in the respective publications reporting the genome analyses.

petMar2

Assembly ID

Petromyzon marinus

Species

Table 1-1. Assembly statistics of the two lamprey genomes.

Molecular Evolution in the Lamprey Genomes

Chapter One

6

To analyze completeness of protein-coding landscape of the two lamprey genomes, we used a program pipeline CEGMA which reports the number of detected genes among 248 conserved genes (CEG, core eukaryotic genes) (Parra et al., 2009). As a result, the L. camtschaticum genome assembly was shown to cover 80% (199/248) of the CEGs, while 69% was detected in the P. marinus genome (172/248) (Table 1-2). This suggests that the L. camtschaticum genome assembly covers more protein-coding genes. The genome consortia of P. marinus and L. camtschaticum reported 26,046 and 17,829 protein-coding genes, respectively (Smith et al., 2013; http://jlampreygenome.imcb.a-star.edu.sg/). These two species are thought to have diverged relatively recently, i.e., 40-10 million years ago (Kuraku & Kuratani, 2006) and possess very similar karyotypic features (reviewed in Caputo Barucchi et al., 2013). It is unlikely that the genomic contents and protein-coding landscape largely differs between the two genomes. Thus, the difference in the number of predicted genes is likely caused by the difference in the completeness of genome assemblies or the difference in gene prediction methods. Table 1-2. Protein-coding landscape in the two lamprey genome assemblies. Species

# CEGs detected by CEGMA Complete Partial All 140 32 172

# of predicted genes 26.046

Petromyzon marinus Lethenteron 141 58 199 17.829 camtschaticum See Parra et al. (2009) for details of the criteria for categorizing genes detected in genome assembly into ‘complete’ and ‘partial’.

GC-content Peculiarity of lamprey genes in terms of GC-content was already reported before the whole genome sequence of lampreys became available (Kuraku & Kuratani, 2006; reviewed in Kuraku, 2008). In the comprehensive analysis of the P. marinus genome consortium, we performed an intensive investigation of its base composition (Smith et al., 2013). The P. marinus genome exhibited relatively high overall GC-content (45.9%), and protein-coding regions, particularly synonymous nucleotide sites, especially had high GC-content (Supplementary Figure 6 in Smith et al., 2013; also see Figure 1-2).

Molecular Evolution in the Lamprey Genomes

7

Figure 1-2. Browser view of GC-content in a selected region in the Petromyzon marinus genome. The graph of GC-content was obtained as GC-percent track at the UCSC Genome Browser, for the P. marinus genomic scaffold GL477094 (base position 170749-189720) containing a homolog of the Lefty gene (PMZ_0009017-RA). Note that the exons of this gene tend to have high GC-content (70-80%). The other ‘unknown’ gene in this view (PMZ_0009018-RA on the right) does not have any obvious homolog in other species and might be a lamprey-specific gene. In such a case, GC-content might serve as an indicator of protein-coding nature of genomic sequences.

Here we have analyzed the Lethenteron camtschaticum genome and compared some characteristics about GC-content with other vertebrates including P. marinus (Table 1-3). The L. camtschaticum genome exhibited markedly higher overall GC-content (48.0%) than the P. marinus genome (45.9%) (Figure 1-3, centerfold page i). Similarly, overall GC-content of protein-coding regions showed a comparable difference between the two species (Table 1-3). The difference of global GC-content in the whole genome sequences of the two lampreys might be caused by either the respective choices of DNA source tissue (liver versus testis, in light of programmed genomic rearrangement), sequencing platform (Sanger versus Roche 454) or assembly methods (Arachne versus Newbler), rather than reflecting the genuine genome compositions. This might also hold for the difference in GC-content of protein-coding regions described above. The lamprey genomes would provide an interesting system to study how epigenetic information is organized in the genome with exceptional GC compartmentalization between coding (GC-rich) and non-coding (GC-poor) regions, as little is known about epigenetic regulation of this group of animals (see Tweedie et al., 1997; Covelo-Soto et al., 2014).

Chapter One

8

Table 1-3. Global and protein-coding GC-content in the two lamprey genomes.

Species Petromyzon marinus Lethenteron camtschaticum

Overall GC %

Genome GC % of 10Kbp non-overlapping windows

Overall GC % of coding regions

45.9

45 ± 3

56.3

48.0

47 ± 4

59.6

Gene model As the lamprey genomes have peculiar features in their protein-coding sequences (see below), gene prediction based on training with those features is expected to enhance its sensitivity and precision. The Petromyzon marinus genome consortium employed the program package MAKER (Cantarel et al., 2008) for genome-wide gene prediction, and it produced a gene typical of vertebrate genomes (Table 1-2) (Smith et al., 2013). In order to predict lamprey genes more precisely, we independently sought to implement lamprey-specific features in gene prediction. First, we built transcriptome assembly using all Sanger sequence reads of P. marinus available in NCBI dbEST (as of March 2008). In the assembled transcript contigs, we inferred open reading frames (ORFs) with identical lengths and high sequence conservation (•70% positive match at the amino acid level, with a methionine corresponding to the putative start codon) in comparison with their jawed vertebrate homologs. Among 828 putative ORFs selected as above, we identified 132 ORF sequences that were contained in the P. marinus genome assembly petMar1 (version 3) with presumably full intronic and 2Kbp-long flanking sequences. Using them, we executed the training module of AUGUSTUS version 2.0.3 (Stanke & Waack, 2003) as instructed in its manual. The resulting parameter files for P. marinus gene model were passed to the developer of AUGUSTUS and are now available in the default species list (with the species identifier ‘lamprey’) of the installable program package (http://bioinf.uni-greifswald.de/augustus/ binaries/) and web server (http://bioinf.uni-greifswald.de/webaugustus/ prediction/create). This alternative gene prediction platform provides a complementary approach to exploit genomic resources of lampreys, although it remains to be

Molecular Evolution in the Lamprey Genomes

9

carefully assessed whether the species parameters for P. marinus performs well for other lamprey species.

Codon usage bias and amino acid composition Before whole genome sequences of lampreys became available, we performed analyses on codon usage bias and amino acid composition with 173 protein-coding genes of Petromyzon marinus that were available in GenBank (Qiu et al., 2011). In this study based on the relatively small data set, we suggested that lampreys have peculiar patterns of codon usage bias and amino acid composition. More recently, with the whole genome sequences of P. marinus, we performed more comprehensive analyses on those characteristics and confirmed that the peculiarity in the sequences of lamprey genes and peptides is genome-wide (Smith et al., 2013; Figure 1-4a and 1-4b, centerfold page ii). In addition, we revealed that GC-content in protein-coding regions is the major factor contributing to the peculiarity of codon usage bias and amino acid composition (Figure 1-4c and 1-4d, centerfold page ii). Our analyses did not support the relevance of codon usage bias to gene expression levels (Supplementary Figure 10 of Smith et al., 2013). It is of particular interest whether this lamprey-specific pattern is shared with other jawless fish genomes.

Homopolymeric amino acid (HPAA) tracts More recently, we focused on homopolymeric amino acid (HPAA) tracts in peptide sequences (or single amino acid repeats, such as ‘QQQQQQQQ’; see Mularoni et al., 2010) and carried out a cross-species comparison of their frequencies (Noro et al., 2015). Our interest originated from a particular case of lamprey Emx genes (reviewed in Kuraku, 2010). Lampreys possess at least two Emx genes (EmxA and EmxB; Tank et al., 2009), and their gene products have a Q-tract and an A-tract at equivalent locations in the sequences of the two Emx gene products (Figure 1-5a, centrefold page iii-iv; Noro et al., 2015). A comparison with their hagfish orthologs without conspicuous HPAA tracts indicates that the insertions of the HPAA tracts occurred in the lamprey lineage after the split of the hagfish lineage (Figure 1-5a, centerfold page iii-iv). Our reanalysis confirmed the result by Tank et al. (2009) supporting lamprey lineage-specific Emx gene duplication, whereas the support was

10

Chapter One

significantly weakened when the HPAA tracts were deleted from the multiple sequence alignment (Noro et al., 2015). Inspired by the example of Emx genes, we performed a genome-wide survey of HPAA tract insertion. Our survey revealed a significant abundance of HPAA tracts in the overall protein-coding landscape of the sea lamprey genome, compared to that in the human and zebrafish (Noro et al., 2015). It also detected significant enrichment of G-tracts and Q-tracts unique to the sea lamprey (Noro et al., 2015; Figure 1-5b, centerfold page iii-iv). It is unknown what biochemical reasons underlie this species difference in HPAA tract insertion. If the trend of HPAA tract insertion reflects similarly on multiple sequences with similar property, namely paralogs, phylogenetic signals in those sequences might be weakened or erased by the secondary effects. This can result in erroneous alignments and molecular phylogeny inferences.

Perspectives Several studies have reported gene duplications in the cyclostome lineage (Fried et al., 2003; Stadler et al., 2004; Tank et al., 2009). More recently, a genome-wide analysis suggested that a duplication event at the genome scale introduced lineage-specific duplicates (Mehta et al., 2013). Our analyses have highlighted several unique aspects of molecular evolution that seem to be characteristic of the cyclostome lineage. Above all, the unique sequence property of lamprey protein-coding genes is remarkable. It possibly drove convergent sequence evolution among ancient paralogs towards unexpected similarity, resulting in erroneous proximity between the paralogs in inferred molecular phylogeny. Molecular phylogeny inference based on amino acid sequences, as often practiced, is apparently supposed to circumvent the effect of GC-content and codon usage bias but is still prone to unfavorable effect of amino acid composition. Some of the gene duplications attributed to the cyclostome lineage so far could be explained by this possible artefact, and it can mislead our interpretation of the timing of whole genome duplications. Thus, extra cautions should be exercised in analyzing gene family trees involving lamprey genes (Figure 1-6). Our knowledge of cyclostome genome compositions is still limited to Northern Hemisphere lampreys. Evolving DNA sequencing technology has enabled economical genome sequencing, and whole genome sequencing of hagfish and Southern Hemisphere lampreys are anticipated. With those

Molecular Evolution in the Lamprey Genomes

11

Figure 1-6. Possible causes of misinterpretation on molecular phylogeny involving lamprey genes. Alternative scenarios regarding the timing of two whole genome duplications (Hypothesis A-C) are shown with species trees and hypothetical gene trees. In the species trees, open circles indicate gnathostome-cyclostome split, and black arrows indicate the timing of whole genome duplication. In the hypothetical gene trees, a black diamond indicates gene duplication giving rise to multiple gnathostome paralogs, while a white diamond represents gene duplication giving rise to multiple cyclostome paralogs. Hypothesis A, with both rounds of whole genome duplications before the split between cyclostomes and gnathostomes, has been supported by a series of recent studies (Hoffmann et al., 2010; Kuraku et al., 2009a; Smith et al., 2013). In reality, some gene families exhibit molecular phylogeny depicted in the right bottom corner (*), with multiple cyclostome genes exclusively clustering with each other. This phylogenetic pattern, with gene duplications after the split between cyclostomes and gnathostomes, is incongruent with Hypothesis A and is rather compatible with Hypothesis C. We propose that the incongruence is, at least partly, caused by convergence of lamprey sequences discussed in this chapter.

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Chapter One

resources, more comprehensive comparison of genomic features is expected to provide an increased understanding of what in the genome makes the phenotypic differences between jawless fishes and other chordates.

Acknowledgements The authors acknowledge Maria Anisimova and Christophe Dessimoz for insightful discussion and Yuichiro Hara and Mitsutaka Kadota for critical reading of the manuscript.

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Kuraku S. 2013. Impact of asymmetric gene repertoire between cyclostomes and gnathostomes. 2013. Seminars in Cell and Developmental Biology 24, 119-127. doi 10.1016/j.semcdb.2012.12.009. Kuraku S. & Kuratani S. 2006. Time scale for cyclostome evolution inferred with a phylogenetic diagnosis of hagfish and lamprey cDNA sequences. Zoological Science 23, 1053-1064. doi http://dx.doi.org/10.2108/zsj.23.1053. Kuraku S. & Meyer A. 2009a. The evolution and maintenance of Hox gene clusters in vertebrates and the teleost-specific genome duplication. The International Journal of Developmental Biology 53, 765-773. doi 10.1387/ijdb.072533km. Kuraku S., Meyer A. & Kuratani S. 2009b. Timing of genome duplications relative to the origin of the vertebrates: did cyclostomes diverge before or after? Molecular Biology and Evolution 26, 47-59. doi 10.1093/molbev/msn222. Kuraku S., Ota K.G. & Kuratani S. 2009c. Jawless fishes (Cyclostomata). In S.B. Hedges & S. Kumar (eds.): The Timetree of Life. Pp. 317-319. New York: Oxford University Press. Mehta T.K., Ravi V., Yamasaki S., Lee A.P., Lian M.M., Tay B.H., Tohari S., Yanai S., Tay A., Brenner S. & Venkatesh B. 2013. Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). Proceedings of the National Academy of Sciences of the United States of America 110, 16044-16049. doi 10.1073/pnas.1315760110. Misof B., Liu S., Meusemann K., Peters R.S., Donath A., Mayer C., Frandsen P.B., Ware J., Flouri T., Beutel R.G., Niehuis O., Petersen M., Izquierdo-Carrasco F., Wappler T., Rust J., Aberer A.J., Aspock U., Aspock H., Bartel D., Blanke A., Berger S., Bohm A., Buckley T.R., Calcott B., Chen J., Friedrich F., Fukui M., Fujita M., Greve C., Grobe P., Gu S., Huang Y., Jermiin L.S., Kawahara A.Y., Krogmann L., Kubiak M., Lanfear R., Letsch H., Li Y., Li Z., Li J., Lu H., Machida R., Mashimo Y., Kapli P., McKenna D.D., Meng G., Nakagaki Y., Navarrete-Heredia J.L., Ott M., Ou Y., Pass G., Podsiadlowski L., Pohl H., von Reumont B.M., Schutte K., Sekiya K., Shimizu S., Slipinski A., Stamatakis A., Song W., Su X., Szucsich N.U., Tan M., Tan X., Tang M., Tang J., Timelthaler G., Tomizuka S., Trautwein M., Tong X., Uchifune T., Walzl M.G., Wiegmann B.M., Wilbrandt J., Wipfler B., Wong T.K., Wu Q., Wu G., Xie Y., Yang S., Yang Q., Yeates D.K., Yoshizawa K., Zhang Q., Zhang R., Zhang W.,

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Zhang Y., Zhao J., Zhou C., Zhou L., Ziesmann T., Zou S., Xu X., Yang H., Wang J., Kjer K.M. & Zhou X. 2014. Phylogenomics resolves the timing and pattern of insect evolution. Science 346, 763-767. doi 10.1126/science.1257570. Mularoni L., Ledda A., Toll-Riera M. & Alba M.M. 2010. Natural selection drives the accumulation of amino acid tandem repeats in human proteins. Genome Research 20, 745-754. doi 10.1101/gr. 101261.109. Noro M., Sugahara F. & Kuraku S. 2015. Reevaluating Emx gene phylogeny: homopolymeric amino acid tracts as a potential factor obscuring orthology signals in cyclostome genes. BMC Evolutionary Biology, 15, 78. doi 10.1186/s12862-015-0351-z. Ohno S. 1970. Evolution by gene duplication. Berlin and New York: Springer-Verlag. Parra G., Bradnam K. & Korf I. 2007. Assessing the gene space in draft genomes. Nucleic Acids Research 37, 289-297. doi 10.1093/nar/ gkn916. Qiu H., Hildebrand F., Kuraku S. & Meyer A. 2011. Unresolved orthology and peculiar coding sequence properties of lamprey genes: the KCNA gene family as test case. BMC Genomics 12, 325. doi 10.1186/1471-2164-12-325. Smith J.J., Antonacci F., Eichler E.E. & Amemiya C.T. 2009. Programmed loss of millions of base pairs from a vertebrate genome. Proceedings of the National Academy of Sciences of the United States of America 106, 11212-11217. doi 10.1073/pnas.0902358106. Smith J.J., Baker C., Eichler E.E. & Amemiya C.T. 2012. Genetic consequences of programmed genome rearrangement. Current Biology 22, 1524-1529. doi 10.1016/j.cub.2012.06.028. Smith J.J., Kuraku S., Holt C., Sauka-Spengler T., Jiang N., Campbell M.S., Yandell M.D., Manousaki T., Meyer A., Bloom O.E., Morgan J.R., Buxbaum J.D., Sachidanandam R., Sims C., Garruss A.S., Cook M., Krumlauf R., Wiedemann L.M., Sower S.A., Decatur W.A., Hall J.A., Amemiya C.T., Saha N.R., Buckley K.M., Rast J.P., Das S., Hirano M., McCurley N., Guo P., Rohner N., Tabin C.J., Piccinelli P., Elgar G., Ruffier M., Aken B.L., Searle S.M., Muffato M., Pignatelli M., Herrero J., Jones M., Brown C.T., Chung-Davidson Y.W., Nanlohy K.G., Libants S.V., Yeh C.Y., McCauley D.W., Langeland J.A., Pancer Z., Fritzsch B., de Jong P.J., Zhu B., Fulton L.L., Theising B., Flicek P., Bronner M.E., Warren W.C., Clifton S.W., Wilson R.K. & Li W. 2013. Sequencing of the sea lamprey

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(Petromyzon marinus) genome provides insights into vertebrate evolution. Nature Genetics 45, 415-421. doi 10.1038/ng.2568. Stadler P.F., Fried C., Prohaska S.J., Bailey W.J., Misof B.Y., Ruddle F. & Wagner G.P. 2004. Evidence for independent Hox gene duplications in the hagfish lineage: a PCR-based gene inventory of Eptatretus stoutii. Molecular Phylogenetics and Evolution 32, 686-694. doi 10.1016/j.ympev.2004.03.015 Stanke M. & Waack S. 2003. Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics 19, Suppl. 2, ii215-225. doi 10.1093/bioinformatics/btg1080 Tank E.M., Dekker R.G., Beauchamp K., Wilson K.A., Boehmke A.E. & Langeland J.A. 2009. Patterns and consequences of vertebrate Emx gene duplications. Evolution & Development 11, 343-353. doi 10.1111/j.1525-142X.2009.00341.x. Tweedie S., Charlton J., Clark V., & Bird A. 1997. Methylation of genomes and genes at the invertebrate-vertebrate boundary. Molecular and Cellular Biology 17, 1469-1475.

CHAPTER TWO MOLECULAR PHYLOGENY AND SPECIATION OF EAST ASIAN LAMPREYS (GENUS LETHENTERON) WITH REFERENCE TO THEIR LIFE-HISTORY DIVERSIFICATION YUJI YAMAZAKI AND AKIRA GOTO

Preface Lampreys have received special attention in evolutionary studies because they are considered to represent primitive vertebrates (Zanandrea 1959; Hardisty & Potter 1971; Yamazaki & Goto 2000a; Osório & Rétaux 2008; Docker 2009). Although more than 40 extant lamprey species are distributed in mid- and high-latitude regions world-wide, East Asian lampreys have received little attention for many years (e.g., Hardisty & Potter 1971; Renaud 2011). However, from the late 1990s, many researchers, for example a group led by the present authors, have attempted to redress this with a wide range of investigations, including taxonomy and phylogeny (Yamazaki & Goto 1997, 1998; Yamazaki et al. 2006b), zoogeography and phylogeography (Yamazaki & Iwata 1997; Yamazaki et al. 1999; Yamazaki et al. 2003, 2005), population genetics (Yamazaki & Goto 1996; Yokoyama et al. 2009; Yamazaki et al. 2011a, 2014), ecology (Yamazaki & Goto 2000b; Yamazaki 2007), reproduction and development (Yamazaki et al. 2001, 2003), and life-history diversification and speciation (Yamazaki et al. 1998; Yamazaki et al. 2011b; Yamazaki & Nagai 2013). These studies were important in elucidating general biological features, life-history evolution, and the phylogenetic relationships and speciation of lampreys over much of their distribution range. East Asian lampreys, especially Lethenteron species, are now considered as a case model for studies on life-history diversification and speciation. At the same time, it has become apparent

Chapter Two

18

that these some lampreys should now be considered endangered, due to human activities (Renaud 1997), and requiring the implementation of conservation programs. Although an overview (in Japanese) of initial studies was given by Yamazaki & Goto (2000), it is now timely to review significant findings to date. Accordingly, this chapter summarizes the present state of knowledge on the evolutionary biology of East Asian lampreys, and indicates future lines of investigation and challenges.

1. Diversity of Lethenteron species Lampreys worldwide Traditional taxonomic classification of lampreys has been based on morphological data, e.g., body proportions, dentition, oral papillae, and number of trunk myomeres (Hardisty & Potter 1970; Vladykov & Kott 1979; Yamazaki & Goto 2000a; Gill et al. 2003; Renaud 2011). Including two new species described recently (Lethenteron ninae Naseka, Tuniyev, & Renaud 2009 from east side of Black Sea Basin and Eudontomyzon graecus Renaud & Economidis 2010 from Greece), three families of lampreys, represented by 10 genera and 40 species worldwide, are presently considered as valid (Renaud 2011). However, the varying degree of morphological variability in lampreys has resulted in some taxonomic problems (Hardisty & Potter 1971; Vladykov & Kott 1979; Yamazaki & Goto 2000a; Gill et al. 2003; Renaud 2011). In particular, the number of recognized genera has fluctuated (see also section 3). However, it is now generally agreed that eight and two genera are represented in the Northern and Southern Hemispheres, respectively (i.e., Lang et al. 2009; Renaud 2011). In recent years, genetic studies have resulted in several implications concerning the validity of lamprey taxonomy, including a suggestion of distinct taxonomic entities within a single species, such as Lampetra species (Boguski et al. 2012) and Entosphenus species (Taylor et al. 2012). On the other hand, genetic continuity has been reported between some species pairs, characterized by different life histories and sympatric distribution (e.g., Docker 2009; Docker et al. 2012; Makhrov et al. 2013). Clearly, some questions remain concerning the present systematic status of lampreys. Taxonomic status of East Asian lampreys In the early 1900s, the classification of lampreys in East Asia was confused (e.g., Hatta 1901; Creaser and Hubbs 1922). Under such

Molecular Phylogeny and Speciation of East Asian Lampreys

19

circumstances, Berg (1931) listed three Lethenteron species from East Asia, viz. Lethenteron camtschaticum (Tilesius) (previously Lampetra japonica), Le. kessleri (Anikin) (previously Lampetra japonica kessleri), and Le. reissneri (Dybowski) (previously Lampetra reissneri). Berg (1931) considered dentition to be one of the most valuable characteristics for discriminating among species, despite of the existence of considerable variability in that character. Subsequently, Iwata et al. (1985) examined three Lethenteron species samples collected from Hokkaido Island, Japan, and reported morphological differences among them, including trunk myomere numbers and body proportions, although the possible extent of geographical variation could not be determined. In order to resolve the taxonomic confusion of East Asian lampreys, genetic studies by our research group have been conducted since the late 1990s. Yamazaki & Goto (1996) conducted allozyme analyses of samples of "Lethenteron reissneri" (sensu Berg 1931) collected from the entire area of the Japanese Archipelago and southern part of the Korean Peninsula. The results indicated two genetically distinct groups, characterized by a lack of hybridization, even in sympatric habitats. The authors concluded that each genetic group should be classified as a distinct species, according to the biological species concept (Mayr 1963), treating them as Lethenteron sp. N (northern group) and L. sp S (southern group) (see section 2; Yamazaki & Goto 1996; Yamazaki et al. 2003). Unfortunately, because of lacks of both description and deposited specimen in the previous studies (e.g. Hatta 1901; Berg 1931), the scientific names of the two species have not been decided. Including such species, genetic studies were subsequently conducted for further taxonomic clarification, including allozyme analysis (Yamazaki & Goto 1998) and mitochondrial DNA (mtDNA) sequencing (Yamazaki et al. 2003, 2006b). The allozyme and mtDNA sequence data showed significant similarities between L. reissneri samples collected from the type locality (Upper Amur River, eastern Eurasia) and L. kessleri samples from the type locality (Ob River), the Lena and middle Amur Rivers (central and eastern Eurasia), and rivers on Hokkaido Island (northern Japan). Similarities in morphological characteristics, such as dentition and the number of trunk myomeres, were also found between L. reissneri and L. kessleri samples (Yamazaki et al. 2006b). Furthermore, those samples were genetically and morphologically different from other species, including L. camtschaticum, L. sp. N, and L. sp. S. Accordingly, L. reissneri and L. kessleri were recognized as being conspecific, the name L. reissneri taking priority. Consequently, four lamprey "species" in the genus Lethenteron, L. camtschaticum. L. reissneri, L. sp. N, and L. sp. S

20

Chapter Two

are currently recognized in East Asia (Table 2-1). Notwithstanding, some recent studies have persisted in using outdated classification, leading to some confusion of results and discussion. For example, an adoption of incorrect scientific name has led to the unnecessary confusion in phylogenetic relationships (e.g., Balakirev et al. 2014). Lethenteron camtschaticum is distributed over the eastern Eurasian Continent, northern part of the Japanese Archipelago, Korean Peninsula and north western North American Continent (Fig 2-1; Berg 1931; Hardisty & Potter 1971; Iwata et al. 1985; Holþík 1986; Renaud 2011; Makhrov et al. 2013; Yamazaki et al. 2014). The range of L. reissneri is also broad, extending from rivers of eastern Eurasia to northern Japan, including Anadyr (Chukotka Autonomous Okrug, Russia), the Kamchatka Peninsula, around the Sea of Okhotsk, Amur (Russian Far East), Sakhalin Island and northern Japan (Fig 2-1; Berg 1931; Hardisty & Potter 1971; Iwata et al. 1985; Holþík 1986; Nikiforov et al. 1994; Yamazaki & Iwata 1997; Chereshnev et al. 2001; Yamazaki et al. 2006b; Renaud 2011). However, Makhrov et al. (2013) indicated that lampreys inhabiting rivers around the Arctic Ocean and treated as L. reissneri were probably fluvial-nonparasitic forms of L. camtschaticum. Lethenteron sp. N has been recorded from rivers in the northern part of the Japanese Archipelago (Fig. 2-1, see section 2; Iwata et al. 1985; Yamazaki & Goto 1996, 1998; Yamazaki et al. 1999, 2003). Lethenteron sp. S is distributed in rivers in the southern part of the Japanese Archipelago and the southern part of the Korean Peninsula (Fig. 2-1; Yamazaki & Goto 1996, 1998; Yamazaki et al. 1999, 2003). These Lethenteron species often coexist in the same habitat during both larval and adult stages (Hardisty & Potter 1971; Yamazaki & Goto 1996, 1998, 2000b; Yamazaki et al. 1999; Yamazaki 2007). Life histories and reproductive characteristics of East Asian lampreys Lamprey individuals exist in a larval form, generally called ammocoetes, for several years, subsequently metamorphosing into the adult stage (e.g., Hardisty & Potter 1971; Holþík 1986; Renaud 2011). Their life histories are classified according to their migration patterns (anadromous, fluvial-lacustrine, or fluvial) and feeding modes (parasitic upon fishes and aquatic mammals or nonparasitic) in the adult stage.

21

Parasitic? Parasitic

Anadromous

Nonparasitic

Nonparasitic

Potadromous?

Fluvial

Lethenteron sp. S

Eudontomyzon morii (Berg 1931) Entosphenus tridentatus (Richardson 1836)

Fluvial

Lethenteron sp. N

Nonparasitic

Nonparasitic

Fluvial

Fluvial

parasitic

Life history

Anadromous

Lethenteron reissneri (Dybowski 1869)

Lethenteron camtschaticum (Tilesius 1811)

Species

38-62

15-20

9-15

8-15

10-23

15-20

30-65

Size at maturation, cm

60-71

68-74

50-62

51-66

63-73

66-70

68-77

Number of trunk myomeres

Northwestern America, Japan

Northern Japan, Eastern Eurasia, Alaska Northern Japan, Kamchatka Eastern Eurasia, Northern Japan Northern and central Japan Southern and central Japan, Southern Korea Korea, Eastern China

Distribution

Table 2-1. East Asian lamprey species with reference to life-history patterns and distribution (after Yamazaki 1998; Renaud 2011 with slight modification).

Molecular Phylogeny and Speciation of East Asian Lampreys

22

Chapter Two

Figure 2-1. Distribution (shaded area) of East Asian lampreys (after Yamazaki et al. 1999, 2006b; Renaud 2011, with slight modifications).

Distribution of East Asian lampreys Of the East Asian lampreys, L. camtschaticum has been thought to have an anadromous-parasitic life history, other Lethenteron species being fluvial-nonparasitic (e.g., Hardisty & Potter 1971; Iwata et al. 1985). Recently, however, some studies have revealed the existence of life-history polymorphism in L. camtschaticum, i.e., anadromous-parasitic, "praecox" (precocious) with a shorter migration period and parasitic (sensu Kucheryavyi et al. 2007), and fluvial-nonparasitic forms (Iwata & Hamada 1986; Yamazaki et al. 1998, 2011b; Kucheryavyi et al. 2007; Makhrov et al. 2013). A fluvial-nonparasitic form of L. camtschaticum has been reported from both northern Japan (see section 4) and Kamchatka Peninsula. Whereas the life span of anadromous-parasitic L. camtschaticum is generally more than seven years [larval period of at least four years, with two or three years of migratory and parasitic behavior following metamorphosis until spawning (Holþík 1986; Kucheryavyi et al. 2007; Renaud 2011)]. A "praecox" form has a life span of about six years due to the shorter period (less than two years) following metamorphosis (Kucheryavyi et al. 2007). The fluvial-nonparasitic form, which spawns in

Molecular Phylogeny and Speciation of East Asian Lampreys

23

the first available season following metamorphosis, has a life span of about five years (Kucheryavyi et al. 2007). Although the life spans of other fluvial-nonparasitic Lethenteron species seem to be equivalent to that of the above (Hardisty & Potter 1971; Holþík 1986; Renaud 2011), the larval period is thought to fluctuate according to individual differences and habitat condition (Yamazaki 1998). The body size at maturation of anadromous-parasitic L. camtschaticum reaches over 400 mm, with high fecundity (adult females produce more than several tens of thousands of eggs) (Hardisty & Potter 1971; Yamazaki et al. 2001). "Praecox" and fluvial-nonparasitic forms of the species attain ca. 200 mm and 150 mm maximum sizes, respectively (Yamazaki et al. 1998; Kucheryavyi et al. 2007). On the other hand, body size at maturation of fluvial-nonparasitic species reaches ca. 150 mm maximum, with lower fecundity (a female having less than three thousand eggs). Yamazaki et al. (2001) reported that egg sizes of anadromous-parasitic L. camtschaticum (ca. 0.8–1.2 mm in long diameter) tended to be smaller than those of fluvial-nonparasitic Lethenteron species (ca. 1.1–1.4 mm). Other East Asian lampreys Two non-Lethenteron lamprey species have been recorded in East Asia (Table 2-1). The Korean lamprey Eudontomyzon morii (Berg) is believed to have a fluvial-parasitic life history (Hardisty & Potter 1971). This species distributed in rivers in northern and central regions of the Korean Peninsula, although its distribution range remains unclear (Berg 1931; Hardisty & Potter 1971; Renaud 2011). The Pacific lamprey Entosphenus tridentatus Gairdner in Richardson, distributed mainly in rivers and coastal areas along western North America, has also been recorded in some East Asian rivers, such as the Yufutsu River, Hokkaido Island, and the Naka River, central Honshu Island, Japan (Hardisty & Potter 1971; Honma & Katoh 1987; Fukutomi et al. 2002; Yamazaki et al. 2005; Lin et al. 2008; Renaud et al. 2009; Renaud 2011). Additionally, this species has been reported from the Khatyrka River, Chukotka, Russia, (Pavlow & Chernov 1998). This lamprey is generally regarded as having an anadromous-parasitic life history (Hardisty & Potter 1971; Renaud 2011). E. tridentatus individuals in Japan had been thought to represent abortive migration attempts, due to fragmentarily occurring (Honma & Katoh 1987). However, natural spawning of the species has recently been recorded in the Naka River (Fukutomi et al. 2002; Yamazaki et al. 2005). Larval E. tridentatus have also been found coexisting with those of L. sp. N in the Naka River (Yamazaki et al. 2005).

24

Chapter Two

2. Cryptic species relationship found between Lethenteron sp. N and sp. S Cryptic East Asian lamprey species Sibling or cryptic species' complexes, reproductively isolated from each other in sympatric areas, nevertheless possess similar morphological features, despite being distinct biological species (Walker 1964; Mayr & Ashlock 1991). Such species complexes are suitable targets for a greater understanding of evolutionary mechanisms (e.g., Futuyma 1998; Yamazaki & Goto 1996, 2000; Gómez et al. 2002; Pfenninger & Schwenk 2007; Yamazaki 2007). In addition, Bickford et al. (2006) reviewed the importance of clarifying the existence of cryptic species in order to fully appreciate biodiversity and conservation necessities. Lethenteron sp. N and L. sp. S showed considerable genetic divergence with each other, and lack of hybridization in regions of sympatry (Yamazaki & Goto 1996, 1998; Yamazaki et al. 2003, 2006b). Of particular interest was the almost complete lack of morphological differences between them, including dentition, body proportions, and numbers of trunk myomeres (Yamazaki & Goto 1997), and should be regarded as cryptic species (sensu Walker 1964). Distribution and habitat utilization Lethenteron sp. N is distributed in rivers of Hokkaido Island and the northern and central parts of Honshu Island, Japan (Fig. 2-1: Yamazaki et al. 1999). On the other hand, L. sp. S is widely distributed throughout most of the southern part of the Japanese Archipelago, as well as the southern region of the Korean Peninsula. Although the distribution ranges of L. sp. N and L. sp. S are biased to the north and south, respectively, their ranges overlap in central Honshu Island, Japan. In this region, the distribution of L. sp. N is primarily restricted to rivers originating from springs, whereas L. sp. S inhabits rivers arising from both surface runoff and springs. However, areas of coexistence are restricted to spring-fed rivers (Yamazaki et al. 1999; Yamazaki 2007). Habitat utilization at the microhabitat scale has been examined for larval L. sp. N and L. sp. S in the Shougawa River, Toyama Prefecture, central Japan (Yamazaki 2007). No significant differences in habitat utilization were found, although densities of individuals of the two species had a positive association with substrate type (smaller particle size and deeper water for large larval individuals of L. sp. N and L. sp. S,

Molecular Phylogeny and Speciation of East Asian Lampreys

25

respectively). It is possibly related to suitability for burrowing (e.g., Beamish & Lowartz 1996). On the other hand, small larval individuals of both species tended to occur at higher densities in areas with a higher component ratio of medium-sized sand substrate and faster current, probably reflecting their preference for respiratory efficiency, as previously reported for other larval lampreys (Beamish & Jebbink 1994). Competition for microhabitat resources may therefore be significant both within and between species. These findings are consistent with previous views (e.g., Lamsa et al. 1980). Such size-dependent differences in habitat utilization by larval forms of the two species may simply represent habitat preference or be a result of behavioral interactions (or both). Further studies are needed to clarify habitat utilization by L. sp. N and L. sp. S larvae in both sympatric and allopatric populations, as well as larval growth patterns (Yamazaki 2007). Reproductive isolation mechanisms Despite the occurrence of sympatric populations of L. sp. N and L. sp. S in central Honshu Island, Japan, no hybrids have been detected based on nuclear allozyme analysis, strongly suggesting complete reproductive isolation between the species (Yamazaki & Goto 1996, 1998, 2000). This result indicates the existence of prezygotic barriers (premating isolating mechanisms) to gene exchange. In general, size assortative mating has been proposed as functioning as a barrier to interbreeding between lamprey species (Hardisty & Potter 1971b; Malmqvist 1983; Docker 2009) (see section 5). However, since body size at maturation is largely similar between Lethenteron sp. N and L. sp. S, even in areas of sympatry (Yamazaki & Goto 2000, Yamazaki et al. 2001), size assortative mating would likely serve no function as a reproductive barrier between them. In the Shougawa River, Toyama Prefecture, Japan, sympatric populations of L. sp. N and L. sp. S are subject to a shift of spawning seasons, the spawning period of L. sp. N being across December to February and that of L. sp. S across May to July (Fig. 2-2: Yamazaki unpubl. data). Thus, temporal isolation may at least be partly responsible for reproductive isolation between the two species at that location. In the Gakko River, Yamagata Prefecture, Japan, on the other hand, the spawning season of L. sp. N (across March to early May) is somewhat closer to that of L. sp. S (across April to June). As a result, overlapping of spawning periods at least in April and May occurs between the two species (Yamazaki & Goto 2000b). In fact, some spawning nests of each species have been observed at the same time on the same day (Yamazaki & Goto

26

Chapter Two

2000b), suggesting that temporal isolation would not act effectively between L. sp. N and L. sp. S in the Gakko River.

Figure 2-2. Spawning seasons of Lethenteron sp. N and L. sp. S in the Gakko and Shougawa Rivers, Japan. Composition of the two species in each spawning nest observed in the Gakko River from April to early May, 1997. Open and solid circles indicate L. sp. N and L. sp. S. M and F indicate male and female. Numbers associated with symbols indicate individual numbers.

Notwithstanding, a field survey of 16 spawning nests in the Gakko River found that in every case, the nests were occupied only by males and females of the same species, suggesting the operation of a premating isolating mechanism between the two species (Yamazaki & Goto 2000b). There were no apparent differences in micro-habitat related to nest construction between the two species, indicating no distinct microhabitat preference for spawning sites by either. In a number of animal species, ethological barriers have been proposed as effective premating isolating mechanisms (e.g., Futuyma 1998). In particular, specific mate recognition has been proposed as the principal basis of ethological isolating mechanisms (Paterson 1985; Futuyma 1998). The species-specific nesting assemblages observed in the sympatric populations of L. sp. N and L. sp. S in the Gakko River strongly suggested

Molecular Phylogeny and Speciation of East Asian Lampreys

27

that each species can recognize species-specific mates, at least in the nesting sites (Yamazaki & Goto 2000b). Such specific mate recognition has been reported as induced by auditory, visual, and/or chemical cues peculiar to each species. For example, species-specific songs and advertisement calls play an important part in the recognition of conspecific individuals in the cricket Allonemobius complex (Mousseau & Howard 1998), frog Rana adenopleura (Matsui & Utsunomiya 1983), and Hyla species complex (Gerhardt et al. 1994). Furthermore, it is well-known that many animals use visual signals and chemical communication (sex pheromones) prior to mating (e.g., Futuyma 1998). Discussing chemical communication in lampreys, Teeter (1980) noted the importance of a specific pheromone in the reproductive behavior of Petromyzon marinus Linnaeus. To date, however, any role of pheromones, as well as auditory and visual cues, for reproductive isolation between L. sp. N and L. sp. S is still to be resolved.

3. Phylogeny and population structure of Lethenteron species Phylogenetic relationships Initially, the phylogenetic relationships of lamprey species, as in many other taxa, were constructed on the basis of morphological characteristics, such as dentition, oral papillae, and the number of trunk myomeres (Hardisty & Potter 1971; Vladykov & Kott 1979; Gill et al. 2003; Monette & Renaud 2005). The relationships of East Asian lampreys were based on the similarity of trunk myomere numbers by invoking the stem-satellite species concept (see section 5): i.e., stem Lethenteron camtschaticum was placed close to satellite L. reissneri (formerly L. kessleri), compared with L. sp. N (formerly L. reissneri) (Vladykov & Kott 1979). Because of the low diversity of morphological features, however, phylogenetic relationships based on morphology have not been fully resolved, resulting in recent emphases on phylogenetic analyses using various genetic markers (e.g., Lang et al. 2009; Renaud 2011). Allozyme analysis of "Lethenteron reissneri" samples, collected from the Japanese Archipelago, by Yamazaki & Goto (1996) led to recognition of two highly divergent groups, L. sp. N and L. sp. S. Subsequently, phylogenetic relationships among East Asian lampreys were examined using allozymes (Yamazaki & Goto 1998), mitochondrial DNA (Yamazaki et al. 2003, 2006b; Lang et al. 2009) and nuclear DNA, such as the ITS (internal transcribed spacers of ribosomal DNA) region (Yamazaki

28

Chapter Two

unpublished data), giving broadly similar results. In addition, molecular phylogenetic analyses based on mitochondrial DNA sequences revealed close relationships among L. camtschaticum, L. reissneri, and L. sp. N. Of these, L. camtschaticum and L. reissneri (including samples formerly classified as L. kessleri) constituted a single cluster, within which no species-specific grouping was detected (Fig. 2-3; Yamazaki et al. 2006b; Artamonova et al. 2011). Allozyme analysis of the two species indicated genetic discontinuities, even in their area of sympatry (Yamazaki & Goto 1998). Thus, Yamazaki et al. (2006b) proposed such inconsistencies between nuclear allozymes and mtDNA markers as having possibly resulted from incomplete lineage sorting of mtDNA genome data between them, as previously reported in some other animal species (e.g., Avise 2000). On the other hand, some authors have hypothesized that L. camtschaticum and L. reissneri are life-history polymorphisms within a single species, L. camtschaticum (Kucheryavyi et al. 2007; Artamonova et al. 2011; Makhrov et al. 2013). These differing opinions clearly require further scrutiny utilizing a broad base of specimen data based on nuclear marker. Over the same period, genetic similarities among species originally determined on the basis of morphological characteristics have been reported in other species complexes, such as Lampetra fluviatilis Linnaeus and L. planeri (Bloch) (e.g., Espanhol et al. 2007), and Ichthyomyzon unicuspis Hubbs & Trautman and I. fossor Reighard & Cummins (Docker et al. 2012). On a molecular phylogenetic tree constructed from mitochondrial DNA sequencing data, Lethenteron sp. S was highly divergent from other Lethenteron species, at a level matching that of Lampetra fluviatilis and Entosphenus tridentatus (Fig. 2-3; Yamazaki et al. 2006b; Lang et al. 2009). Although the determination of “genus” rank in lampreys has been based on morphological characteristics, mainly dentition, disagreements exist between traditional classification and those derived from molecular phylogenetic data (Fig. 2-3; Docker 1999; Lang et al. 2009). Therefore, the phylogenetic data have demonstrated the necessity of redefining the generic position of "Lethenteron sp. S". Among East Asian lampreys, Eudontomyzon morii has been included in a cluster comprising Lethenteron species (Fig. 2-3; Lang et al. 2009; Renaud 2011; Yamazaki et al. unpublished data). Also, this result suggests that E. morii is a valid species belonging to the genus Lethenteron.

Molecular Phylogeny and Speciation of East Asian Lampreys

29

Fig. 2-3. Phylogenetic relationships among haplotypes of East Asian lamprey species, plus Lampetra fluviatilis, Petromyzon marinus and Ichthyomyzon unicuspis, based on the partial mtDNA CO I gene (after Yamazaki et al. 2006b, with slight modifications). Bootstrap probabilities (%) with 1000 replications shown for each cluster.

30

Chapter Two

Population structure An understanding of the population structure provides important insights into the historical, present and future status of the species, including information on evolution and speciation (e.g., Futuyma 1998). Numerous studies on lamprey population structures have been conducted as understanding of genetic markers and analysis methods have advanced. Genetic population structures for some East Asian lamprey species have been based on allozyme, mitochondrial DNA and microsatellite DNA analyses (Yamazaki & Goto 1996, 1998; Yamazaki et al. 2003, 2006b, 2014). Lethenteron camtschaticum, characterized by life-history polymorphism (see section 4), has revealed a population structure depending upon life-history type. Both allozyme and microsatellite DNA analyses have indicated low genetic divergence among almost all L. camtschaticum populations broadly sampled from rivers in the Far East region, including Japan. It suggests reciprocal gene flow among populations, mainly due to their anadromous life history (Yamazaki & Goto 1998; Yamazaki et al. 2014). On the other hand, fluvial-nonparasitic populations of L. camtschaticum, landlocked following dam construction in the upper reaches of rivers in northeastern Japan, have been recently found (see section 4; Yamazaki et al. 2011b). Microsatellite analysis revealed these populations to be genetically divergent from anadromous-parasitic populations, with a divergence time following isolation from the ancestral anadromous-parasitic population of some 17.9–428.2 years, easily encompassing the dates of initial dam construction, ca. 90 years ago. The greater genetic divergence found between landlocked fluvial-nonparasitic and anadromous-parasitic populations of L. camtschaticum probably resulted from the founder effect and subsequent genetic drift in the former population (Yamazaki et al. 2011b). This is strong evidence for the very recent establishment of landlocked populations, and subsequent broadening of divergence (see section 4). Among the fluvial-nonparasitic Lethenteron species in East Asia, the degree of genetic divergence among populations of L. reissneri is low compared with those of L. sp. N or L. sp. S, in spite of the extensive distribution of L. reissneri over eastern Eurasia, Sakhalin and Hokkaido islands (Yamazaki & Goto 1998; Yamazaki et al. 2006b). Lethenteron reissneri is thought to have recently originated from L. camtschaticum or its ancestral species (see section 5; Yamazaki & Goto 1998). Subsequently, its distribution should have radiated rapidly. Similar divergence patterns of intraspecific populations have been reported in some fluvial fishes, such as

Molecular Phylogeny and Speciation of East Asian Lampreys

31

grayling Thymallus arcticus (Froufe et al. 2005), alpine bullhead Cottus poecilopus (Yokoyama et al. 2008), and pond minnow Rhynchocypris perenurus mantschuricus (Sakai et al. 2014). Yokoyama et al. (2008) suggested that the distribution of these fishes, including L. reissneri, rapidly expanded via refugia existing in the interior region of eastern Eurasia during the Pleistocene. Thus, these fishes are thought to have insufficient time for genetic differentiation among intraspecific populations. Both L. sp. N and L. sp. S are characterized by considerable genetic divergence among intraspecific populations (Yamazaki & Goto 1996, 1998; Yamazaki et al. 2003, 2006b). Phylogeographic analyses have revealed several phylogenetic groups within L. sp. S that have arisen in geographically close localities, although barely apparent in L. sp. N (Yamazaki & Goto 1996; Yamazaki et al. 2003). The phylogeographical pattern in the former is thought to have been caused by long-term extrinsic barriers to genetic exchanges among populations (e.g., Avise 2000). Geographical dispersal and gene flow in Lethenteron sp. S may have been prevented or restricted by physical barriers, such as mountains between larger rivers or seas resulting from glacial-interglacial climate changes (Yamazaki et al. 2003). Notwithstanding, gene flow could have occurred within each river, as has been shown in some freshwater fishes distributed mainly in south-western Japan and Korea, such as Misgurnus anguillicaudatus (Khan & Arai 2000), Cobitis biwae (Kimizuka & Kobayashi 1983), Oryzias latipes (Sakaizumi et al. 1983; Matsuda et al. 1997), Odontobutis obscura (Sakai et al. 1998), and Rhinogobius flumineus (Shimizu et al. 1993). On the other hand, L. sp. N populations do not show clear geographic groups comparing with the instance of L. sp. S (Yamazaki et al. 2003). Thus, the geographical dispersal of L. sp. N to its present distribution area may have occurred recently. Alternatively, since L. sp. N has a seemingly relict distribution on Honshu Island, Japan (Yamazaki & Goto 1996; Yamazaki et al. 1999), the original genetic population structure may have been lost as a result of genetic drift in each region or the extinction of local populations (Yamazaki et al. 2003).

32

Chapter Two

4. Life-history polymorphism and maintenance mechanisms in Lethenteron camtschaticum Life-history polymorphisms have been reported in many freshwater fish groups, including lampreys, salmonids, smelts, sticklebacks, and Japanese dace (Berg 1931; Yamazaki et al. 2011 for lampreys, Johnson 1980; Gross 1985; Morita et al. 2014 for salmonids, Hamada 1961; Taylor & Bentzen 1993 for smelts, McPhail 1994; Kitamura et al. 2006 for sticklebacks, Sakai 1995 for Japanese dace). Among them, two types of life-history polymorphisms exist, one type occurring in both sexes, such as in lampreys, smelts, Japanese dace, and sticklebacks, and the other which is found only in males, such as in many salmonid species (Johnson 1980; Thorpe 1987; Kato 1991; Yamamoto et al. 1999) (Table 2-2). In general, these life history polymorphisms in freshwater fishes are thought to have evolved as conditional strategies, such as if individual fishes are small or have low social status they should adopt migratory life history tactics so as to increase in fitness (Gross 1985; Gross & Repka 1998; Morita et al. 2014). In lampreys, it is possible that the stem species of stem-satellite species sensu Vladykov & Kott (1979), such as Lethenteron camtschaticum, had already been subject to a polymorphic life history, namely anadromous-parasitic and fluvial-nonparasitic forms, when they diverged from their ancestral type, due to all extant lamprey species being restricted to freshwater spawning. The stem species of each paired species in several genera, such as Ichthyomyzon, Lampetra, and Lethenteron (all Petromyzontidae), generally include two such alternative forms (Zanandrea 1959; Vladykov & Kott 1979; Hardisty & Potter 1981; Yamazaki & Goto 2000a; Salewski 2003; Docker 2009). Life-history polymorphism in Lethenteron camtschaticum In L. camtschaticum, life-history polymorphism consisting of an anadromous-parasitic and fluvial-nonparasitic forms is found in Hokkaido and the northeastern part of Honshu Island, Japan (Iwata & Hamada 1986; Yamazaki et al. 1998, 2011, 2014). In the Ohno River, located in the most southern part of Hokkaido, the former form measured from 35 to 43 cm in total length in the spawning period, the latter form measuring from 14 to 16 cm (Photo 2-1, centerfold page vii; Yamazaki et al. 1998; Yamazaki & Goto 2000a).

Molecular Phylogeny and Speciation of East Asian Lampreys

33

Table 2-2. Examples of life history polymorphisms in fishes. Family Salmonidae

Petromyzontidae Osmeridae Salangidae

Salmonidae

Cyprinidae Gasterosteidae

Species Oncorhynchus mykiss Oncorhynchus masou Oncorhynchus kisutch Oncorhynchus nerka Lethenteron camtschaticum Lampetra fluviatilis Hypomesus nipponensis Osmerus mordax Salangichthys microdon Salvelinus malma malma Salvelinus leucomaenis Salvelinus alpinus Salmo salar Salmo trutta Tribolodon hakonensis Gasterosteus aculeatus Gasterosteus nipponicus Anadromous and freshwater forms of G. aculeatus

The life-history divergence of the two forms apparently occurs in 3rd or 4th year ammocoete larvae in both sexes (Yamazaki & Goto 1998, 2000a). Subsequently, small larval individuals may metamorphose into silvery larvae, migrating to the sea, whereas larger individuals probably metamorphose directly into adult eyed lampreys, which eventually attain sexual maturation (Fig. 2-4, centerfold, page v; Fukayama & Takahashi 1983; Yamazaki & Goto 2000a). Silvery young adults, on the other hand, remain as parasites in the sea for 2 or 3 years, using adult salmonid fishes and walleye Pollock, Theragra chaloogramma as their hosts, before migrating upstream for spawning (Sato 1951; Yamazaki & Goto 2000).

34

Chapter Two

Genetic support for plastic life-history polymorphism in Lethenteron camtschaticum It is necessary to evaluate whether or not the alternative life-history forms in L. camtschaticum were the product of genetic polymorphism or phenotypic plasticity, changes in the phenotypic expression of a single genotype as a function of the environment. Yamazaki et al. (2011b) examined population structures and gene flow among anadromous-parasitic, landlocked, and fluvial-nonparasitic populations, using polymorphic microsatellite loci (Fig. 2-5). Abundant gene flow was evident on multi-temporal scales between potentially sympatric populations (Fig. 2-6; anadromous-parasitic ISH, UZR, MOG, AGA, and JNZ populations among which no genetic differences were detected and a single natural fluvial-nonparasitic OHN population). This result suggested that ongoing gene flow had resulted from imperfect size-assortative mating between those populations and plastic determination of differential life histories. On the other hand, landlocked fluvial-nonparasitic populations in regions upstream of dams were genetically divergent from other anadromous-parasitic populations as well as a natural fluvial-nonparasitic population. Thus, temporal heterogeneity of gene flow, i.e., low contemporary gene flow but significant over the long-term, was found between the landlocked fluvial-nonparasitic populations and all other populations.

Molecular Phylogeny and Speciation of East Asian Lampreys

35

Figure 2-5. Sampling sites of Lethenteron camtschaticum population in Japan for estimation of gene flow among populations (after Yamazaki et al. 2011b). Potential barriers to migration, i.e., dams, are shown. Star indicates localities in which fluvial-nonparasitic individuals previously occurred. ISH: Ishikari River, UZR: Uzura River, MOG: Mogami River, JNZ: Jinzu River, AGA: Agano River, INA: OHN: Ohno River, Ina River, TAT: Tateiwa River.

This view was supported by Artamonova et al. (2011), who examined population structures of anadromous and resident forms of L. camtschaticum from Kamchatka Peninsula, Russia, using mitochondrial cytochrome oxidase subunit I (COI) genes, and concluded that there are no genetic differences between the two forms. Present genetic data strongly suggests that the alternative life-history forms in L. camtschaticum have evolved through phenotypic plasticity (Scheiner 1993; Klemetsen et al. 2003; Dewitt & Scheiner 2004; Chapman et al. 2011).

36

Chapter Two

Figure 2-6. Long-term and contemporary gene flow among populations (see Figure 2-5) estimated for Lethenteron camtschaticum; anadromous-parasitic (ż) and fluvial-nonparasitic (Ɣ ) life-history forms. Direction and thickness of arrows indicate direction and relative abundance of gene flow, respectively.

Adaptive significance of life-history polymorphism and its maintenance mechanisms Phenotypic plasticity has broad significance, embracing genetics, development, ecology and evolution, and including aspects of physics, physiology and behavioral science (Scheiner 1993; Dewitt & Scheiner 2004). Within a population, environmentally induced phenotypic traits may be a more important source of polymorphic life-histories than those derived from genetic differentiation (Adams et al. 2014; Sandlund & Jonsson 2014). Genetically based intraspecific variation may arise if a population has a sub-structure of reproductively isolated sub-units (Hendry et al. 1999), or is sufficiently small to undergo genetic drift. Three main sources of polymorphic life-histories within a fish population have been proposed: (1) body size determines many reproductive traits in fishes (e.g.,

Molecular Phylogeny and Speciation of East Asian Lampreys

37

Wootton 1990); (2) environmental conditions can influence size-adjusted reproductive traits (Jonsson et al. 1996; Lobon-Cervia et al. 1997; Morita et al. 1999; Baker & Foster 2002); and (3) breeding dates have been shown to be correlated with reproductive traits (Bagenal 1971; Ware 1975; Candolin 1998; Hendry et al. 1999; Poizat et al. 1999). These patterns have been variously described as morphological constraints, proximal environmental effects or adaptations (Poizat et al. 2002). Su ch life-history polymorphism is a variation in natural life-history tendencies observed in usual anadromous-parasitic forms, the latter thus standing as a starting point in its evolution. Typically, low food availability (= poor growth condition) for juvenile individuals in streams tends to result in the anadromous-parasitic form, while high food availability (= good growth condition) leads to the fluvial-nonparasitic form. In general, such polymorphic life-history variation is regarded as an adaptation for unpredictably changing stream environments on spatial and temporal scales (Gross 1985; Fujisaki 1991; Gross & Repka 1998; Morita et al. 2014). In the case of L. camtschaticum, the source of polymorphic life-histories is possibly the influence of environmental conditions on size-adjusted reproductive traits (2 above), because of differing adult body sizes between the anadromous-parasitic and fluvial-nonparasitic forms, the former being about twice the total length of the latter (Iwata & Hamada 1986; Yamazaki et al. 1998, 2011b). Accordingly, a high frequency of form-assortative mating was observed. However, anadromous-parasitic females appear to mate not only with males of the same form (Photo 2-2, centerfold, page viii ) but also with males of the fluvial-nonparasitic form, due to sneaking behavior of the latter. This has been evidenced not by direct observations of mating behavior so much as by gene flow between the two potentially sympatric forms detected by analysis of microsatellite DNA data (Fig. 2-6; Yamazaki et al. 2011b). In general, life history polymorphisms are maintained when multiple life-history solutions provide equivalent fitness returns, or as a result of conditional strategies (Sinervo & Calsbeek 2006; Chapman et al. 2011). It is plausible that the life-history polymorphism in L. camtschaticum is maintained as a conditional strategy. The ultimate mechanisms are responsible for maintenance of such polymorphism resulting from the optimum outcome for an individual, being dependent upon the phenotype (Swingland & Lessells 1979; Lundberg 1988). In L. camtschaticum, mean individual fitness of the anadromous-parasitic and fluvial-nonparasitic forms is ordinarily greater in the latter, due to its earlier maturation (Goto & Yamazaki unpubl. data). In the Ohno River, mature anadromous-parasitic individuals ranged between 353 to 431 mm (n= 4) and 385 to 399 mm (n=

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4) total length in females and males, respectively, being usually 5 or 6 years old (both sexes), compared with fluvial-nonparasitic individuals which ranged between 149 to 154 mm (n= 2 ) and 151 to 161 mm (n= 4) total length, respectively, and were generally 3-4 years old (Yamazaki et al. 1998, 2001). Under the conditional strategy, however, fitness balancing between anadromous-parasitic and fluvial-nonparasitic strategies is not entirely necessary, as both forms can evolve as the “best of a bad job” (Lundberg 1987). For example, white spotted charr Salvelinus leucomaenis migrants (anadromous individuals) have a higher yearly growth probability whereas residents (fluvial residency) have increased reproductive success (Morita et al. 1999, 2014). Overall, the increase in migrant growth is insufficient to offset the reproductive deficit, migration therefore being the “best of a bad job” in this species. A similar situation appears to exist between anadromous-parasitic and fluvial-nonparasitic forms of L. camtschaticum (Fig. 2-7, centerfold, page vi; Goto & Yamazaki unpubl. data).

5. Processes and causes of speciation in Lethenteron species with reference to the stem-satellite species hypothesis General speciation concept applicable to lampreys Generally, lamprey genera include complexes consisting of parasitic species having an anadromous or fluvial life history, and nonparasitic species with a fluvial life history, such being referred to as "paired species" (Zanandrea 1959; Hardisty & Potter 1971). Vladykov & Kott (1979), however, referred to such species complexes as "stem-satellite species", due to some genera comprising one parasitic species and two or more nonparasitic species. Zanandrea (1959) was first to propose the lamprey speciation concept, i.e., nonparasitic species originated from parasitic ones within a "paired" or "stem-satellite" species complex. This concept, especially the direction of the life history shift, has been broadly accepted (Hardisty & Potter 1979; Vladykov & Kott 1979; Beamish 1985; Yamazaki & Goto 2000a; Salewski, 2003; Docker 2009). As the verification study of the direction, Yamazaki et al. (2001) conducted a histological comparison of intestines in parasitic and nonparasitic Lethenteron species. The authors found that parasitic L. camtschaticum possessed functional mucosal folds around the inner layer of the intestine at the metamorphosed stage, reflecting an adaptive feature for parasitic feeding after metamorphosis. On the other hand, although nonparasitic

Molecular Phylogeny and Speciation of East Asian Lampreys

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species are generally thought to cease feeding around metamorphosis, nonparasitic L. reissneri and L. sp. N also exhibited mucosal folds, albeit in a degenerative condition, indicating the likely presence of functional or at least rudimentary mucosal folds in an ancestral (parasitic) species. As a first step of lamprey speciation, the occurrence of life-history polymorphism within a species, i.e., a fluvial-nonparasitic form directly derived from an anadromous-parasitic form or via a fluvial-parasitic form (Beamish 1985, 1987; Docker 2009), has often been assumed (Fig. 2-8A; Zanandrea 1959; Hardisty & Potter 1971; Yamazaki & Goto 2000). Currently, different life-history forms coexist within a species in some lamprey genera (see section 4; Beamish 1997; Yamazaki et al. 1998; Kucheryavyi et al. 2007; Docker 2009). Speciation can be expressed as the establishment of reproductive isolation between populations having a common origin (e.g., Futuyma 1998). Ecological factors, such as spacial and temporal spawning preferences, can also serve as reproductive isolation mechanisms (Hardisty & Potter 1971). Furthermore, ethological barriers have been proposed as possible reproductive isolation mechanisms between cryptic Lethenteron sp. N and L. sp. S (see section 3; Yamazaki & Goto 2000b). On the other hand, the main mechanism of reproductive isolation in lampreys is thought to be differences in body size between anadromous-parasitic and fluvial-nonparasitic forms, i.e., size assortative mating (Hardisty & Potter 1971; Malmqvist 1983; Docker 2009). For example, a traditional field study reported no examples of interspecific pairing of Lampetra fluviatilis and L. planeri, which spawn in the same area, even in the same redds (Hardisty & Potter 1971). Beamish & Neville (1992) also believed that reproductive isolation between L. ayresi and L. richardsoni might have developed through size assortative mating. However, recent studies have indicated some less than fully effective cases of interspecific reproductive barriers based on body size differences. Cochran et al. (2008) suggested the occurrence of gene flow between Ichthyomyzon gagei Hubbs and Trautman and I. castaneus Girard via alternative reproductive behavior, such as satellite males. Similarly, Hume et al. (2013b) showed experimentally that alternative male reproductive strategies of Lampetra fluviatilis and L. planeri had resulted in mating by interspecific sneaking, thereby establishing gene flow between the species due to the lack of post-zygotic barriers (Hume et al. 2013a). Furthermore, abundant gene flow was found between potentially sympatric fluvial-nonparasitic and anadromous-parasitic populations of Lethenteron camtschaticum (Yamazaki et al. 2011b).

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Figure 2-8. Schematic diagram showing speciation process of fluvial lampreys from anadromous lampreys. Open and solid arrows indicate life range of anadromous and fluvial forms, respectively.

These results should request the physical barriers and subsequent genetic divergence on the establishment of reproductive isolation, such as landlocked by dam constructions (Fig. 2-8B and C; Yamazaki et al. 2011b). Such physical isolation could be established by the geological events (see below). Through a series of the above processes, lamprey speciation could be established (Fig. 2-8D). Speciation process of East Asian lampreys East Asian lampreys have been the subject of empirical speciation studies. Molecular phylogenetic analysis of East Asian lampreys revealed the monophyly of parasitic Lethenteron camtschaticum, and nonparasitic L. reissneri and L. sp. N, supporting their treatment as a "stem-satellite species complex" (Yamazaki et al. 2006b). Additionally, paraphyletic relationships were found between L. reissneri and L. sp. N, each being more closely related to L. camtschaticum compared with another

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nonparasitic species, thereby indicating the likely parallel evolution of a nonparasitic form from a parasitic one in the "stem-satellite species complex" (Yamazaki & Goto 1998, 2000a; Yamazaki et al. 2006b). Parallel evolution of life histories has been reported in other lampreys (Hardisty & Potter 1971; Vladykov & Kott 1979), as well as in some bony fishes, such as the amphidromous sculpin Cottus amblystomopsis and fluvial sculpin C. nozawae (Goto & Andoh 1990), and the sockeye and kokanee forms of the salmon Oncorhynchus nerka (Wood & Foote 1996). In the stem-satellite species complex, both L. reissneri, which showed relatively shallow divergence from L. camtschaticum, and L. sp. N, which represented deep divergence, would have been derived from respective populations of a fluvial-nonparasitic form of L. camtschaticum or its ancestor, which occurred under multi-temporal and multi-spatial conditions (Yamazaki et al. 2006b, 2011b). Although the present landlocked fluvial-nonparasitic populations were probably the result of dams, the founder populations of L. reissneri and L. sp. N may have become established due to different events, such as the formation of falls and cataracts. These circumstances may result in the subsequent prevention of gene flow from anadromous-parasitic individuals inhabiting the lower region of the physical barrier. Environmental disturbances in the glacial epoch, such as isolation to refugia or restriction of migration due to a cold dry climate, have also been thought to have operated as physical barriers leading to speciation in lampreys, such as occurred for Lampetra species in Europe (Hardisty & Potter 1971; Espanhol et al. 2007). The influence of glaciation might have been greater for L. reissneri, which is widely distributed in high latitudes in eastern Eurasia, than for L. sp. N, which has limited mid-latitude distribution in the Japanese Archipelago (Yamazaki et al. 2011a). In addition, rising seawater temperatures are suggested to have caused a latitudinal shift in the northward migration route of anadromous-parasitic lampreys (Yamazaki et al. 2014). Subsequently, fluvial-parasitic or nonparasitic individuals, if any, are likely to have become isolated from the former in the southern area, probably leading to fluvial-nonparasitic L. sp. N (Yamazaki et al. 2011b). In essence, climatic change may have triggered speciation in lampreys.

6. Conservation of endangered Lethenteron species Overview of conservation status At least half of the Northern Hemisphere lamprey species are endangered in some way (Renaud 1997). Thirty-one species are listed in

Chapter Two

42

IUCN (2014), including extinct species. Of them, both Lethenteron camtschaticum and L. reissneri are categorized as being of "Least Concern" (Table 2-3; IUCN 2014). In Japan, on the other hand, L. camtschaticum, L. sp. N, and L. sp. S have all been categorized as "Vulnerable", and L. reissneri as "Near Threatened" (Japan Ministry of the Environment 2014), with local authorities also recognizing threats to the species on a local scale. Table 2-3. List of threatened lamprey species in East Asian. Species Lethenteron camtschaticum Lethenteron reissneri Lethenteron sp. N Lethenteron sp. S

IUCN (2014)

Japan Ministry of the Environment (2014)

Least Concern

Vulnerable

Least Concern Not Evaluated Not Evaluated

Near Threatened Vulnerable Vulnerable No distribution in Japan Threatened local population

Eudontomyzon morii

Not Evaluated

Entosphenus tridentatus

Not Evaluated

In general, reductions in lamprey numbers result mainly from habitat degradation through water pollution and stream regulation (Renaud 1997; IUCN 2014). Lamprey larvae require a sandy substrate with a suitable particle size for their habitat, such being formed by natural river dynamics (Giller & Malmqvist 1998; Yamazaki et al. 2006a; Yamazaki 2007). Lampreys also require rich riparian vegetation which directly and/or indirectly supplies their feeding requirements, such as algae and detritus. However, water pollution and stream regulation have often impacted upon such habitat conditions. Additionally, obstruction of migration or isolation by damming or irrigation projects likely lead to a decrease in genetic diversity and/or population viability, sometimes resulting in population extinction (Beamish & Northcote 1989; Miller et al. 1989; Renaud 1997; Yamazaki et al. 2011a, b). The direct impact of excessive capture of adult and larval individuals for commercial purposes is a further cause of population decline (Renaud 1997; Yazawa 1998; IUCN 2014). Because of the critical situation now existing for many lamprey species, efforts for their conservation have been made in many parts of the world, including, for example, North America (Mesa & Copeland 2009; Renaud et al. 2009) and the Iberian Peninsula (Mateus et al. 2012a).

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Conservation status of Lethenteron species in Japan Lethenteron camtschaticum has long been significant for Japanese people, the species having been utilized as medicine for night blindness for the past ca.300 years. Currently, it is utilized in northern Japan, especially, in a wide variety of dishes and as medicine. However, L. camtschaticum catches have drastically declined, especially since the late 1980s (Yazawa 1998). In the Ishikari River, Hokkaido Island, the main fishing ground for L. camtschaticum, present-day catches have declined to 2 % of the highest catches about 30 years ago. Major causes of the decline are believed to be river modification and dredging, which have continued widely in the river throughout the period, resulting in a significant loss of suitable habitat for lampreys, especially larvae. A further cause for the decline in population size has been a drastic change in fishing methods, the traditional fish trap (“dou”) having been replaced by highly effective set nets since the mid-1980s, resulting in over fishing. Similar decreases in L. camtschaticum populations have been observed elsewhere throughout Japan in recent years, due to similar causes. In addition, the effects of increasing ocean temperatures due to global warming cannot be ignored, with a likely habitat shift northward in the future. Recently, Yamazaki et al. (2014) determined from microsatellite DNA analysis of L. camtschaticum samples collected across East Asia, that the species has considerable ability to migrate long distances in the sea. For resource management of L. camtschaticum, therefore, it is necessary to maintain unconstrained gene flow across all present populations. Recently, lower genetic diversity has been found within landlocked fluvial-nonparasitic populations of L. camtschaticum, compared with that of anadromous-parasitic populations (Yamazaki et al. 2011b). Habitat fragmentation by dams plays a significant role in promoting genetic differentiation between the resulting upper and lower populations due to the reduction of gene flow (Yamamoto et al. 2004; Barluenga et al. 2006; Palkovacs et al. 2008). The founder effect and subsequent genetic drift should also result in lower genetic diversity within populations, as shown for many fishes (Kawamura et al. 2007; Frankham et al. 2012). The landlocked fluvial-nonparasitic L. camtschaticum populations appear to be analogous to founder populations in terms of lamprey speciation (see section 5). In order to avoid species and population extinctions and to maintain the possible evolutionary trend of future life-history diversification and adaptive ability to differing environments, conservation programs for East Asian Lethenteron species, including local populations, must be undertaken and enforced in the near future.

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Essential information for determining conservation status for L. reissneri broadly lacks. IUCN (2014) categorized L. reissneri (hitherto classified as L. kessleri) as being of "Least Concern", whereas the Japan Ministry of the Environment (2014) listed the species as "Near Threatened", despite inadequate information on distribution, habitat condition and population viability. For conservation of L. sp. N and L. sp. S, both of which were categorized as "Vulnerable" by the Japan Ministry of the Environment (2014) because of reductions in their population sizes and range (Yamazaki et al. 1999; Yamazaki & Goto 2000a). Precise methods for species identification were recognized as necessary due to their morphological similarity (Yamazaki & Goto 1997). The genetic methods recently developed and adopted for identification of population samples (Yamazaki et al. 2003; Yamazaki 2007; Yokoyama et al. 2009) have highlighted areas of concern for ongoing conservation of L. sp. N and L. sp. S. For example, these species revealed the restricted distribution, especially the relict-like distribution of L. sp. N in spring water areas, in central Japan, as well as lower genetic diversity within populations (Yamazaki et al. 1999, 2003). Such places clear emphasis on the importance of habitat restoration, so as to reverse the pattern of increasing habitat destruction due to human activities. Yamazaki et al. (2011a) also estimated the gene flow disturbance in L. sp. N populations inhabiting a paddy water system, at the micro-scale level based on microsatellite DNA data. Fluvial lampreys often undergo short upstream migration after metamorphosis so as to find a suitable spawning ground (Hardisty & Potter 1971; Malmqvist 1986). However, artificial stream reconstruction, especially dams and sluice gates constructed across the streams, can be expected to limit upstream migration of lampreys (Fig. 2-9). Microsatellite DNA analysis indicated that historical gene flow among the subpopulations of L. sp. N inhabiting the paddy water system was both frequent and bidirectional. However, contemporary gene flow was unidirectional, being downstream only (Yamazaki et al. 2011a), indicating that the associated sluice gates had obstructed the migration of individuals between different subpopulations. Continued limitation of migration between subpopulations and likely reduced gene flow will probably result in a loss of genetic diversity.

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Figure 2-9. Directional gene flow among populations of Lethenteron sp. N based on long-term and contemporary gene flow estimates. Arrows indicate directionality and relative abundance of gene flow (after Yamazaki et al. 2011a).

Invasion of alien genotypes threaten wild populations with genetic pollution or extinction via hybridization (e.g., Rhymer & Simberloff 1996; Frankham et al. 2002). Amongst East Asian lampreys, disturbance of the indigenous gene pool via intraspecific introgression has been found in L. sp. S populations in the Jinzu River, central Japan (Yokoyama et al. 2009). Exogenous genotypes were concluded as having originated from lamprey individuals from geographically distant water bodies, probably resulting from unintentional introductions together with commercial fishes. Yokoyama et al. (2009) also revealed various genetic disturbance phases

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of exogenous genotypes in the Jinzu River. Further clarification of the impact of such alien individuals and genotypes on survival, fecundity and other features of the indigenous lamprey population is necessary, not to mention impact on the ecosystem overall. Lampreys are important organisms for understanding the evolutionary history of vertebrates, as well as being an important element of their ecosystem (Hardisty & Potter 1971; Yamazaki & Goto 2000a; Osório & Rétaux 2008; Docker 2009). East Asia is one of the "hot spots" for lamprey diversification, although some biodiversity components have become critical. In order to protect lampreys from their vulnerability to habitat change, as well as allowing for the possibility of future evolutionary trends, diverse environments for nursery and spawning habitats with opportunities for unrestricted gene flow within the indigenous gene pool and surrounding ecosystem should be maintained.

Acknowledgments We are grateful to V. G. Sideleva, H. Sakai, K. Takata, M. Nishida, R. Yokoyama, and T. Nagai for their invaluable support of our ecological field surveys and suggestions on various phases of the research program, and to H. K. Byeon, K. Iguchi, O. Inaba, S. R. Jeon, D. Pitruk, H. Shirakawa, H. Sugiyama, H. Takahashi, and S. Zolotukhin for their assistance in sample collection. We are grateful to G. Hardy for his invaluable suggestions and great help with the English. The field surveys and fish collections carried out in this study complied with local by-laws and regulations.

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implications for fitness and population dynamics in a salmonid fish. Journal of Animal Ecology 83, 1268–1278. Mousseau T.A. & Howard D.J. 1998. Genetic variation in cricket calling song across a hybrid zone between two sibling species. Evolution 52, 1104–1110. Naseka A.M., Tuniyev S.B. & Renaud C.B. 2009. Lethenteron ninae, a new nonparasitic lamprey species from the north-eastern Black Sea basin (Petromyzontiformes: Petromyzontidae). Zootaxa 2198, 16–26. Nikiforov S.N., Makeev S.S. & Belovolvo V.F. 1994. The freshwater ichthyofauna of southern Sakhalin and its origin. Journal of Ichthyology 34, 24–41. Osório J. & Rétaux S. 2008. The lamprey in evolutionary studies. Development Genes and Evolution 218, 221–235. Palkovacs E.P., Dion K.B., Post D.M. & Caccone A. 2008. Independent evolutionary origins of landlocked alewife populations and rapid parallel evolution of phenotypic traits. Molecular Ecology 17, 582–597. Paterson H.E.H. 1985: The recognition concept of species. In Vrba E.S. (ed.): Species and Speciation. Pp. 21–29. Pretoria: Transvaal Museum. Pavlov D. S. & Chernov Y.I. 1998. Annotated check-list of cyclostomata and fishes of the continental waters of Russia. Moscow: Nauka. Pfenninger M. & Schwenk K. 2007. Cryptic animal species are homogeneously distributed among taxa and biogeographical regions. BMC Evolutionary Biology 7, 1–6. Poizat G., Rosecchi E. & Crivelii A.J. 1999. Empirical evidence of a trade-off between reproductive effort and expectation of future reproduction in female three-spined sticklebacks. Proceedings of the Royal Society of London, Series B 266, 1543–1548. Poizat G., Rosecchi E. & Crivelii A.J. 2002. Life-history variation within a three-spined stickleback population in the Camargue. Journal of Fish Biology 60, 1296–1307. Potter I.C. & Gill H.S. 2003. Adaptive radiation of lampreys. Journal of Great Lakes Research 29 (Supplement 1), 95–112. Renaud C.B. 1997. Conservation status of Northern Hemisphere lampreys (Petromyzontidae). Journal of Applied Ichthyology 13, 143–148. Renaud C.B. 2011. Lampreys of the world: an annotated and illustrated catalogue of lamprey species known to date. Rome: Food and Agriculture Organization of the United Nations. Renaud C.B. & Economidis P.S. 2010. Eudontomyzon graecus, a new nonparasitic lamprey species from Greece (Petromyzontiformes: Petromyzontidae). Zootaxa 2477, 37–48.

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Renaud C.B., Docker M.F. & Mandark N.E. 2009. Taxonomy, distribution, and conservation of lampreys in Canada. American Fisheries Society Symposium 72, 293-309. Riva-Rossi C., Pascual M.A., Babaluk J.A., Garcia-Asorey M. & Haldenk N.M. 1970. Intra-population variation in anadromy and reproductive life span in rainbow trout introduced in the Santa Cruz River, Argentina. Journal of Fish Biology 70, 1780–1797. Sakai H. 1995. Life-history and genetic divergence in three species of Tribolodon (Cyprinidae). Memories of the Faculty of Fisheries, Hokkaido University 42, 1–98. Sakai H., Yamamoto C. & Iwata A. 1998. Genetic divergence, variation and zoogeography of a freshwater goby, Odontobutis obscura. Ichthyological Research 45, 363–376. Sakai H., Ueda T., Yokoyama R., Safronov S.N. & Goto A. 2014. Genetic structure and phylogeography of northern Far Eastern pond minnows, Rhynchocypris perenurus sachaliensis and R. p. mantschuricus (Pisces, Cyprinidae), inferred from mitochondrial DNA sequences. Biogeography 16, 87–109. Salewski V. 2003. Satellite species in lampreys: a worldwide trend for ecological speciation in sympatry? Journal of Fish Biology 63, 267–279. Sandlund O.T. & Jonsson B. 2014. Life history plasticity: migration ceased in response to environmental change? Ecology of Freshwater Fish. doi: 10.1111/eff.122304. Sato S. 1951. Studies on the lampreys of Hokkaido. Bulletin of the Faculty of Fisheries, Hokkaido University 1, 54–62. Shimizu T., Taniguchi N. & Mizuno N. 1993. An electrophoretic study of genetic differentiation of a Japanese freshwater goby, Rhinogobius flumineus. Japanese Journal of Ichthyology 39, 329–343. Shinervo B. & Calsbeek R. 2006. The developmental. physplogical, natural and genetical causes and consequences of frequency-dependent selection in the wild. Annual Review of Ecology and Evolutionary Systematics 37, 581–610. Taylor E.B. & Bentzen P. 1993. Evidence for multiple origin and sympatric divergent of trophic ecotypes of smelts (Osmerus) in northeastern North America. Evolution 47, 813–832. Taylor E.B., Garris L.N., Spice E.K. & Docker M.F. 2012. Microsatellite DNA analysis of parasitic lamprey (Entosphenus spp.) populations: implications for evolution, taxonomy, and conservation of a Canadian endemic. Canadian Journal of Zoology 90, 291–303. Teeter J. 1980. Pheromone communication in sea lamprey (Petromyzon

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CHAPTER THREE UKRAINIAN BROOK LAMPREY EUDONTOMYZON MARIAE (BERG): PHYLOGENETIC POSITION, GENETIC DIVERSITY, DISTRIBUTION, AND SOME DATA ON BIOLOGY BORIS LEVIN, ANDREY ERMAKOV, OLEG ERMAKOV, MARINA LEVINA, OLGA SARYCHEVA AND VLADIMIR SARYCHEV

Introduction The genus Eudontomyzon Regan, 1911 with five to seven valid species (Renaud 2011; http://researcharchive.calacademy.org/research/ichthyology /catalog/fishcatmain.asp) and some putative undescribed species (Kottelat and Freyhof 2007; Geiger et al. 2014) is the most diverse genus of lampreys in Europe. The Ukrainian brook lamprey Eudontomyzon mariae (Berg, 1931) is a non-parasitic species stated as the most widelydistributed lamprey in Europe being found in drainages of the Azov, Black, Baltic, Aegean, Adriatic and Caspian Seas (Holþík and Renaud 1986; Levin and Holþík 2006; Renaud 2011). Wide distribution and great phenotypic plasticity coupled with the divergent opinions of taxonomists as to the delimitation of the species suggests a complex taxonomic structure to E. mariae (Holþík and Renaud 1986; Blank, Jürss, and Bastrop 2008; Lang et al. 2009; Renaud 2011). With the progress in molecular approaches, the phylogenetic relationships among lamprey lineages, the taxonomic ranks of genera and species, the paired species concept and our knowledge of the population diversity were significantly revised (Docker et al. 1999; Yamazaki et al.

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2006; Espanhol, Almeida, and Alves 2007; Lang et al. 2009; Pereira, Almada, and Doadrio 2011; Artamonova, Kucheryavyy, and Pavlov 2011; April et al. 2011; Mateus et al. 2013; Geiger et al. 2014). The genetic structure within the genus Eudontomyzon, the phylogenetic position of certain species, and their taxonomic allocation have changed dramatically due to the results of recent molecular studies (Blank, Jürss, and Bastrop 2008; Lang et al. 2009; Geiger et al. 2014). Different studies have produced divergent results and opinions as to the phylogenetic relationships of the Ukrainian brook lamprey. For instance, Blank, Jürss, and Bastrop (2008) explored several mtDNA markers in Lampetra species as well as in E. mariae, and recommended reintegration of the latter into the genus Lampetra. On the other hand, in their phylogenetic analysis Lang et al. (2009) recognized the genus Eudontomyzon as being monophyletic while excluding E. hellenicus and E. morii, which they classified into the genera Caspiomyzon and Lethenteron respectively. It should be stressed that lampreys have the low diversity of morphological features. Phylogenetic relationships based on morphology have not been fully resolved, while phylogenetic analyses using various genetic markers have significantly clarified phylogeny (e.g., Lang et al. 2009; Renaud 2011). The Ukrainian brook lamprey E. mariae was described by Berg (1931) from the Kharkov R., drainage of the Don River. Until now, no DNAsamples have been studied from this population. Moreover, recent findings of Ukrainian brook lamprey in the Caspian Sea basin (Levin and Holþík 2006) raise the question as to their correct identification. These significant gaps prevent us from resolving the phylogenetic position and genetic diversity of the most widespread lamprey species in Europe. In the current study we aim: i) to clarify the phylogenetic relationships of the Ukrainian brook lamprey by including the populations from all of the main drainages as well as closely-related species using several mtDNA markers; ii) to update data on the current distribution of the species; and iii) to make a contribution to our knowledge on the biology of the species.

Phylogenetic position and genetic diversity Three loci of mtDNA, cytochrome b (cyt b), barcoding cytochrome c oxidase subunit I (COI), and control region1 (CR1) were selected to estimate relationships between species and populations. These markers were successfully used in previous phylogenetic analyses including E. mariae (Espanhol, Almeida, and Alves 2007; Blank, Jürss, and Bastrop 2008; Lang et al. 2009). Phylogenetic analyses were done on sequences of

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cyt b (1164 bp) and COI (652 bp) (see data in Table 3-1 and in Fig. 3-1, centerfold, page ix). All cyt b and COI sequences of E. mariae deposited to GenBank (http://www.ncbi.nlm.nih.gov/) and Barcode of Life Database (BOLD - http://www.boldsystems.org/) were also included (see Table 31). All published sequences of other members of the genus Eudontomyzon were also included with the exception of the Korean lamprey ‘Eudontomyzon’ morii that was shown to be a member of the genus Lethenteron (Lang et al. 2009). Species of the genus Lampetra were also included in the analysis as well as the closely related lineage ‘Lethenteron’ zanandreai. Species E. hellenicus and E. graecus were recently shown to be a sister group of the genus Caspiomyzon (Lang et al. 2009; Geiger et al. 2014). This clade Caspiomyzon + ‘Eudontomyzon’ hellenicus + ‘Eudontomyzon’ graecus is phylogenetically distant from the true Eudontomyzon and not considered here. CR1 sequences of Eudontomyzon are very scarce in the GenBank database which makes difficult the use of this marker for reconstruction of the phylogenetic position of E. mariae. However CR1 is more variable than protein-coding loci and suitable to estimate intrarelationships of the Ukrainian brook lamprey. For that purpose we analyzed CR1 in 71 specimens of E. mariae from 22 localities in the Don (n = 23), Dnieper (n = 11) and Volga (n = 37) basins (Table 3-1, Fig. 3-1, centerfold, page ix). Also we included the two CR1 sequences of E. mariae deposited to GenBank (EU404077-78 from the Gail River, tributary of the Drava River, Danube system). We extracted the total genomic DNA from ethanol-preserved tissues using the salt method (Aljanabi & Martinez 1997). Double-stranded DNA was amplified in 25 μl reactions (2 μl 1x buffer, 2 μM MgCl2, 0.25 mM of each primer, 0.2 μM dNTP of each nucleotide, 18.5 μl ddH20, 1 μl template DNA, and 1U Taq polymerase (Sileks, Moscow). PCRs were performed according to Espanhol, Almeida, and Alves (2007) for cyt b, Ivanova et al. (2007) for COI, and Almada et al. (2008) for CR. The primers used are listed in Table 3-2. The PCR products were visualized by mini-gel electrophoresis using ethidium bromide staining on 1.5% agarose gels. The PCR products were purified with double volume of ethanol 96% and sodium acetate (3M) precipitated after 10 min centrifugation at 13200 rpm, and centrifuged twice with 70% alcohol. Both strands were sequenced on Applied Biosystems 3500 DNA sequencer following the manufacturer’s instructions. DNA sequences were deposited in GenBank (accession numbers are indicated in Table 3-1). Homologous regions were aligned manually against previously published sequences of the Ukrainian brook lamprey (Blank et al. 2008;

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Lang et al. 2009; April et al. 2011). Chromatograms and alignments were visually checked and verified; there were no gaps in the cyt b and COI gene alignments. Sequences of CR include 3-4 conserved repeats by 39 bp and some short indels throughout the locus. The Caspian lamprey Caspiomyzon wagneri was used as an outgroup in both cyt b and COI analyses (GQ206152 and HQ579131 respectively) and as a representative of an evolutionarily distant lineage (Lang et al. 2009). The Akaike Information Criterion (AIC) implemented in jModelTest 2.1.5 (Darriba et al. 2012) was used to determine the evolutionary model that best fits the data set. The models selected were used for subsequent analyses. Bayesian inference (BI) was performed with Mr Bayes 3.2.3 (Ronquist et al. 2012) by simulating four simultaneous Markov chain analyses for 5 000 000 generations each for cyt b and 3 000 000 for COI to estimate the posterior probabilities distribution. The first 25 % of trees were discarded as burnin. Topologies were sampled every 1000 generations. The tree was constructed using FigTree v.1.2.3. The best-fit model selected following the Akaike Information Criterion was the TIM3+G for cyt b and TIM2+I for COI. Rate matrix was as follows: R(a)[A-C] = 2.04, R(b)[A-G] = 30.61, R(c)[A-T] = 1.00, R(d)[CG] = 2.04, R(e)[C-T] = 17.81, R(f)[G-T] = 1.00 for cyt b and R(a)[A-C] = 2.93, R(b)[A-G] = 17.73, R(c)[A-T] = 2.93, R(d)[C-G] = 1.00, R(e)[C-T] = 17.73, R(f)[G-T] = 1.00 for COI. Among-site variation approached the gamma distribution shape parameter (a) = 0.177 for cyt b, and 0.187 for COI. Base frequencies were A=0.287, C=0.229, G=0.133, T=0.351 for cyt b gene (1164 bp), A=0.276, C=0.255, G=0.153, T=0.316 for COI gene (652 bp), and A=0.313, C=0.246, G=0.153, T=0.288 for CR1 gene (611-653 bp). For cyt b, 259 characters were variable, 114 sites were parsimony informative, for COI – 136 and 84 sites respectively, and for CR – 30 and 28 sites respectively. The CR1 sequence contains 3-4 conserved repeats of 39 bp (for more details see the Results section). The number of variable sites of the CR1 sequence was calculated by considering each 39 bp-repeat as one event (one site). P-distances were counted in MEGA6 (Tamura et al. 2013). The haplotype diversity, nucleotide diversity and average number of differences were counted using Network 5.10.01 (Librado & Rozas, 2009).

Chapter Three

1. Don River at Lebedyan’ town; Lipetsk Region 2. Bystraya Sosna River, tributary of Don River near Talitsa vil.; Lipetsk Region 3. Semenyok River, tributary of Krasivaya Mecha River, Don system; Lipetsk Region 4. Sineomutovka River, tributary of Khoper R., Don system; Penza Region 5. Tauza River, tributary of Medveditsa R., Don system; Saratov Region 6. Nyan’ga River, tributary of Bol’shoy Chembar, Vorona-Don system; Penza Region 7. Devitsa R., tributary of Don River; Voronezh Region

Sample no., locality

Cyt b

KP135557

KP135511KP135515 KP135488KP135492 KP135527KP135531 KP135550KP135551

5 (A)

5 (A)

5 (Q)

2 (Q)

KP135471

-

KP135464KP135467

-

KP135475

Eudontomyzon mariae Azov Sea basin KP135552KP135472KP135555 KP135474 KP135556 KP135476KP135477

Control region1

2 (Q)

2 (A)

4(Q)

Number of samples studied

-

-

-

-

-

KP135454KP135457 -

COI

This study

This study

This study

This study

This study

This study

This study

Reference

Table 3-1. Localities, sample size and GenBank/BOLD accessions numbers for specimens examined in this study (Cyt b – cytochrome b, COI – cytochrome oxidase I subunit, A – adults, Q – ammocoetes).

62

15. Chardym River, tributary of Uza River, Sura-Volga system; Penza Region 16. Chardym River, tributary of Volga River; Saratov Region 17. Ardym River, tributary of Penza River, Sura-Volga system; Penza Region 18. Teshnyar’ River, tributary of Sura River, Volga system; Penza

8. Vyaz’ma River, tributary of Dnieper River, Smolensk Region 9. Bolva River, tributary of Desna River, Dnieper system, Kaluga Region 10. Ivianka River, tributary of Teterev River, Dnieper system; Ukraine 11. Gail River, tributary of Drava River, Danube system; Austria 12. Turiec brook, tributary of Váh River, Danube system; Slovakia 13. Morava drainage, Danube system; Czech Republic 14. Danube River (no specifications) -

1 (?)

1 (?)

KP135516KP135521

6 (A)

5 (A)

KP135532KP135533 KP135498KP135502

2 (A)

5 (A)

AM051061

-

EU404063 EU404062 -

GQ206162

-

-

-

Caspian Sea basin KP135493KP135468KP135497 KP135470

EU404077 EU404078 -

2 (Q)

3 (?)

-

Black Sea basin KP135539KP135478KP135545 KP135482 KP135535KP135538

1 (?)

4 (Q)

7 (A)

-

-

-

-

EU404068 EU404067 JN026603 JN026604 JN026605 IFCZE83811.COI-5P -

JN026606

KP135458KP135460 -

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

This study

This study

This study

This study

Espanhol, Almeida, and Alves (2007)

Halaþka et al. (unpubl.)

Blank, Jürss, and Bastrop (2008) April et al. (2011)

Lang et al. (2009) April et al. (2011)

This study

This study

63

Region 19. Ilim-Kadada River, tributary of Kadada River, Sura-Volga system; Penza Region 20. Shnayovka River, tributary of Sura River, Volga system; Penza region 21. Yulovka River, tributary of Sura R., Volga system; Penza Region 22. Muromka River, tributary of Moksha River, Oka-Volga system; Penza Region 23. Ugra River, tributary of Oka River, Volga drainage; Kaluga Region 24. Rudyanka River, tributary of Ugra River; Oka-Volga system; Kaluga Region 25. Sigosa River, tributary of Ugra River, Oka-Volga system; Smolensk Region 26. Zhelov’ River, tributary of Oka River, Volga system; Kaluga Region 27. Skniga River, tributary of Oka River, Volga system; Moscow Region

64

KP135503KP135504 KP135510

KP135522KP135526 KP135505KP135509 KP135549

KP135546

KP135547KP135548 KP135534

KP135558

2 (A)

1 (A)

5 (A)

5 (Q)

1 (Q)

1 (Q)

3 (Q)

1 (A)

1 (Q)

-

-

KP135484KP135486

KP135483

KP135487

-

-

-

-

Chapter Three

KP135461

-

-

-

-

-

-

-

-

This study

This study

This study

This study

This study

This study

This study

This study

This study

1 (?)

29. Zeta River, tributary of Moraca River, Adriatic Sea basin; Montenegro 30. Ohrid Lake drainage; Makedonia

-

-

2 (?)

1 (?)

2 (?)

-

-

1 (Q)

32. Ilz River drainage, Danube basin; Germany 33. Vardar drainage, Aegean Sea basin; Greece

2 (Q)

-

1 (A?)

34. Linda River, left tributary of Volga River; Nizhny Novgorod Region 35. Zhizdra River, left tributary of Oka River, Volga system; Kaluga Region 36. Ugra River, left tributary of Oka River, Volga system; Kaluga Region

-

-

-

Lampetra planeri Caspian Sea basin KP135462KP135463

-

Eudontomyzon sp. -

Eudontomyzon vladykovi GQ206161

-

Eudontomyzon stankokaramani GQ206189

Eudontomyzon danfordi GQ206158

31. Studenec brook, Danube River system; Slovakia

2 (?)

1 (?)

28. Zdychava River, Tisza River basin, Danube system; Slovakia

JN026956*

HQ579120* JN026957*

-

KJ553314, KJ553433

HQ955744

-

KJ553549 KJ553550

JN026607

-

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

April et al. (2011)

April et al. (2011)

This study

Geiger et al. (2014)

Neumann (unpubl.)

Lang et al. (2009)

Geiger et al. (2014)

Lang et al. (2009), April et al. (2011)

Lang et al. (2009)

65

44. Baltic Sea near Barth; Germany 45. Beke stream, Warnow River drainage; Germany 46. Neva River, Baltic Sea tributary; Russia

42. Tiber River drainage, Tyrrhenian Sea; Italy 43. Tagus drainage; Atlantic Ocean basin; Portugal

40. Stepenitz Stream, Elbe River drainage; Germany 41. Kalte Moldau River, tributary of Vltava River, Elbe River drainage; Germany

37. Nebel stream, Warnow River drainage; Germany 38. Althofener Bach, tributary of Baltic Sea; Germany 39. Oder drainage; Czech Republic

66

-

1 (?)

-

1 (A)

1 (A)

1 (A)

-

EU404059

Lampetra fluviatilis Baltic Sea basin EU404060

-

Mediterranean Sea basin -

GQ206149

North Sea basin EU404061

-

EU404061

Baltic Sea basin EU404061

1 (?)

-

1 (?)

-

1 (Q)

-

-

1 (A)

1 (A)

-

1 (A)

Chapter Three

HQ579125

-

-

KJ553986

KJ554002

-

-

HQ960780

-

-

Blank, Jürss, and Bastrop (2008) Blank, Jürss, and Bastrop (2008) April et al. (2011)

Geiger et al. (2014)

Geiger et al. (2014)

Blank, Jürss, and Bastrop (2008) Lang et al. (2009)

Blank, Jürss, and Bastrop (2008) Blank, Jürss, and Bastrop (2008) Halaþka et al. (unpubl.)

KJ554056 HQ579127 JN026955

-

-

-

-

Lethenteron zanandreai -

2 (?)

1 (?)

1 (?)

1 (?)

1 (?)

1 (?)

1 (?)

50. Sado River drainage, Atlantic Ocean basin; Portugal

51. Nabão River drainage, Tagus River system, Atlantic Ocean basin; Portugal

52. Vouga River drainage, Atlantic Ocean basin; Portugal

53. Atlantic Ocean basin; Portugal

54. Po River drainage, Adriatic Sea basin; Italy 55. Vipava River, Adriatic Sea basin; Slovenia

KJ553926

Lampetra alavariensis -

GQ206184

Geiger et al. (2014) Lang et al. (2009)

-

Mateus et al. (2011)

Geiger et al. (2014)

Geiger et al. (2014)

Geiger et al. (2014)

Lang et al. (2009) April et al. (2011)

Geiger et al. (2014)

Blank, Jürss, and Bastrop (2008)

KJ553679

-

KJ554063

Lampetra auremensis -

Lampetra sp. FN641832

KJ554076

Lampetra lusitanica -

GQ206176

Lampetra lanceolata

2 (?)

-

48. Sapanca Lake drainage; Turkey 49. Ykizdere brook, tributary of Black Sea; Turkey

North Sea basin EU404060

1 (A)

47. Elbe River near Brunsbüttel; Germany

-

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg) 67

-

-

1 (?)

Lethenteron reissneri -

CR1 (611-653 bp)

COI (652 bp)

Fragment Cyt b (1164 bp)

Okada et al. (2010)

April et al. (2011)

April et al. (2011)

Almada et al. (2008)

Ivanova et al. (2007) Messing (1983)

Reference Espanhol, Almeida, and Alves (2007)

AB565771

JN027076

HQ579005

Primer LA: 5’-GCGACTTGAAAAACCACCGTT-3’ PRO: 5’-TAGATACAGAGGTTTGAATCCC-3’ Internal primers: LB: 5’-CTGCAGCTACTGCTTTCGTTGG-3’ CB2H: 5’-CCCTCAGAATGATATTTGCCCTCA-3’ FF2d 5’-TTCTCCACCAACCACAARGAYATYGG-3’ FR1d 5’-CACCTCAGGGTGTCCGAARAAYCARAA-3’ Additional sequencing primer M13 (í21): 5’-TGTAAAACGACGGCCAGT-3’ LampFor: 5’-ACACCCAGAAACA GCAACAAA-3’ LampRev: 5’-GCTGGTTTACAAGACCAGTGC-3’

Table 3-2. Fragment used and primer sequences.

-

Lethenteron camtschaticum -

1 (?)

1 (?)

Chapter Three

* - these sequences belong to E. mariae, see discussion in section I.1.

58. Senju River, Fukuoka; Japan

56. Sukhona River, tributary of Severnaya Dvina, White Sea basin; Russia 57. Lower Chena River, Alaska; USA

68

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

69

Phylogenetic relationships of the Ukrainian brook lamprey based on sequences of coding mtDNA markers Based on the two protein-coding mitochondrial markers examined in this study, cyt b and COI, BI analysis highly supported the monophyly of the European Eudontomyzon species included in this study (Figs. 3-2 and 3-3) conrming the results of a recent mitochondrial phylogenetic study (Lang et al. 2009). The genus Lampetra restricted to European species only was a sister group to the genus Eudontomyzon. The most divergent member of the genus Eudontomyzon was the lineage of E. stankokaramani from the Adriatic Sea drainage (Fig. 3-3, centerfold, page xi). This lineage was the sister group to all other Eudontomyzon (Figs. 3-2 and 3-3, centerfold, pages x and xi). Phylogenetic analyses recovered three main lineages inside the widespread Eudontomyzon mariae s. lato: 1) the Eudontomyzon mariae s. stricto from the Don drainage and the upper reaches of two right tributaries of the Volga River, the Oka and the Sura; 2) its sister clade, the Dnieper lineage of Eudontomyzon that we assigned here as Eudontomyzon sp. ‘Dnieper’ based on the divergence level between sister lineages – 2.5 % p-distances in cyt b and 1.4 % in COI sequences and based on its distribution being restricted to the Dnieper drainage as well as adjacent tributaries of the Oka-Volga basin, and 3) the Danubian lineage of E. ‘mariae’ that probably contains the closely-related E. danfordi judging from the available cyt b sequences (Fig. 3-2, centerfold, page x) as well as putative new species from Aegean Sea drainage according to COI analysis (Fig. 3-3, centerfold, page xi). This potentially new species was discovered in the previous study of Geiger et al. (2014). Though the cladogenetic event of separation of a Danubian lineage and Eudontomyzon mariae s. stricto from the Don combined with its sister Dnieper clade is only moderately supported (post. prob. = 66 in cyt b and 68 in COI tree), divergence of Don and Dnieper lineages is highly supported in both trees (post. prob. = 99). Both the Don and Dnieper lineages penetrated into the Volga drainage and coexist in some of those rivers (see section ‘Distribution’ for more details). We should note the anomalous position of some specimens, the sequences of which were taken from GenBank. Two specimens of ‘Lampetra planeri’ (HQ579120 and JN026957) from the Zhizdra R., tributary of the Oka R. (Volga drainage), inserted within the lineage of Eudontomyzon sp. ‘Dnieper’ and one specimen of ‘Lampetra planeri’ (JN026956) from the Ugra R., also a tributary of the Oka R., inserted within the lineage of E. mariae s. str. of the COI tree (underlined with blue in Fig. 3-3, centerfold, page xi). There are two explanations for these mismatches. The specimens

Chapter Three

70

Table 3-3. P-distances between lineages of E. mariae s. lato. COI Dnieper Don

Danube 0.025 ±0.006 0.021 ±0.006

Dnieper 0.014 ±0.004

Cyt b Danube Dnieper 0.024 ±0.006 0.039 0.025 ±0.008 ±0.005

CR1* Danube Dnieper 0.038 ±0.007 0.032 0.024 ±0.007 ±0.005

* - The CR1 sequences contain 3-4 conserved repeats by 39 bp; p-distances for CR1 were calculated taking each 39 bp-repeat as one event (mutation).

might be misidentified as L. planeri while in fact they belong to the genus Eudontomyzon. Records of E. mariae in the Volga’s right tributaries have been previously reported (Levin 2001; Margolin & Chernikov 2001; Levin & Holþík 2006; Reshetniko et al. 2012). Another explanation for these mismatches may be attributed to hybridization between L. planeri and E. mariae as previously documented by Rembiszewski (1968). These mismatches notwithstanding the European brook lamprey Lampetra planeri does inhabit some tributaries of the Middle Volga (Alekseev 1982; Reshetnikov 2003). We included two specimens of L. planeri from a left tributary of the Middle Volga in our study and found that they clustered together with other members of this genus in the cyt b tree (Fig. 3-2, centerfold, page x) compared to the aforementioned ‘L. planeri’ that inserted themselves within the Eudontomyzon subtree in the COI tree (Fig. 3-3, centerfold, page xi). It is also doubtful that the sample of E. mariae (AM051061) from the Danube R. (without more specifications on sampling) is nested inside the Dnieper lineage (E. sp. ‘Dnieper’) of the cyt b tree (Fig. 3-2, centerfold, page x). Genetic distance between the Danube and Dnieper lineages is high in all three studied markers (Table 3-3). We suppose that the positioning of that specimen within the Dnieper lineage resulted from mislabeling. Relationships among Eudontomyzon inhabiting the Danube drainage, E. danfordi, E. vladykovi and local E. ‘mariae’ needs further investigation. Non-coding mtDNA sequence: control region1 analysis The control region1 of the Ukrainian brook lamprey contains 3-4 repeats of 39 bp (Ermakov et al. 2014) (Fig. 3-4A), similar to the condition found in other lampreys (Lee & Kocher 1995; White & Martin 2009; Okada et al. 2010; Pereira et al. 2010). Variation in the number of repeats (R) of E. mariae is intrapopulational. Even syntopic specimens have 3-4 R (Fig. 3-4B). Interestingly, the ratio between individuals with four and three repeats (4R/3R) in two of the recognized lineages is more or less stable

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

71

and close to 3/2: 34/21 (62/38%) specimens in the E. mariae s. str., and 10/6 (63/37%) in E. sp. ‘Dnieper’. Two individuals from the Danube drainage had only 3R. Sixteen haplotypes were found in the 73 samples studied (Table 3-4). Table 3-4. Sample size (n), number of haplotypes (NH), nucleotide diversity and average number of differences in CR1. Average number of differences Don-Volga 55 10 0.7926 0.00137 0.835 Dnieper 16 4* 0.6917 0* 0 Danube 2 2 1 0.00327 2 * - number of haplotypes is four but nucleotide diversity is zero because of indels. Lineage

n

NH

Haplotype diversity

Nucleotide diversity

Three haplogroups were detected corresponding to the lineages discovered in the phylogenetic analyses (Fig. 3-5, centerfold, page xii). Divergence between CR1 haplogroups is rather high and similar or higher to divergence observed in cyt b (Table 3-3). Therefore, the Ukrainian brook lamprey is a complex species comprised of at least three divergent lineages: E. mariae s. str. (the Don lineage), E. sp. ‘Dnieper’ (the Dnieper lineage), and the Danube lineage. The Don and Dnieper lineages are sister clades represented two distinct species, which should be properly described. The Danube lineage may include closely related E. sp. (formerly called E. mariae), the Danubian brook lamprey E. vladykovi and the Carpathian lamprey E. danfordi. Relationships between these forms require further investigation. Remarkably, the parasite form E. danfordi is nested within the clade comprising Eudontomyzon mariae s. lato, but in a basal position (Fig. 3-2) confirming the generally accepted hypothesis of the origin of non-parasitic forms from parasitic lampreys (Zanandrea 1959; Vladykov & Kott 1979; Docker 2009). The lamprey population from the Kuban River drainage (Black Sea basin) was reported previously as E. mariae (Berg 1948; Abakumov 1966). We refrain from making any statement at this time regarding its taxonomic position until genetic analysis is performed. The Kuban fish fauna exhibits high endemicity and the Kuban lamprey may represent a distinct lineage based on the opinion of N.G. Bogutskaya (reported as a personal communication in: Renaud 2011).

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Chapter Three

Fig. 3-4. (A) Nucleotide sequence of the mtDNA control region1 of E. mariae (GenBank No. KP135490), specimen from the Tauza River, Don drainage. In this specimen, three 39 bp repeats are observed and a fourth one occurred in some other specimens showed as asterisk. (B) Haplotypes of the Eudontomyzon mariae s. lato (the Don, Dnieper, and Danube lineages).

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

73

Distribution and colonization of the eastern part of the range, Caspian Sea drainage The Ukrainian brook lamprey was thought to be the most widely distributed lamprey species in Europe. The species was previously reported from the widespread territory comprising the Danube, Dnieper, Dniester, Prut, Don and Kuban’ drainages (Azov and Black Sea basins), the Oder, Vistula, and Neman drainages (Baltic Sea basin), rivers discharging into the eastern part of the Black Sea, the Vardar River drainage (Aegean Sea), and some right tributaries of the Middle Volga (Zhukov 1969; Holþík & Renaud 1986; Levin & Holþík 2006; Renaud 2011). However, based on a recent study, the small eastern tributaries of the Black Sea are inhabited by the Western Transcaucasian lamprey Lethenteron ninae Naseka, Tuniyev et Renaud, 2009. According to Li (2014) this species is phylogenetically closer to the genus Lampetra (i.e., L. lanceolata). Even excluding ‘Lethenteron’ ninae, E. mariae remains a species complex in need of further taxonomic resolution (see genetic section above). We do not consider here the distribution of the Danube lineage of E. ‘mariae’, which in our opinion belongs to another species. Eudontomyzon mariae s. str. inhabits the Don riverine system and has recently occupied right tributaries of the Middle Volga (Levin 2001; Margolin & Chernikov 2001; Levin & Holþík 2006). We have updated the data on the distribution of E. mariae s. str. based on the records published during the last 15 years (Fig. 3-6, centerfold, page xiii). Discovery of the Ukrainian brook lamprey in the basin of the Caspian Sea was surprising. Further studies showed that E. mariae is relatively abundant in the upper reaches of right tributaries of the Middle Volga (Levin & Holþík 2006; Artaev et al. 2013; Ermakov et al. 2013). Moreover, it also occurs in a Lower Volga tributary (Zavialov et al. 2007). Despite a rather wide expansion of the Ukrainian brook lamprey in the Middle and Lower Volga basin, there is a discontinuity in its distribution. All records until the present are associated with the upper streams bordering the Don or Dnieper basin watersheds. Such a pattern of distribution and the absence of a genetic divergence between the originating populations and the established populations in the Volga suggest recent colonizations of this basin. Based on the geographic position of the records (Fig. 3-6, centerfold, page xiii), we suggest that the Volga basin was colonized by the Ukrainian brook lamprey in different ways:  from the Upper Khoper (left tributary of the Don) to the Upper Sura (right tributary of the Volga) basin;

74

Chapter Three

 from the Upper Medveditsa (left tributary of the Don) to the Upper Sura tributaries and to small tributary of Lower Volga, the Chardym River, that discharges into the Volgograd Reservoir near Saratov.  from the Upper Vorona (the Khoper system) to the Upper Moksha (right tributary of the Oka);  from the Upper Don in Tula Region to the tributaries of the Oka River. Additionally the Oka basin was invaded by the Dnieper lineage (E. sp. ‘Dnieper’) based on genetic evidence (Figs. 3-2 – 3-3, 3-5). Therefore both lineages co-occur in the Upper Oka (at least in the Ugra River) as inferred from COI phylogenetic analysis (see Fig. 3-3, centerfold, page xi). Seemingly, Eudontomyzon sp. ‘Dnieper’ lineage has even wider distribution because our COI sequence from the Vyaz’ma River (KP135458) is identical to Eudontomyzon sp. from the Vistula basin, Baltic Sea drainage (Matthias Geiger, personal communication).This raises the question whether the Dnieper lineage also inhabits the adjacent drainages of the Neman and Zapadnaya Dvina rivers (Baltic Sea), the Southern Bug, Dniester and Prut rivers (Black Sea) or maybe some of these drainages are occupied by the Danube lineage, or even a combination of lineages as in the Volga drainage. The Ukrainian brook lamprey was also reported in the Kuban basin (Berg 1948; Abakumov 1966). The Kuban lamprey may represent a distinct endemic lineage and we require genetic evidence before making a statement as to its affinities.

Biology Data on the biology of E. mariae s. str. (the Don lineage) are scarce in the literature. Here we provide some data on the ecology of spawning, spawning behavior and some other information on the biology of the Ukrainian brook lamprey based on recent published data and our own observations.

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

75

Spawning period In the rivers of the Upper Don the spawning period occurs during late April - early May (Berg, 1931; our observations). Spawning begins at 1012 °C in the Upper Don and ended at 13.5-14 °C. We observed the same spawning temperature range in the Middle Volga. Probably the thermal regime of the rivers significantly affects the period of spawning. We registered late spawning in the tributary of the Uza River, the Chardym River (Volga system - 52.642 N 45.776 E). Spawning in the Chardym River occurs in mid-of-June on a spawning site located around 500 m from the mouth of the river compared to a 40-45 days earlier spawning in the Uza River located at 800 m upstream of the mouth of the Chardym River. The water in the latter heats more slowly due to an abundance of cold spring inputs, and spawning temperature of the water was attained about one month later than in the Uza River. Spawning sites The spawning substrate is typically gravel and sand; however, we have also detected spawning on a hard white clay bottom in both the Upper Khoper (Levin & Holþík 2006) and the Upper Don. Spawning usually occurs at shallow depths, 0.1-0.2 (rarely 0.4) meters, which is similar to the depths reported for E. ‘mariae’ of the Danube lineage (Holþík & Renaud 1986). Spawning sites are rather diverse in size, position in the river and in the density of spawners. Provisionally, we subdivide spawning sites for several types to show their diversity: 1) Localized spawning sites with small number of individuals in the center or to one side of the river. This is the most common type of spawning sites, often encountered under bridges. 2) Localized spawning sites situated in very restricted areas, such as in the submerged roots of trees where some current still exists. Such sites are occupied by many individuals, up to 40-60 per square meter by our estimate. 3) Spawning sites located linearly on top of a gravel ridge. These sites may span the width of the river often at an angle. 4) Widespread spawning sites occupying the entire riffle area of the river with nests more or less equally distant from each other. This type of site occurs more frequently in large rivers such as the Don R., Krasivaya Mecha R., and Nepryadva R.

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Chapter Three

Breeding habits Adult lampreys begin their spawning migration just after spring flooding in April. We observed such a migration in the Don River at Donskoe village (Lipetsk Region). Similar migrations were previously observed for other lineages of the Ukrainian brook lamprey by Zhukov (1965) and Rembiszewski (1967). Males begin to build nests when temperature rises to 10-12 °C. They dig a shallow, oval or circular nest using their suctorial disc to move pebbles as well as move their tail to deepen the nest. We also observed cooperative nest building by several lampreys. Using their suctorial disc they carry pebbles up to 5 cm in diameter to the edge of a prospective nest. When the nest was almost free of pebbles, the individuals each took their turn to deepen it by movements of the tail. The size of such collectively constructed nests varied from 1025 ɫɦ in diameter. Sometimes we observed the lampreys labouring together in the construction of the nest attack other lampreys not involved in the cooperative activity that were trying to approach the nest. Eudontomyzon mariae s. str. manifests the same breeding habits described previously for E mariae s. lato (Rembiszewski 1967). When the nest is ready, the male attaches its suctorial disc behind the head of the female and wraps around her body. The partners then spasmodically quiver during several seconds. At that moment reproductive products are released. Rarely, both the male and female were attached to the stones located side by side and were still engaged in a spawning. One male can spawn with several females located in the nest. Observations on the day and night activity of the spawning lampreys were done at the mouth of the Sukhaya Lubna River, a left tributary of the Don, during 13-14 May 2006 (Sarycheva 2006). The weather conditions during the observations were cloudless and warm. Five spawning sites were monitored. Sunrise was at 05:22, sunset was at 21:30, the daylight duration was therefore 16:08. The moonrise was at 22:30 and the moonset was at 04:51. The moon was full. Spawning activity changed significantly over the period of observation. Two peaks of spawning activity were detected (Fig. 3-7). The highest number of spawners (161) in the nests was recorded at 13:00. A gradual decrease in the number of spawners was observed until 21:30 (35). The number of spawning individuals then increased significantly at 23:00 (80) and continued to increase until 02:00 (91). After 02:00 the number of spawners decreased drastically. The minimal activity on the spawning sites was recorded in the morning between 06:00-10:00 (1-5 specimens), after which the number of spawners increased abruptly. The spawning activity did not directly correlate with

Ukrainian Brook Lamprey Eudontomyzon mariae (Berg)

77

water temperature during a period of observation. Maximum temperature of the water was at 16:00 (13 °C) and the minimum was at 06:00 (8.5 °C). It appears that light intensity taking into consideration more intensive light at full moon has a greater effect on the spawning activity (Holþík et al. 1965 in Holþík & Renaud 1986; Abakumov 1966).

Figure 3-7. Change in the number of spawners at the spawning sites in the Sukhaya Lubna River during the day and night (compiled using data from Sarycheva 2006).

Lampreys as a prey Fishes, birds and reptile species were recorded to consume Ukrainian brook lamprey in the Don and Volga basins. The burbot Lota lota (L.), sterlet sturgeon Acipenser ruthenus L., chub Squalius cephalus (L.), European perch Perca fluviatilis L. feed on both larvae and adults, while the stone loach Barbatula barbatula (L.) and Eurasian minnow Phoxinus phoxinus (L.) consume the lamprey’s eggs in nests (Levin & Holþík 2006; our observations). Many fishermen reported that larvae of Ukrainian brook lamprey are the preferred bait for predators such as the pike-perch Sander lucioperca (L.), the wels catfish Silurus glanis L., and to a lesser degree for the northern pike Esox lucius L. Some corvid birds, particularly the hooded crow Corvus cornix L. and the rook C. frugilegus L., were found to be feeding on lampreys in shallow spawning sites of the Sosna River, tributary of the Don River (Lipetsk Region, Russia). Terns (genus Sterna L.) actively fed on larval lampreys in the Voronezh Region when these

78

Chapter Three

aggregated close to the water surface, and their leaving of the substrate was probably caused by a polluted water event (A. Klyavin, personal communication). We encountered a grass snake Natrix natrix (L.) with a still alive and half-swallowed lamprey adult at the spawning site in the Sukhaya Lubna River, a Don tributary, on 6 May 2006. In the village of Donskoe (the Lipetsk Region, Russia) people until the 1990s consumed adults of the Ukrainian brook lamprey preferring females with eggs (our data based on an interview of local people).

Acknowledgments We are grateful to Nikolai Mugue for his valuable advice in the laboratory and to Alexey Bolotovskiy, Vladimir Il’in, Valery Korolev, Alexander Levin, Dmitry Mednikov, Yuri Reshetnikov, Dmitry Smirnov, Vladimir Salnikov, and Eugene Zavialov for their help with the collecting of samples. We acknowledge Ana Pereira and Claude Renaud for their reviews of the manuscript that significantly improved it. Also we are grateful to Dmitry Ⱥ. Pavlov and Claude Renaud for linguistic corrections. At the final stage this study was partially supported by Russian Science Foundation (No. 15-14-10020).

References Abakumov V.A. 1966. Systematics and ecology of Ukrainian lamprey (Lampetra mariae Berg). Voprosy Ikhthiologii 6, 609-618 (in Russian). Alekseev S.S. 1982. Occurrence of larval lampreys Lampetra planeri (Bloch) (Petromyzonidae) in the Moscow District. Voprosy Ikhtiologii 22, 502–503 (in Russian). Aljanabi S.M. & Martinez I. 1997. Universal and rapid salt-extraction of high genomic DNA for PCR-based techniques. Nucleic Acids Research 25, 4692–4693. Almada V.C., Pereira A.M., Robalo J.I., Fonseca J.P., Levy A., Maia C. & Valente A. 2008. Mitochondrial DNA fails to reveal genetic structure in sea-lampreys along European shores. Molecular Phylogenetics and Evolution 46, 391–396. April J., Mayden R.L., Hanner R.H., & Bernatchez L. 2011. Genetic calibration of species diversity among North America's freshwater fishes. Proceedings of the National Academy of Sciences 108, 1060210607. Artaev O.N., Ermakov A.S., Ruchin A.B., Ermakov O.A., & Levin B.A. 2013. Distribution of Ukrainian lamprey Eudontomyzon mariae (Berg,

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1931) on north-eastern border of range. Vestnik Tambovskogo Universiteta. Seriya Estestvennye I Tekhnicheskie Nauki 18, 29752978 (in Russian). Artamonova V.S., Kucheryavyy A.V. & Pavlov D.S. 2011. Nucleotide sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene of lamprey classified with Lethenteron camtschaticum and the Lethenteron reissneri complex show no species-level differences. Doklady Biological Sciences 437, 113-118. Berg L.S. 1931. A review of the lampreys of the northern hemisphere. Ezhegodnik Zoologicheskogo Muzeya Akademii Nauk USSR 32, 87116 + 8 pls. Berg L.S. 1948. Freshwater Fishes of the USSR and adjacent countries. V. 1. Moscow: IzdateĐstvo Akademii Nauk SSSR (in Russian). Blank M., Jürss K., & Bastrop R. 2008. A mitochondrial multigene approach contributing to the systematics of the brook and river lampreys and the phylogenetic position of Eudontomyzon mariae. Canadian Journal of Fisheries and Aquatic Sciences 65, 27802790. Darriba D., Taboada G. L., Doallo R. & Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature methods 9: 772-772. Docker M.F. 2009. A review of the evolution of nonparasitism in lampreys and an update of the paired species concept. American Fisheries Society Symposium 72, 71-114. Docker M.F., Youson J.H., Beamish R.J., and Devlin R.H. 1999. Phylogeny of the lamprey genus Lampetra inferred from mitochondrial cytochrome b and ND3 gene sequences. Canadian Journal of Fisheries and Aquatic Sciences 56, 2340-2349. Ermakov A.S., Artaev O.N., Il’in I.V., Ermakov O.A., Ruchin A.B. & Levin B.A. 2013. Distribution of the Ukrainian brook lamprey Eudontomyzon mariae (Berg, 1931) in the Sura River and Moksha River drainages. Proceedings of the Mordovian State Reserve 11, 263269 (in Russian). Ermakov A.S., Levin B.A. & Ermakov O.A. 2014. Genetic diversity of Ukrainian brook lamprey Eudontomyzon mariae on north-eastern border of range inferred from mtDNA control region. XXI Century: Resumes of the Past and Challenges of the Present plus 17, 17-21 (in Russian). Espanhol R., Almeida P.R. & Alves J. 2007. Evolutionary history of lamprey paired species Lampetra fluviatilis (L.) and Lampetra planeri

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(Bloch) as inferred from mitochondrial DNA variation. Molecular Ecology 16, 1909-1924. Geiger M.F., Herder F., Monaghan M.T., Almada V., Barbieri R., Bariche M., Berrebi P., Bohlen J., Casal-Lopez M., Delmastro G.B. Denys G.P.J., Dettai A., Doadrio I., Kalogianni E., Kärst H., Kottelat M., Kovaþiü M., Laporte M., Lorenzoni M., Marþiü Z., Özulu÷ M., Perdices A., Perea S., Persat H., Porcelotti S., Puzzi C., Robalo J., Šanda R., Schneider M., Šlechtová V., Stumboudi M., Walter S. & Freyhof J. 2014. Spatial heterogeneity in the Mediterranean biodiversity hotspot affects barcoding accuracy of its freshwater fishes. Molecular ecology resources. doi: 10.1111/1755-0998.12257. Holþík J. and Renaud C. B. 1986. Eudontomyzon mariae (Berg, 1931). In Holþík J. (ed.): The Freshwater Fishes of Europe. 1/I. Petromyzontiformes. Pp. 165–185. Wiesbaden: AULA-Verlag. Ivanova N.V., Zemlak T.S., Hanner R.H. & Hebert P.D.N. 2007. Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7, 544–548. Kottelat M. & Freyhof J. 2007. Handbook of European freshwater fishes. Cornol and Berlin: Publications Kottelat. Lang N.J., Roe K.J., Renaud C.B., Gill H.S., Potter I.C., Freyhor J., Naseka A.M., Cochran P., Pérez H.E., Habit E.M., Kuhajda B.R., Neely D.A., Reshetnikov Y.S., Salnikov V.B., Stoumboudi M.T. & Mayden R.L. 2009. Novel relationships among lampreys (Petromyzontiformes) revealed by a taxonomically comprehensive molecular data set. American Fisheries Society Symposium 72, 41–55. Lee W.J. & Kocher T.D. 1995. Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139, 873-887. Levin B.A. 2001. Finding of the Ukrainian lamprey Eudontomyzon mariae (Petromyzontidae) in the Volga basin. Journal of Ichthyology 41, 810– 811. Levin B.A. & Holþík J. 2006. New data on the geographic distribution and ecology of the Ukrainian Brook Lamprey, Eudontomyzon mariae (Berg, 1931). Folia Zoologica 55, 282-286. Li Y. 2014. Phylogeny of the lamprey genus Lethenteron Creaser and Hubbs 1922 and closely related genera using the mitochondrial cytochrome b gene and nuclear gene introns. MS dissertation, University of Manitoba. Librado P. & Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451-1452.

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Mateus C.S., Almeida P.R., Quintella B.R. & Alves M.J. 2011. MtDNA markers reveal the existence of allopatric evolutionary lineages in the threatened lampreys Lampetra fluviatilis (L.) and Lampetra planeri (Bloch) in the Iberian glacial refugium. Conservation Genetics 12, 1061-1074. Mateus C., Alves J., Quintella B. & Almeida, P.R. 2013. Three new cryptic species of the lamprey genus Lampetra Bonnaterre, 1788 (Petromyzontiformes: Petromyzontidae) from the Iberian Peninsula. Contributions to Zoology 82, 37-53. Margolin V.A. & Chernikov M.A. 2001. Study of lampreys of Kaluga oblast. In Proceedings of VIII Regional Scientific Conference “Voprosy arkheologii, istorii, kul’tury I prirody Verkhnego Pooch’ya”. Pp. 318–320, Kaluga: PoligrafInform (in Russian). Naseka A.M., Tuniyev S.B. & Renaud C.B. 2009. Lethenteron ninae, a new nonparasitic lamprey species from the north-eastern Black Sea basin (Petromyzontiformes: Petromyzontidae). Zootaxa 2198, 16–26. Okada K., Yamazaki Y., Yokobori S. & Wada H. 2010. Repetitive sequences in the lamprey mitochondrial DNA control region and speciation of Lethenteron. Gene 465, 45-52. Pereira A.M., Robalo J.I., Freyhof J., Maia C., Fonseca J.P., Valente A. & Almada V.C. 2010. Phylogeographical analysis reveals multiple conservation units in brook lampreys Lampetra planeri of Portuguese streams. Journal of Fish Biology 77, 361-371. Pereira A.M., Almada V.C.& Doadrio I. 2011. Genetic relationships of brook lamprey of the genus Lampetra in a Pyrenean stream in Spain. Ichthyological Research 58, 278-282. Renaud C.B. 2011. Lampreys of the World. Rome: FAO. Rembiszewski J.M. 1967 [Contribution to the knowledge of the lampreys (Petromyzontidae) of the genus Lampetra Gray in Poland. I. Lampetra (Eudontomyzon) mariae Berg]. Fragmenta Faunistica 13, 249-260 (In Polish). Rembiszewski J.M. 1968. Observations on hybrids of Lampetra (Lampetra) planeri (Bloch, 1784) x Lampetra (Eudontomyzon) mariae Berg, 1931. VČstník ýeskoslovenské Spoleþnosti Zoologicke 32, 390– 393. Reshetnikov Yu.S. (ed). 2003. Atlas of freshwater fishes of Russia. V. 1. Moscow: Nauka (in Russian). Reshetnikov Y.S., Dyakina T.N. & Korolev V. V. 2012. Changes in the structure of ichthyofauna in water bodies of Kaluga oblast over the past few decades. Russian Journal of Ecology 43, 52-61.

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Ronquist F., Teslenko M., van der Mark P., Ayres D.L., Darling A., Höhna S., Larget B., Liu L., Suchard M.A. & Huelsenbeck J.P. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61, 539-542. Sarycheva O.V. 2006. Day and night activity of the Ukrainian brook lamprey Eudontomyzon mariae (Berg, 1931) during spawning. In Voprosy Estestvoznaniya: Proceedings of 14 Interuniversity Scientific Conference of Teachers, PhD Students and Undergraduates. Pp. 4143, Lipetsk (in Russian). Sarycheva O.V., Ivanchev V.P., Sarychev V.S. & Ivancheva E.Y. 2014. Distribution and numbers of Ukrainian lamprey Eudontomyzon mariae (Petromyzontidae) in the upper Don basin. Journal of Ichthyology 54, 566-575. Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 2725-2729. Vladykov V.D. & Kott E. 1979. Satellite species among the holarctic lampreys (Petromyzonidae). Canadian Journal of Zoology 57, 860– 867. White M.M. & Martin H.R. 2009. Structure and conservation of tandem repeats in the mitochondrial DNA control region of the Least Brook lamprey (Lampetra aepyptera). Journal of Molecular Evolution 68, 715-723. Yamazaki Y., Yokoyama R., Nishida M. & Goto A. 2006. Taxonomy and molecular phylogeny of Lethenteron lampreys in Eastern Eurasia. Journal of Fish Biology 68, 251-269. Zanandrea G. 1959. Speciation among lampreys. Nature 184, 380. Zavialov Ye.V., Schlyakhtin G.V., Ruchin A.B., Mosolova E.Yu., Yakushev N.N. & Tabatchishin V.G. 2007. To distribution and biology of lampreys (Petromyzontidae) on the north of Lower Volga. In Proceedings of Scientific Conference “Ichthyological research in inland water bodies”. Pp. 47-50, Saransk (in Russian). Zhukov P.I. 1965. Distribution and evolution of freshwater lampreys in the waters of the Belorussian Soviet Republic. Voprosy Ikhtiologii 5, 240– 244 (in Russian). Zhukov P.I. 1969. New data on the biology of freshwater lampreys in Belorussia. Voprosy Ikhtiologii 9, 181-186 (in Russian).

CHAPTER FOUR DIVERSITY AND DISTRIBUTION OF THE HAGFISHES AND LAMPREYS FROM CHILEAN WATERS GERMÁN PEQUEÑO AND SYLVIA SÁEZ

Which are the Chilean Myxiniformes and Petromyzontiformes species and their current conservation status? The Myxiniformes and Petromyzontiformes are the oldest living groups of cranial chordates (Berg 1947, Olson 1971, Long 1995), in terms of their fossil history (Bond 1996). They are few species compared to other groups of chordates with 14 marine Myxiniformes and two Petromyzontiformes in Chile. (Pequeño 1989, 1997) (Table 4-1). The Chilean forms have been studied as a group only once (De Buen 1961) however, there are a number of studies that have contributed to form a more complete picture of their taxonomic composition and geographic distribution, as well as providing a better basis to understand possible relations among the species (Fowler 1941, 1951; Neira 1984; Wisner & McMillan 1988, 1995; Ruiz &Marchant 2004).In the last 30 years these studies provided a considerable advance in the knowledge of the two taxa that has changed the panorama related to them in the southeast Pacific Ocean, especially the Myxiniformes in Chilean waters. These studies show that the southeast Pacific Ocean may hold key information about the origin and distribution of these two groups.

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Table 4-1. Systematic list of the hagfishes and lampreys living in Chilean waters, with basic conservation information (for Myxiniformes, extracted from Knapp et al., 2011). Taxa Order Myxiniformes Family Myxinidae Subfamily Myxininae Myxini affinis Günther, 1870 Myxine australis Jenyns, 1842 Myxine circifrons Garman, 1899 Myxine debueniWisner & McMillan, 1995 Myxine fernholmi Wisner & McMillan, 1995 Myxine hubbsi Wisner & McMillan, 1995 Myxine hubbsoides Wisner & McMillan, 1995 Myxine pequenoi Wisner & McMillan, 1995 Myxine tridentiger (Garman, 1899) Subfamily Eptatretinae Eptatretus bischoffi (Schneider, 1880) Eptatretus laurahubbsae McMillan & Wisner 1984 Eptatretus nanii Wisner & McMillan, 1988 Eptatretus polytrema (Girard, 1855) Eptatretus strickrotti Moller & Jones, 2007 Order Petromyzontiformes Family Geotriidae Geotria australis Gray, 1851 Family Mordaciidae Mordacia lapicida (Gray, 1851)

Conservation status

LC LC LC DD LC LC DD DD DD DD LC DD DD LC

* *

DD = Data deficient; LC = Least concern; * = In study

The Chilean Myxiniformes and Petromyzontiformes and their geographical distribution Myxyniformes Myxine affinis inhabits waters from Madre de Dios Island (45º36’S) to the southernmost tip of South America (Wisner & McMillan 1995).

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Myxine australis lives from 45º36’S to the Magellan Strait (52°30´S, 75°0´W) (Fowler 1941, 1951; Wisner & McMillan 1995; Pequeño 1997, 2000; Sielfeld et al.2006). It is also found in the Argentinean coast between 48ºS and 50º S and southern Brazil (Mincarone & Soto 2001). Myxine circifrons lives from 38º 00’N (California waters) to 33º17’ S (near Valparaíso), with apparently continuous distribution, at 700-1860 m depth (Wisner & McMillan 1995, Pequeño 1997). Myxine debueni is endemic to Chilean waters. Known only from two specimens captured at 53º 39’ S (Magellan Strait) at about 300 m depth (Wisner & McMillan 1995). Myxine fernholmi lives from off Valparaíso (33º 39´S) to near the Falkland/Malvinas Islands (53º 00’S), and also probably lives in the Chilean channels and around Cape Horn (55°50´S, 67°30´W), at 135 to 1480 m depth (Wisner & McMillan 1995; Pequeño 1997). Myxine hubbsi lives from about 33°N (California) to south off Valparaíso (33°S), between 1100-2440 m depth (Wisner & McMillan 1995; Pequeño 1997). Myxine hubbsoides is endemic to Chile, between Coquimbo (31°00’S, 71°20´W) and Topocalma Point (34°10°S´, 72°2’W), at 735-880 m depth (Wisner & McMillan 1995; Pequeño 1997). Myxine pequenoi. Is endemic to Chile, known only from off the Valdivia coast, between 40°44’S and 41°29’S, between 185-215 m depth (Wisner & McMillan 1995; Pequeño 1997). Notomyxine tridentiger. The holotype comes from Punta Arenas, Strait of Magellan (53°10´S, 71°0´W), captured between 11 and 106 m depth (Fowler 19411951; Mann 1954; Fernholm 1998). Also off the Argentinean coast (Nani & Gneri 1951) Eptatretus bischhoffi. Endemic to Chile, between Caldera (27°05´S, 70°55’W) and Puerto Montt (41°28°S´, 73°0´W), in shallow waters from 8 to 50 m depth (Wisner & McMillan 1988, Meléndez et al. 1993; Pequeño 1997, Wisner 1999). Eptatretus laurahubbsae. Is endemic to Chile. Known only from near Robinson Crusoe Island (Juan Fernandez Archipelago), off central Chile, at about 2400 m depth (McMillan & Wisner 1984; Meléndez et al. 1993; Pequeño 1997). Eptatretus nanii. Is endemic to Chile. Inhabits the bottom near Valparaíso, between 33°22’S and 36°26.5’S, from 100-470 m depth (Wisner & McMillan 1988, Kong & Meléndez 1991, Meléndez et al., 1993, Pequeño 1997, Acuña et al. 2005). Eptatretus polytrema. Is endemic to Chile, from Bahía Inglesa (about 27°S) to Concepción (36°50´S, 73°00´W), in shallow water, 10-350 m

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depth (Wisner & McMillan 1988; Meléndez et al. 1993; Pequeño 1997Fowler 1951; Mann 1954; De Buen 1961). Eptatretus strickrotti. Is endemic to Chile in the East Pacific Rise (37° 47,363’S; 110°54.905’W) off the central coast of Chile, very close to the Motu Motiro Hiva Chilean Marine Park, at about 2111 m depth (Moller & Joe Jones 2007) . Petromyzontiformes Geotria australis. Present in Chile (from about 33° S to the southernmost tip of South America), coast of Argentina from about Buenos Aires to the southern border, including the Falkland/Malvinas Islands, South Georgia Is., New Zealand, southern Australia. Inhabits freshwater, estuaries and also shallow littoral marine waters (Fowler 1941 1951; Mann 1954; De Buen 1961; Ivanova-Berg 1968; Potter et al. 1980; Neira 1984; Pequeño 1989, 2007; Meléndez et al.1993; Ruiz & Marchant 2004; Renaud 2011). Mordacia lapicida. Is endemic to Chile. Inhabits freshwater as well as estuarine and coastal shallow marine waters of central and southern Chile (Fowler 1941, 1951; De Buen 1961; Neira 1984; Pequeño 1989; Ruiz & Marchant 2004; Habit et al. 2006; Renaud 2011; Carrasco-Lagos et al. 2012).

Comments on the endemism in Chilean Myxiniformes and Petromyzontiformes, and other aspects of these groups All the species of Myxine found off the coast of Chile live near the western South American coast and belong to the family Myxinidae which also has many species in the Northern Hemisphere. Some authors have reported Myxine glutinosa from Chilean waters (Mann, 1954; De Buen 1961, among others), but other investigators (Fowler 1941, Wisner & MacMillan 1995, and Fernholm 1998), consider that the species has been confused with M. australis and that M. glutinosa is found only in the Northern Hemisphere.There are a large number of endemic species of Myxiniformes found off the coast of Chile (Table 4-2). All five species in the genus Eptatretus and five of the eight species of Myxine are endemic. The number of species and their high degree of endemism is of considerable interest to biogeographers because it may mean that their location in the eastern South Pacific is closely related to the area of origin of the group and that they are very old. Only one of the fourteen species known (Eptatretus laurahubbsae) lives in the surroundings of Robinson

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Crusoe Island (Juan Fernandez Archipelago, off central Chile), meanwhile E. strickrotti was caught in the East Pacific Rise, in a point very near the Motu Matiro Hiva Chilean National Marine Park. The Chilean species of Eptatretus are completely (100%) endemic and deserves attention, because it seems to be a unique case in the world. The high degree of endemism of a given taxon is considered of much interest by biogeographers, beause it may mean that the area is related to the possible origin of the group (Briggs 1984, Bueno Hernandez & Llorente Bousquets2000, Zunino & Zullini 2003). All the species of Myxine listed here live near the western South American coast. The understanding of the evolutionary age of the various species will be improved with the results of the current phylogenetic studies. The results most likely will show a linkage to the history of the South American continent. There is no defined state of conservation on the majority of the Chilean Myxiniformes, mostly because of the scarcity of information. There is little known about the spawning sites of the Southern Hemisphere lampreys; it is possible that there has been heavy predation by birds (very abundant in South America) on the larval and juvenile states, that have been commonly found in shallow water (Pequeño 1989).Chile and Argentina host the highest number of Data Deficient species of Myxiniformes in the world (Knapp et al. 2011) (Table 1). Eptatretus polytrema was probably caught when bottom trawling fishery was strong in Chile, but now that fishery has decreased and the species may be more abundant (Mincarone 2013). For example, the first author of this chapter has often seen large numbers that were caught and discarded from bottom trawls. It is not known how many of the discarded fish survive, but it is important to determine the impact of bottom trawling on the population. Hagfishes are considered as “cleaners” which help maintain a healthy ocean bottom ecosystem. The jawless craniata linked to the continental or freshwaters of Chile currently consists of Geotria australis and Mordacia lapicida (Table 1). Both are anadromous, and spawn in rivers and lakes, but remain part of the year in the marine coast, mainly close to or within estuaries. Two theories have proposed for the origin of the current distribution of lampreys. The oldest theory suggests that a hypothetical ancestral form probably lived in the tropical ocean, from which it spread to interior waters and towards both poles; later the original tropical forms became extinct (Berg 1933). However, Hubbs & Potter (1971) observed that the geographic distribution was inconsistent with this concept and attributed it to crossing the tropics during the Pleistocene. The suggestion of Hubbs (1952) that one of the possible causes of the disappearance of lampreys from the tropics may be

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the richness of the fauna that developed there, is not consistent with the fact that the rivers, estuaries and lakes of Chile are currently much poorer in megafauna than the equivalent areas of the Northern Hemisphere. There is no evidence to suggest that the Southern Hemisphere fresh waters were much richer in megafauna in past epochs. It is possible that an old stock with a preferentially austral distribution was separated from the more northern populations due to the warming of the tropics after the cooling during the Pleistocene, followed by the diversification of genera of Petromyzontidae in the Northern Hemisphere, with the consequent development of the forms that Hubbs (1952) found to be similar to those of the Southern Hemisphere. It is also possible that two subgroups, predecessors of the Geotridae and Mordaciidae, were formed due to effects to be yet unknown before the separation of Australia from South America. Table 4-2. Number of genera and species of hagfishes in Chilean waters, with comparison to world fauna total and percentage of endemics

Genera

Eptatretus Notomyxine Myxine Total

Total species world number 46 1 19 66

Number of species in Chile

Endemics/Genus

%

5 1 8 14

5 1 5 11

100 100 62.5 78.6

Although Mordacia lapicida appears to be present in the Magellan Strait, the majority of the records of Geotria are evidently more southern. The fact that two species of Mordacia and the genus Geotria are present in Australia (Potter et al. 1958; Potter et al. 1979) suggests that the latter genus spread from Australia to New Zealand. Moreover, if we accept that at an earlier time when South America and Australia were close Geotria spread from the former continent to the latter, this would be consistent with the fact that the genus is found in a greater area and latitudinal range in South America and surroundings. It may have adapted to cold temperatures because its ancestors lived in this type of zone, and in the case of the Chilean coast reach lower latitudes than in other regions of the world due to the favorable influence of the Humboldt Current which is superficially cold, accompanied by winds that are also cold. During autumn and winter, this phenomenon affects especially the south-central

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coast of Chile, and also projects farther north. The coast of Chile also has numerous short rivers, rich in runoff water from the Andes whose temperatures are not that cold, favouring the reproduction of ammocoetes, similar to what happens in Australia. It is in the southeastern Pacific that the origin of many groups of fish (Mead 1970), the Myxiniformes and the Petromyzontiformes may be found. It is necessary, for future studies, to have the following key to identify Chilean genera of those groups.

Key for determining the genera of hagfishes and lampreys of Chile According to different authors (De Buen 1961; McMillan & Wisner 1984; Neira 1984; Wisner & McMillan 1988, 1995), we prepared the following key for determining the genera. 1a. Unique nasal opening in the tip of the head, surrounded by four barbels. Mouth narrow, with one pair of barbels. Vestigial eyes. No dorsal fins…........................Order Myxiniformes. ………………………………2 1b. Unique nasal opening in the dorsal part of the head, without barbels. Mouth wide, without barbels, but with many fleshy fringes around the oral circle. Conspicuous eyes. One or two dorsal fins.........……........... .......……………………………..Order Petromyzontiformes…………….4 2a. One single pair of gill apertures.............Subfamily Myxininae .......................................................……….………………………….……3 2b. Five to 14 pairs of gill apertures.................Subfamily Eptatretinae ...................................................… Eptatretus (5 species in Chilean waters) 3a. Pharyngocutaneous duct opening not confluent with the gill aperture on the left side .......………………………………….. Notomyxine (monotypic, with N . tridentiger) 3b. Pharyngocutaneous duct opening confluent with the gill aperture on the left side…......……….............. Myxine (7 species in Chilean waters) 4a. Odontoids separated in one plate, odd. Second dorsal fold separated from tail fin ........................................…………Geotria (Geotria australis) 4b Odontoids separated in two symmetrical plates. Second dorsal fold united with caudal fin...................................Mordacia (Mordacia lapicida)

Acknowledgments To the late Roberto Meléndez (Museo Nacional de Historia Natural de Chile), who provided information about jawless Chordata specimens under

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his care. Dr. Lafayette Eaton gave support preparing the manuscript in American English style. Two referees greatly helped improving the manuscript. The Office for Research and Development of the Universidad Austral de Chile, gave support to several projects of the main author.

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sharks in Argentina. Contributions to the collaboration of the National Action Plan. Pp. 1-58. Buenos Aires: Consejo Federal Pesquero (In Spanish). Fowler H.W. 1941. Fishes of Chile. Systematic Catalog. Part I. Lancelets. Lampreys, sharks, rays, chimaeras. RevistaChilena de Historia Natural 45, 22-57. Fowler H.W. 1951. Analysis of the Fishes of Chile. Revista Chilena de Historia Natural 51-53, 263-326. Habit E., Dyer B & Vila I. 2006. Current state of knowledge of freshwater fishes of Chile. Gayana 70, 100-113 (In Spanish). Hubbs C.L & Potter I.C. 1971. Distribution, phylogeny and taxonomy. In Hardisty M.W & Potter I.C. (eds.): The biology of lampreys. Vol. 1. Pp. 1-65. London & New York: Academic Press. Hubbs C.L. 1952. Antitropical distributions of fishes and other organisms. Proceedings of Seventh Pacific Science Congress 3, 324-329. Knapp L., Mincarone M.M., Harwell H, Polidoro B., Sanciangco J. & ɋarpenter K. 2011. Conservation status of the world’s hagfish species and the lost of phylogenetic diversity and ecosystem function. Aquatic Conservation: Marine and Freshwater Ecosystems 21, 401-411. Kong I. & Meléndez R. 1991. Taxonomic and systematic research into the fish caught in deep sea between Arica and Isla Mocha (18º30’ – 38º30’ Lat. S). Estudios Oceanológicos 10, 1-81 (In Spanish). Long J.A. 1995. The rise of fishes, 500 million years of evolution. Baltimore and London: The Johns Hopkins University Press. Macey D.J. & Potter I.C. 1978. Lethal temperatures of ammocoetes of the Southern Hemisphere lamprey, Geotria australis Gray. Environmental Biology of Fishes 3, 241-243. Mann G. 1954. The life of the fish in Chilean waters. Santiago: Instituto de Investigaciones Veterinarias y Universidad de Chile. McMillan C. & Wisner R.L. 1984. Three new species of seven-gilled hagfishes (Myxinidae, Eptatretus) from the Pacific Ocean. Proceedings California Academy of Sciences 43, 249-267. Mead G.W. 1970. A history of South Pacific fishes. In Wooster W.S. (ed.): Scientific Exploration of the South Pacific. Pp. 236251.Washington, D.C.: National Academy of Sciences. Meléndez R., Gálvez O. & Cornejo A. 1993. Catalogue of collection of fish deposited in the National Museum of Natural History of Chile. Museo Nacional de Historia Natural de Chile, Publicación Ocasional 47, 1-224 (In Spanish).

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Mincarone M.M. & Soto J.M.R. 2001. First record of the southern hagfish Myxine australis (Myxinidae) in Brazilian waters. Mare Magnum 1, 125-127. Mincarone M.M. 2013. Eptatretus polytrema. The IUCN Red List of Threatened Species. Version 2014.3. . Møller P.R. & Joe Jones W. 2007. Eptatretus strickrotti n.sp. (Myxinidae): first hagfish captured from a hydrotermal vent. Biological Bulletin 212, 55-66. Nani A. & Gneri F.S. 1951. Introduction to the study of South American hagfishes (class Myxini, family Myxinidae). 1. A new genus of "sea slug", "Notomyxine". Revista Museo Argentino de Ciencias Naturales Bernardino Rivadavia, Buenos Aires (Ciencias Zoológicas) 2, 183-224 (In Spanish). Neira F.J. 1984. Biomorphology of Chilean parasitic lampreys Geotria australis Gray, 1851 and Mordacia lapicida (Gray, 1851) (Petromyzontiformes). Gayana (Zool.) 48, 3-40 (In Spanish). Olson E.C. 1971. Vertebrate Paleozoology. New York: Wiley Interscience. Pequeño G. 1989. Fishes of Chile. Systematic list Revised and Commented. Revista de Biología Marina 24, 1-132 (In Spanish). Pequeño G. 1997. Fishes of Chile. Systematic list Revised and Commented: Addendum. Revista de Biología Marina y Oceanografía 31, 77-94 (In Spanish). Pequeño G. 2000. Fishes of the CIMAR Fjords 3 Cruise to the southern channels of the Magellan Province (ca. 55°S), Chile. Ciencia y Tecnología del Mar 23, 83-94 (In Spanish). Potter I.C., Hilliard R.W. & Bird D.J. 1980. Metamorphosis en the Southern Hemisphere lamprey, Geotria australis. Journal of Zoology 190, 405-430. Potter I.C., Lanzing W.J.R. & Strahan R. 1968. Morphometric and meristic studies on populations of Australian lampreys of the genus Mordacia. Journal of the Linnean Society (Zoology) 47, 533-546. Potter I.C., Prince P.A. & Croxhall J.P. 1979. Data on the adult marine and migratory phases in the life cycle of the Southern Hemisphere lamprey, Geotria australis Gray. Environmental Biology of Fishes 4, 65-69. Renaud C.B. 2011. Lampreys of the World. An annotated and illustrated catalogue of lamprey species known to date. Rome: FAO. Ruiz V.H. & Marchant M. 2004. Fish fauna of inland waters of Chile. Concepción: Departamento de Zoología, Universidad de Concepción.

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Sielfeld W., Guzmán G. & Amado N. 2006. Distribution of rocky shore fishes of the east Patagonian channels (48º37´S – 53º34´S). Anales Instituto de la Patagonia, Chile 34, 21-32. Wisner R.L. & McMillan C. 1988. A new species of hagfish, genus Eptatretus (Cyclostomata, Myxinidae), from the Pacific Ocean near Valparaíso, Chile, with new data on E. bischoffii and E. polytrema. Transactions San Diego Society of Natural History 21, 227-244. Wisner R.L. & McMillan C. 1995. Review of the New World hagfishes of the genus Myxine (Agnatha, Myxinidae) with descriptions of nine new species. Fishery Bulletin 93, 530-550. Wisner R.L. 1999. Descriptions of two new subfamilies and a new genus of hagfishes (Cyclostomata: Myxinidae). Zoological Studies 38, 307313. Zunino M. & Zullini A. 2003. Biogeography, the spatial dimension of evolution. Mexico: Fondo de Cultura Económica (In Spanish).

CHAPTER FIVE HAGFISHES OF MEXICO AND CENTRAL AMERICA: ANNOTATED CATALOG AND IDENTIFICATION KEY ARTURO ANGULO AND LUIS FERNANDO DEL MORAL-FLORES

Introduction Hagfishes (Myxiniformes: Myxinidae) are evolutionarily significant organisms that are phylogenetically unique (Fernholm 1998; Knapp et al. 2011; Fernholm et al. 2013). They are characterized by having naked and eel-like bodies, vestigial eyes, three paired sets of sensory barbels, two sets of laterally everting and biting-scraping keratinous cusps (or teeth) attached to a dental plate (in turn attached to the anterior end of the dental muscle), a series of slime pores located along the lower side of the body and by lacking paired fins, among other characters (Fernholm 1998; Nelson 2006). Although in their general organization these fishes follow a vertebrate pattern, they are in many aspects totally different. This is even more evident if other than morphological properties are considered (Fernholm 1998; Nelson 2006). Currently, about 81 species, 6 genera and 3 subfamilies (Myxininae, Eptatretinae and Rubicundinae) of hagfishes are recognized (Fernholm et al. 2013; Eschmeyer & Fong 2015). These fishes are widely distributed in all oceans except the Polar seas, occurring at depths ranging from 15 to 5000 m (Nelson 2006; Fernholm et al. 2013). Despite having a wide distribution, hagfish species richness is naturally low, with only one or few species known to be present along the majority of coastlines (Knapp et al. 2011). Principal factors determining their distributional patterns, which are

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marked by a high degree of endemism and vicariance (Cavalcanti & Gallo 2008), appear to be salinity, temperature, substrate preference and depth (see Martini 1998, for an overview). In the Pacific coasts of Mexico and Central America hagfishes are relatively rare (Mincarone & McCosker 2004). In fact, the extensive deepwater collections made by the United States Fish Commission Streamer Albatross in 1891 (Garman 1899) in this area resulted in but three hagfish specimens; which were captured over a rocky bottom at 1335 m in the southern end of the Gulf of Panama and described as Myxine circifrons by Garman (1899). Until recently, new material from this region has been collected and new species and new records also were described and reported (i.e., Wisner & McMillan 1990, 1995; Cruz-Mena & Angulo 2015). In contrast to the Albatross, the successful capture of hagfishes by the recent expeditions can be explained by the difficulty that the former expedition had in trawling over unusually rocky terrain, and by the undeniable benefits provided by manned submersibles in exploring and collecting in complex deepwater habitats (Mincarone & McCosker 2004; Fernholm et al. 2013). In the Gulf of Mexico and the Caribbean Sea hagfishes appear to be comparatively more abundant and diverse; however, this could be an effect of the sampling effort. Extensive deepwater fish collections in the area were made between 1950 and 1980 by the United States National Marine Fisheries research vessels Oregon I and II. Additionally, in the last years, several scientific expeditions (see Mok et al. 2001; Mincarone & Sampaio 2004; Polanco Fernandez & Fernholm 2014) also have been collected an important amount of material, principally in the Gulf of Mexico and the southern Caribbean Sea. As result of this considerable sampling effort several hagfish specimens were obtained and several new species were described (Fernholm & Hubbs 1981; Fernholm 1982; Mok et al. 2001; Mincarone & Sampaio 2004; Polanco Fernandez & Fernholm 2014). Because most hagfish species are relatively rare in scientific collections, usually known only from a few specimens, and have a relatively small and often imprecise literature, the information available on their biology is still very limited (Mincarone & McCosker 2004; Fernholm et al. 2013). Even the most basic aspects of the life histories, such as growth rate, age at sexual maturity, and longevity, remain unknown for several species. Compilations and overviews on the available information include those published by Brodal & Fänge (1963), Hardisty (1979) and more recently by Jørgensen et al. (1998).

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In order to provide a tool that improves the identification process at the regional scale, as basis for future applied works, this Catalogue includes a key to and accounts for all species of hagfishes know to be present in the Pacific and Atlantic coasts of Mexico and Central America, as well as contiguous waters (Galápagos Islands, West Indies, and the Colombian Pacific and Caribbean coasts). A comprehensive catalogue of hagfishes, like the one herein presented, can serve, in this sense, as an invaluable educational tool to the fisheries workers, to hagfish researches not specializing in taxonomy, and to the interested public at large as a guide to the taxonomic literature and to the numerous name changes and additions of species since the last comprehensive works were published.

Plan of the catalogue Most factual information in this review is derived from published sources, but especially for the Pacific coasts of Mexico and Costa Rica, some unpublished material has been used. In the following catalog, genera and species are arranged alphabetically. Species accounts include: scientific name with author(s), year of description and pagination, following Eschmeyer (2015); popular, technical and/or vernacular names in English (En) and Spanish (Sp), when available, following Page et al. (2013) and Froese & Pauly (2015); references to the original description, type locality, and holotype information; a brief description with distinctive characters; known distribution; documented records in Mexican and/or Central American waters (museum specimens); conservation status; major threats; literary references; and other pertinent information (“Remarks”). An asterisk before the species name indicates that there are no confirmed records of the species in Mexican or Central American waters. A “P” after the number of specimens by lot indicates that there are paratypes. Because most of body proportions are often strongly affected by preservation, counts have been emphasized in taxonomy. Both, measurements and counts (tables 5-1 and 5-2, respectively), follow Fernholm & Hubbs (1981) and Wisner & McMillan (1990). Length of the specimens is given as total length (TL). Morphological nomenclature and technical terms used were compiled from Fernholm & Hubbs (1981), McMillan & Wisner (1984), Wisner & McMillan (1995), and Mok (2001). Institutional abbreviations follow Sabaj Pérez (2014). The “Literature” section includes only those references arbitrarily considered by us to have taxonomic, behavioral, or distributional value and not mere usage of the specific names herein treated.

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Key to species of Myxinidae from Mexico and Central America 1a. Efferent branchial ducts open by a common external aperture on each side (i.e. only one pair of branchial apertures); caudal fin rays not bifurcated ..................................................................................................... 2 1b. Efferent branchial ducts open separately to the exterior with 5–14 external gill apertures; caudal fin rays bifurcated at tip ............................... 7 2a. Two fused cusps on both anterior and posterior multicusps (2/2); gill pouches 6, rarely 7 ....................................................................................... 3 2b. Three fused cusps on anterior and two on posterior multicusps (3/2); gill pouches 5............................................................................................... 5 3a. Body color light to dark purplish black, rarely with pale blotches, head usually pale anteriorly but generally of the same color of the body; unicusp in anterior row 5–9; unicusp in posterior row 5–8; total cusps 32– 42 (eastern Pacific) ................................................................ Myxine hubbsi 3b. Body color bluish gray to brown or reddish brown to dark purple, with the head white .............................................................................................. 4 4a. Unicusp in anterior row 8–10; unicusp in posterior row 8–10; total cusps 42–48 (northeastern Gulf of Mexico and Caribbean Sea); body color bluish gray to brown .............................................................. M. mcmillanae 4b. Unicusp in anterior row 4–8; unicusp in posterior row 5–8; total cusps 29–41 (north Atlantic Ocean); body color reddish brown to dark purple ...... ...................................................................................................M. glutinosa 5a. Single nasal sinus papillae (eastern Pacific) ....................... M. circifrons 5b. Paired nasal sinus papillae (southern Caribbean Sea)............................ 6 6a. Unicusp in anterior row 6–9; unicusp in posterior row 7–10; total cusps 36–48 ....................................................................................... M. mccoskeri 6b. Unicusp in anterior row 11–13; unicusp in posterior row 11–12; total cusps 56–58 ........................................................................... M. robinsorum 7a. Nostril tubular, elongated; body color reddish; gill pouches 5, total cusps about 36, and total slime pores about 88 (eastern Pacific, Galápagos Islands) ........................................................................ Rubicundus lakeside 7b. Nostril short; body color usually grey or brown (reddish brown in E. mcconnaugheyi and E. sinus); combination of characters not as described above ........................................................................................................... 8 8a. Gill pouches 5–8; three fused cusps on anterior and two on posterior multicusps (3/2) or two or three fused cusps on both anterior and posterior multicusps (3/3 or 2/2)................................................................................. 9 8b. Gill pouches 9–14; three fused cusps on anterior and two on posterior multicusps (3/2) ......................................................................................... 19

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9a. Body depth with finfold about 3.2% of TL; body depth without finfold about 2.4% of TL; total slime pores about 174 (southern Caribbean Sea) ........................................................................................... Eptatretus aceroi 9b. Body depth with finfold 4.2–11.6% of TL; body depth without finfold 4.5–11.0% of TL; total slime pores 73–93 ................................................ 10 10a. Gill pouches 5–6 ................................................................................ 11 10b. Gill pouches 7–8 ................................................................................ 17 11a. Three fused cusps on anterior and two on posterior multicusps (3/2) ................................................................................................................... 12 11b. Three fused cusps on both anterior and posterior multicusps (3/3) ................................................................................................................... 15 12a. Unicusp in anterior row about 13; unicusp in posterior row about 12; total cusps about 60; gill apertures nonlinear and crowded (southern Caribbean Sea).................................................................................E. ancon 12b. Unicusp in anterior row 9–11; unicusp in posterior row 8–9; total cusps about 43–52; gill apertures linear .................................................... 13 13a. Gill pouches 5; prebranchial slime pores about 24; branchial slime pores about 2; trunk slime pores 38–40; tail slime pores about 9; total slime pores 73–75 (southern Caribbean Sea) ................................. E. wayuu 13b. Gill pouches 5–6; prebranchial slime pores about 12–19; branchial slime pores 3–6; trunk slime pores 44–57; tail slime pores 11–15; total slime pores 76–92 ...................................................................................... 14 14a. Total cusps about 44; total slime pores 76–77 (eastern Pacific, Galápagos Islands)....................................................................... E. grouseri 14b. Total cusps 48–52; total slime pores 77–92 (Gulf of Mexico) .................................................................................................... E. springeri 15a. Trunk slime pores 52–55; tail slime pores 17–20; total slime pores 88–93 (Caribbean Sea) ..............................................................E. multidens 15b. Trunk slime pores 41–48; tail slime pores 11–15; total slime pores 74–82 ......................................................................................................... 16 16a. Unicusp in anterior row 11–13; unicusp in posterior row 10–12; total cusps 56–61; body color bluish gray (Caribbean Sea)............... E. mendozai 16b. Unicusp in anterior row 8–11; unicusp in posterior row 8–10; total cusps 46–54; body color gray or brown pale, with a thin, light middorsal stripe (Gulf of Mexico) .................................................................... E. minor 17a. Gill pouches 7; total cusps 54–58; total slime pores 79–85 (Caribbean Sea) .......................................................................................... E. caribbeaus 17b. Gill pouches 8; total cusps 44–51; total slime pores 72–76 (eastern Pacific)....................................................................................................... 18 18a. Three fused cusps on anterior and two on posterior multicusps (3/2); total cusps about 44; total slime pores about 76; body color brownish

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black, head slightly lighter, face nearly all white, including area around mouth and base of labial barbels ............................................. E. bobwisneri 18b. Three fused cusps on both anterior and posterior multicusps (3/3); total cusps 48–51; total slime pores 72–74; body color brownish black with head region slightly lighter than body color, face dark except for small white area around the mouth ........................................... E. mccoskeri 19a. Prebranchial length usually less than branchial length, very rarely equal to or even slightly greater; tail length always less than branchial length (eastern north Pacific) ........................................... E. mcconnaugheyi 19b. Prebranchial and tail lengths always greater than branchial length ... 20 20a. Prebranchial slime pores 4–10 (eastern north Pacific) .............. E. deani 20b. Prebranchial slime pores 10–17 ......................................................... 21 21a. All barbels large, robust; third barbel 42–59% of preocular length (eastern north Pacific, Guadalupe Island) .......................................... E. fritzi 21b. All barbels small, not robust; third barbel 31–37% of preocular length ................................................................................................................... 22 22a. Gill apertures usually 12 (10–14); ventral finfold prominent, with wide pale margin (eastern Pacific) ..................................................E. stoutii 22b. Gill apertures usually 10 (9–12); ventral finfold vestigial or absent (eastern north Pacific, Gulf of California) ........................................ E. sinus

Species account 1) *Eptatretus aceroi Polanco Fernandez & Fernholm, 2014. Eptatretus aceroi Polanco Fernandez & Fernholm, 2014: 530 (Original description; type locality: 10°44'04.1"–10°43'26.0"N, 75°37'15.2"– 75°37°46.0"W, off Cartagena, Colombia, Caribbean Sea, depth 705 m; holotype: INVE PEC8257, 584 mm TL, sex indeterminate). Distinctive characters: Fused cusps 2–3/2; total cusps 58; total slime pores 174; one single dorsal nasal sinus papillae; gill apertures linear; last gill apertures and pharyngocutaneous duct separated; eyespots absent; ventral fin fold and caudal fin moderately developed; body color dark brown in fresh specimens, light to medium brown in preserved specimens, head whitish. Distribution: Off Cartagena, Colombia, Caribbean Sea; occurring at depths of about 705 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Not assessed (IUCN 2014). Major threats: None known (IUCN 2014). Literature: Polanco Fernandez & Fernholm (2014: 530–533). Remarks: Known only from the holotype (Polanco Fernandez & Fernholm 2014; Eschmeyer 2015).

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2) *Eptatretus ancon (Mok, Saavedra-Diaz & Acero P., 2001). Quadratus ancon Mok, Saavedra-Diaz & Acero P., 2001: 1031 (Original description; type locality: 11°29'31.8"–11°29'47.4"N, 73°26'44.4"– 73°27'6.6"W, off La Punta de los Remedios, Colombia, Caribbean Sea, depth 470–488 m; holotype: INV PEC2412, 220.3 mm TL, sex indeterminate). Distinctive characters: Gill pouches 6; fused cusps 3/2; total cusps 60; total slime pores 80; afferent branchial arteries at the median ventral aorta 3; ventral aorta bifurcates at gill pouches 4–5; gill apertures nonlinear and crowded; last gill apertures and pharyngocutaneous duct separated; eyespots present; ventral fin fold low, caudal fin fold present but not well developed; body color light brown. Distribution: North coast of Colombia, Caribbean Sea; occurring at depths between 476 and 705 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Data deficient (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Mok et al. (2001: 1026–1033), Møller & Jones (2007: 63, 65), Knapp et al. (2011: 405). Remarks: Known only from the holotype and five additional specimens deposited at the Instituto de Investigaciones Marinas y Costeras – INVEMAR, Colombia (INV PEC8264 and INV PEC8265; data from INVEMAR 2015). 3) *Eptatretus bobwisneri Fernholm, Norén, Kullander, Quattrini, Zintzen, Roberts, Mok & Kuo, 2013; Wisner’s eight-gilled hagfish (En). Eptatretus bobwisneri Fernholm, Norén, Kullander, Quattrini, Zintzen, Roberts, Mok & Kuo, 2013: 6 (Replacement for Eptatretus wisneri McMillan, 1999 (see Remarks); type locality: 0°28.0'S, 91°37.2'W, Galápagos Islands, Ecuador, eastern Pacific, depth 563 m; holotype: CAS 86429, 563 mm TL, female). Distinctive characters: Gill pouches 8; fused cusps 3/2; total cusps 44; total slime pores 76; gill pouches at end of the dental muscle 3; ventral aorta bifurcates at gill pouch 5; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots distinct, large and irregularly shaped; ventral fin fold vestigial or absent, caudal fin fold well developed; body color brownish black, head slightly lighter, face nearly all white, including area around mouth and base of labial barbels. Distribution: Galápagos Islands, Ecuador, eastern Pacific; occurring at depths between 512 and 563 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Not assessed (IUCN 2014). Major threats: None known (IUCN 2014). Literature: McMillan (1999: 110–117), Mok et al. (2001: 1026), Mincarone & McCosker (2004: 164), Møller & Jones

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(2007: 64, 66), Cavalcanti & Gallo (2008: 1261, 1264, 1266), Mincarone & Fernholm (2010: 798), McCosker & Rosenblatt (2010: 187), Fernholm et al. (2013: 296, 302), Mincarone & McCosker (2014: 349). Remarks: (1) Kuo et al. (1994) describe Paramyxine wisneri from six specimens collected from off Fukan, Taiwan, western Pacific, while McMillan (1999) describes E. wisneri from two specimens collected from the Galápagos Islands, Ecuador, eastern Pacific. Fernholm et al. (2013) synonymized the genera Paramyxine and Quadratus with Eptatretus, creating a homonymy between both species. In order to resolve this nomenclatural dilemma Fernholm et al. (2013) renamed E. wisneri McMillan, 1999 as E. bobwisneri maintaining its afliation to Robert L. Wisner (1921–2005). (2) Known only from the holotype and the paratype (SIO 97-76, ex CAS 86430, 328 mm TL, male, collected on 16 November 1995, 00°17.5'S, 91°38.9'W, depth 512 m) (McMillan 1999; Eschmeyer 2015). 4) Eptatretus caribbeaus Fernholm, 1982; Caribbean hagfish (En); Bruja caribeña (Sp). Eptatretus caribbeaus Fernholm, 1982: 435 (original description; type locality: 16°55'N, 81°12'W, Oregon station 1886, Caribbean Sea, depth 500 m; holotype: MCZ 40409, 331 mm TL, sexually immature female). Distinctive characters: Gill pouches 7; fused cusps 3/3; total cusps 54–58; total slime pores 79–85; dorsal nasal sinus papillae absent; gill apertures linear; eyespots weak; ventral fin fold low, caudal fin fold absent; body color light tan. Distribution: Caribbean Sea; occurring at depths between 365 and 500 m. Documented records in Mexican and/or Central American waters (Museum specimens): 5 specimens. Holotype; CNPE-IBUNAM 9826 (1), 305 mm TL, Gulf of Campeche, Mexico; UF 27894 (1 P), 345 mm TL, 14°08'N, 81°55'W; UF 27895 (1 P), 364 mm TL, 16°50'N, 81°21'W; USNM 218405 (1 P), 364 mm TL. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: Fernholm (1982: 434–438), Hensley (1985: 866), Fernholm (1991: 117), Fernholm (1998: 34), Mincarone (2000: 815, 819), Mok (2001: 355–363), Mok et al. (2001: 1026), Fernholm (2003: 355), Mok & McMillan (2004: 740, 743), Mincarone & Stewart (2006: 227), Møller & Jones (2007: 63, 65), Fernholm & Mincarone (2010: 1002), Knapp et al. (2011: 404). 5) Eptatretus deani (Evermann & Goldsborough, 1907); Black hagfish (En); Bruja pecosa (Sp). Polistotrema deani Evermann & Goldsborough, 1907: 225 (original description; type locality: 55°48'22''N, 131°42'18''W, Albatross station 4238, Near Yes Bay, Behm Canal, off Nose Point, Alaska, United States of America (USA), northeastern Pacific, depth 419–

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422 m; holotype: USNM 57820, poor condition). Distinctive characters: Gill pouches 10–12; fused cusps 3/2; total cusps 37–46; total slime pores 67–80; dorsal nasal sinus papillae absent; gill pouches at end of the dental muscle 5; afferent branchial arteries at median ventral aorta 5; ventral aorta bifurcates at gill pouch 6; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots present; ventral fin fold weakly developed, occasionally absent, caudal fin fold vestigial or absent; body color usually black or purplish blue, rarely pinkish. Distribution: Northern and eastern Pacific, from southeastern Alaska, USA, to central Baja California, Mexico; occurring at depths between 107 and 2743 m. Documented records in Mexican and/or Central American waters (Museum specimens): 358 specimens. SIO 63-23 (4), 390–490 mm TL, 28°54.1'N, 118°12.7'W; SIO 66-36 (127), 270–500 mm TL, 29°30.0'N, 117°17.0'W; SIO 66-37 (55), 290–480 mm TL, 29°29.3'N, 117°15.2'W; SIO 63-177 (23), 250–400 mm TL, 28°51.7'N, 118°13.1'W; SIO 66-535 (21), 31°59.5'N, 117°6.1'W; SIO 67-60 (2), 320–360 mm TL, 28°9.0'N, 118°16.2'W; SIO 67-64 (20), 380–510 mm TL, 29°26.5'N, 117°15.6'W; SIO 70-2 (61), 31°56.6'N, 118°22.1'W; SIO 70-3 (1), 31°55.5'N, 118°20.1'W; SIO 70-11 (1), 31°6.3'N, 118°54.5'W; SIO 70-16 (3), 31°18.6'N, 118°44.9'W; SIO 71-115 (29), 28°21.8'N, 115°44.3'W; SIO 71127 (11) specimens, 30°16.6'N, 116°9.7'W. Conservation status: Data deficient (Knapp et al. 2011). Major threats: Target of fisheries (Knapp et al. 2011). Literature: Evermann & Goldsborough (1907: 225–226), Böhlke (1953: 8), Miller & Lea (1972: 32), Smith & Hessler (1974: 72– 73), Jespersen (1975: 189–198), Theisen (1976: 167–173), Fernholm (1981: 137–145), Eschmeyer & Herald (1983: 10), Wisner & McMillan (1990: 793–795), McAllister (1990: 25), Fernholm (1998: 34), Mok (2001: 355–363), Mecklenburg et al. (2002: 55), Mok & McMillan (2004: 737, 740, 743, 745), Love et al. (2005: 1), Møller & Jones (2007: 63, 65), Cavalcanti & Gallo (2008:1259, 1260, 1264, 1266), Knapp et al. (2011: 404), Reyes-Bonilla et al. (2011: 199). 6) Eptatretus fritzi Wisner & McMillan, 1990; Guadalupe hagfish (En); Bruja de Guadalupe (Sp). Eptatretus fritzi Wisner & McMillan, 1990: 791 (original description; type locality: 28°51'N, 118°14'W, vicinity of Guadalupe Island, Mexico, eastern Pacific, depth 512 meters; holotype: SIO 66-26, 550 mm TL, male). Distinctive characters: Gill pouches 10– 12; fused cusps 3/2; total cusps 38–46; total slime pores 74–85; dorsal nasal sinus papillae absent; gill pouches at end of the dental muscle 5; afferent branchial arteries at median ventral aorta 3; ventral aorta bifurcates at gill pouch 9; gill apertures linear; last gill apertures and

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pharyngocutaneous duct fused; eyespots prominent the margins irregular; ventral fin fold usually absent, caudal fin fold variably thin to thick; body color dark purple to blackish in most specimens, dark brown in others. Distribution: Known only from the immediate vicinity of Guadalupe Island, Mexico, northeastern Pacific; occurring at depths between 182 and 2743 m. Documented records in Mexican and/or Central American waters (Museum specimens): 451 specimens. Holotype; CAS 63201 (15), 29°52'N, 118°14'W; LACM 44407-1 (15 P), 375–495 mm TL, 28°51'N, 118°14'W; SIO 63-177 (20 P), 324–522 mm TL, 28°52'N, 118°14'W; SIO 66-22 (36 P), 232–521 mm TL, 29°06'N, 118°17'W; SIO 66-23 (195 P), 286–556 mm TL, 28°54'N, 118°13'W; SIO 66-36 (2 P), 354–405 mm TL, 29°30'N, 117°17'W; SIO 67-60 (120 P), 281–498 mm TL, 29°09'N, 118°16'W; SIO 68-424 (13), 250-430 mm TL, 29°10.3'N, 118°16.2'W; SIO 68-664 (2 P), 425–535 mm TL, 29°6.3'N, 118°23.3'W; SIO 70-43 (13), 29°9.6'N, 118°16'W; SIO 72-294 (4 P), 207–541 mm TL, 29°10'N, 118°16'W; USNM 296318 (15 P), 350–570 mm TL, 29°19'N, 112°50'W. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: Wisner & McMillan (1990: 787–804), Fernholm (1998: 34), Mok (2001: 355–363), Mok et al. (2001: 1026), Love et al. (2005: 1), Møller & Jones (2007: 63, 65), Cavalcanti & Gallo (2008: 1260, 1266), Reyes-Bonilla et al. (2011: 199). 7) *Eptatretus grouseri McMillan, 1999. Eptatretus grouseri McMillan, 1999: 114, (original description; type locality: 0°14.6'S, 91°26.6'W, Galápagos Islands, Ecuador, eastern Pacific, depth 722 m; holotype: CAS 86428, 378 mm TL, female). Distinctive characters: Gill pouches 5–6; fused cusps 3/2; total cusps 44–48; total slime pores 71–79; gill pouches at end of the dental muscle 1–2; ventral aorta bifurcates at gill pouch 5; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots prominent, nearly round; ventral fin fold vestigial, caudal fin fold well developed; body color brownish black, head region dark brown. Distribution: Galápagos Islands, Ecuador, eastern Pacific; occurring at depths between 648 and 722 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: McMillan (1999: 110–117), Mok & McMillan (2004: 740, 743), Møller & Jones (2007: 63, 65), Cavalcanti & Gallo (2008: 1260, 1266), Knapp et al. (2011: 404). Remarks: Known only from the holotype, the paratype (SIO 97-77, ex CAS 864280, 142 mm TL, male, taken with the holotype) and two

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additional specimens deposited at CAS (CAS 201882, collected on 17 November 1995, 00°21.7'S, 95°15'W) (McMillan 1999; Eschmeyer 2015). 8) Eptatretus mcconnaugheyi Wisner & McMillan, 1990; Shorthead hagfish (En); Bruja de cabeza chica (Sp). Eptatretus mcconnaugheyi Wisner & McMillan, 1990: 790 (original description; type locality: 32°32'00"N, 117°21'07"W, off southern California, USA, northeastern Pacific, depth 148 m; holotype: SIO 69-231E, 482 mm TL, female). Distinctive characters: Gill pouches 12–14; fused cusps 3/2; total cusps 38–45; total slime pores 67–84; dorsal nasal sinus papillae absent; gill pouches at end of the dental muscle 9; afferent branchial arteries at median ventral aorta 1–3; ventral aorta bifurcates at gill pouch 11; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots rather prominent; ventral fin fold usually prominent, often low, caudal fin fold prominent, wide; body color uniformly dark, reddish brown in life. Distribution: Southwestern coast of the USA and Mexico, northeastern Pacific; occurring at depths between 43 and 689 m. Documented records in Mexican and/or Central American waters (Museum specimens): 74 specimens. CICIMAR-CI 4814 (1), 25°34'N, 113°9'W; CICIMAR-CI 4817 (1), 25°34'N, 113°9'W; SIO 68-109 (1), 390 mm TL, 25°48.4'N, 110°44.8'W; SIO 68-110 (6), 270–350 mm TL, 25°47.8'N, 110°45.2'W; SIO 68-111 (1), 230 mm TL, 25°47.2'N, 110°46.3'W; SIO 68-124 (1), 249 mm TL, 25°48.3'N, 110°49.7'W; SIO 68-125 (19), 239–247 mm TL, 25°48.7'N, 110°50.1'W; SIO 68-126 (5), 355–409 mm TL, 25°49.1'N, 110°50.6'W; SIO 68-127 (3), 315–440 mm TL, 25°49.6'N, 110°51.1'W; SIO 69-225C (1), 392 mm TL, 31°48.3'N, 116°50.2'W; SIO 69-225D (6), 175–300 mm TL, 31°47.6'N, 116°50.4'W; SIO 69-228B (14 P), 185–400 mm TL, 32°5.9'N, 117°4.4'W; SIO 69-228C (1), 443 mm TL, 32°6.0'N, 117°5.4'W; SIO 71-114 (7 P), 360–440 mm TL, 28°21.0'N, 115°43.0'W; USNM 296320 (7), 29°54.5'N, 113°1.02'W. Conservation status: Data deficient (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Wisner & McMillan (1990: 787–804), Fernholm (1998: 34), Mok (2001: 355–363), Mok & McMillan (2004: 737, 740, 742–745), Love et al. (2005: 1), Møller & Jones (2007: 63, 65), Cavalcanti & Gallo (2008: 1260, 1264, 1266), Knapp et al. (2011: 404), Del Moral-Flores et al. (2013: 187). Remarks: This species appears to consist of two disjunctive populations, one from Santa Monica Bay, California, USA, to the Cedros and San Benito Islands, Mexico, and the other one apparently restricted to the lower portion of the Gulf of California, Mexico (Wisner & McMillan 1990). These two populations differ in the number of trunk and total slime pores, with the

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higher counts occurring in the southern population (Wisner & McMillan 1990). Collecting efforts between the Cedros and San Benito Islands and the mouth of the Gulf of California have failed to take this species (Wisner & McMillan 1990). 9) *Eptatretus mccoskeri McMillan, 1999. Eptatretus mccoskeri McMillan, 1999: 115 (original description; type locality: 1°06.3'S, 89°06.9'W, Galápagos Islands, Ecuador, eastern Pacific, depth 215 m; holotype: CAS 86431, 320 mm TL, male). Distinctive characters: Gill pouches 8; fused cusps 3/3; total cusps 48–51; total slime pores 72–74; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 4–6; afferent branchial arteries at median ventral aorta 2; ventral aorta bifurcates at gill pouch 6–7; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots visible but not prominent; ventral fin fold absent or vestigial, caudal fin fold present; body color brownish black with head region slightly lighter than body color, face dark except for small white area around the mouth. Distribution: Galápagos Islands, Ecuador, eastern Pacific; occurring at depths about 215 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: McMillan (1999: 110–117), Mok (2001: 355–363), Mincarone & McCosker (2004: 164), Mok & McMillan (2004: 740, 741), Møller & Jones (2007: 64, 65), Cavalcanti & Gallo (2008: 1259–1261, 1264–1266), McCosker & Rosenblatt (2010: 187), Mincarone & Fernholm (2010: 797), Knapp et al. (2011: 404), Mincarone & McCosker (2014: 348). Remarks: Known only from the holotype, the paratypes (SIO 97-75 (2), 283–300 mm TL, males, and USNM 344905 (1), 298 mm SL, taken with the holotype) and one additional specimen deposited at CAS (CAS 86431, 1º6'18.9"S, 89º6'56.34"W). 10) *Eptatretus mendozai Hensley, 1985. Eptatretus mendozai Hensley, 1985: 866 (original description; type locality: 17°50.4'N, 66°59.8'W, southwestern coast of Puerto Rico, Caribbean Sea, depth 1100 m; holotype: USNM 268923, 450 mm TL, female). Distinctive characters: Gill pouches 6; fused cusps 3/3; total cusps 56–61; total slime pores 77–82; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 2–4; afferent branchial arteries at median ventral aorta 0; ventral aorta bifurcates at gill pouch 5–6; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots present; body color bluish gray. Distribution: Puerto Rico, Dominican Republic and Haiti,

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Caribbean Sea; occurring at depths between 720 and 1100 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: Hensley (1985: 865–869), Fernholm (1998: 34), McMillan (1999: 115), Mincarone (2000: 816), Mok (2001: 355–363), Fernholm (2003: 355), Mok & McMillan (2004: 743), Møller & Jones (2007: 64, 65), Cavalcanti & Gallo (2008; 1260, 1262), Mincarone & Fernholm (2010: 797), Knapp et al. (2011: 404). 11) Eptatretus minor Fernholm & Hubbs, 1981. Eptatretus minor Fernholm & Hubbs 1981: 78 (original description; type locality: 24°34'N, 83°34'W, Gulf of Mexico, Oregon station 1009, Atlantic Ocean, depth 370 m; holotype: USNM 164119, 359 mm TL, female). Distinctive characters: Gill pouches 5–6; fused cusps 3/3; total cusps 46–54; total slime pores 74–82; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 3–4; afferent branchial arteries at median ventral aorta 0–1; ventral aorta bifurcates at gill pouch 5–6; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots inconspicuous; ventral fin fold inconspicuous, caudal fin fold inconspicuous; body color gray or brown pale, with a thin, light middorsal stripe. Distribution: Gulf of Mexico, Atlantic Ocean; occurring at depths between 300 and 400 m. Documented records in Mexican and/or Central American waters (Museum specimens): 6 specimens. CNPEIBUNAM 2615 (6), 248–277 mm TL, 24°05.4'N, 88°16.4'W. Conservation status: Data deficient (Knapp et al. 2011). Major threats: Bycatch (Knapp et al. 2011). Literature: Fernholm & Hubbs (1981: 69– 83), Fernholm (1991: 117), Fernholm (1998: 34), McEachran & Fechhelm (1998: 32), McMillan (1999: 115), Mincarone (2000: 816), Mok (2001: 355–363), Fernholm (2003: 355), Mok & McMillan (2004: 740, 743, 744, 746), Møller & Jones (2007: 64, 66), Cavalcanti & Gallo (2008; 1259, 1260), McEachran (2009: 1256), Mincarone & Fernholm (2010: 797), Knapp et al. (2011: 404). 12) *Eptatretus multidens Fernholm & Hubbs, 1981. Eptatretus multidens Fernholm & Hubbs 1981: 80 (original description; type locality: 12°52'N, 70°43'W, Caribbean Sea, Oregon II station 11299, Atlantic Ocean, depth 510 m; holotype: USNM 218401, 600 mm TL, male). Distinctive characters: Gill pouches 6; fused cusps 3/3; total cusps 52– 57; total slime pores 87–91; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 2–3; afferent branchial arteries at median

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ventral aorta 0; ventral aorta bifurcates at gill pouch 6; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; on paler specimens the eyespots can barely be discerned, while on the darker the light skin overlying the eyes is clearly visible; body color pale brown to medium brown, without a middorsal light stripe. Distribution: Caribbean Sea and off northeastern Brazil, Atlantic Ocean; occurring at depths between 239 and 770 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats: Bycatch (Knapp et al. 2011). Literature: Fernholm & Hubbs (1981: 69– 83), Fernholm (1991: 117), Fernholm (1998: 34), McMillan (1999: 115), Mincarone (2000: 816), Mok (2001: 355–363), Fernholm (2003: 355), Mincarone & Sampaio (2004: 33), Mok & McMillan (2004: 740, 744, 747), Møller & Jones (2007: 64, 66), Cavalcanti & Gallo (2008; 1260, 1264), Mincarone & Fernholm (2010: 797), Knapp et al. (2011: 404). 13) Eptatretus sinus Wisner & McMillan, 1990; Cortez hagfish (En); Bruja de Cortés (Sp). Eptatretus sinus Wisner & McMillan, 1990: 792 (original description; type locality: 25°49'N, 110°44'W, Gulf of California, Mexico, eastern Pacific, depth 708 m; holotype: SIO 68-108, 307 mm TL, female). Distinctive characters: Gill pouches 9–12; fused cusps 3/2; total cusps 34–46; total slime pores 66–82; dorsal nasal sinus papillae absent; gill pouches at end of the dental muscle 2; afferent branchial arteries at median ventral aorta 6; ventral aorta bifurcates at gill pouch 5–6; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots prominent, with the margins well defined; ventral fin fold low, occasionally absent, caudal fin fold thickened ventrally, thinner around tail; body color reddish brown, without pale markings or spotting. Distribution: Gulf of California, Mexico, eastern Pacific; occurring at depths between 198 to 1330 m. Documented records in Mexican and/or Central American waters (Museum specimens): 426 specimens. Holotype; CAS 63202 (15 P), 270–400 mm TL, 25°49'N, 110°44'W; LACM 44408-1 (14 P), 270–400 mm TL, 25°49'N, 110°44'W; SIO 62-240 (3), 270–380 mm TL, 28°59'N, 113°28.7'W; SIO 68-94 (29 P), 129–346 mm TL, 29°20'N, 113°10'W; SIO 68-97 (7), 223–308 mm TL, 29°06'N, 113°17.3'W; SIO 68-98 (17), 276–338 mm TL, 29°00'N, 113°21.5'W; SIO 68-99 (11), 263–420 mm TL, 29°00'N, 113°23'W; SIO 68-100 (60 P), 267–464 mm TL, 29°20'N, 113°25'W; SIO 68-101 (16), 264–355 mm TL, 29°00'N, 113°27.5'W; SIO 68-108 (31 P), 230–425 mm TL, taken with the holotype; SIO 69-201 (1), 190 mm TL, 28°40'N, 112°57'W; SIO 69-202 (84), 202–464 mm TL, 29°41.5'N, 112°58.5'W; SIO 69-203 (11), 381–630

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mm TL, 28°40'N, 113°53'W; SIO 69-206 (54), 363–430 mm TL, 28°28.4'N, 112°40.5'W; SIO 69-207 (57), 259–404 mm TL, 28°29.5'N, 112°37'W; USNM 296319 (15 P), 275–420 mm TL, 25°49'N, 110°44'W. Conservation status: Least concern (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: Fernholm (1998: 34), Fernholm (1998: 34), Mok (2001: 355–363), Mok & McMillan (2004: 737–747), Møller & Jones (2007: 64, 66), Cavalcanti & Gallo (2008; 1260, 1266), Knapp et al. (2011: 404), Palacios-Salgado et al. (2012: 156), Del Moral-Flores et al. (2013: 187). 14) Eptatretus springeri (Bigelow & Schroeder, 1952); Gulf hagfish (En); Bruja del Golfo (Sp). Paramyxine springeri Bigelow & Schroeder 1952: 5 (original description; type locality: 27°44'N, 85°09'W, Gulf of México, Oregon station 489, Atlantic Ocean, depth 465 m; holotype: USNM 161512, 590 mm TL, sex indeterminate). Distinctive characters: Gill pouches 6; fused cusps 3/2; total cusps 48–52; total slime pores 84– 92; gill pouches at end of the dental muscle 1–3; afferent branchial arteries at median ventral aorta 1; ventral aorta bifurcates at gill pouch 4–5; gill apertures linear; caudal fin fold well developed; body color greyish brown. Distribution: Gulf of Mexico, Atlantic Ocean; occurring at depths between 400 and 730 m. Documented records in Mexican and/or Central American waters (Museum specimens): 11 specimens. CNPEIBUNAM 17984 (1), 535 mm TL, 23°37'61''–23°38'62''N, 97° 14'50''– 97°13'57''W; CNPE-IBUNAM 20046 (9), 370–530 mm TL, Gulf of Mexico (Mexico); MCZ 40218 (1), 415 mm TL, 16°41'N, 82°40'W (Honduras). Conservation status: Least concern (Knapp et al. 2011). Major threats: Bycatch (Knapp et al. 2011). Literature: Bigelow & Schroeder (1952: 1–10), Fernholm & Hubbs (1981: 74), Hensley (1985: 866), Fernholm (1998: 34), McEachran & Fechhelm (1998: 33), Mok (1999: 61), Wisner (1999: 312), Mincarone (2000: 815), Mok et al. (2001: 1026), Fernholm (2003: 355), Mok & McMillan (2004: 737–747), Møller & Jones (2007: 64, 66), Cavalcanti & Gallo (2008; 1259, 1260, 1265), Knapp et al. (2011: 405). 15) Eptatretus stoutii (Lockington, 1878); Pacific hagfish (En); Bruja del Pacífico (Sp). Bdellostoma stoutii Lockington, 1878: 793 (original description; type locality: no coordinates were provided, Humboldt Bay, California, USA, eastern Pacific; holotype: none known; the author did not designate his specimen as a holotype or state that it was deposited anywhere); Wisner & McMillan, 1990: 796 (type designation; type locality: no coordinates were provided, two miles southwest of whistler

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buoy at entrance to Humboldt Bay, California, USA, northeastern Pacific, depth 38–44 m; Neotype: SIO 68-426, 530 mm TL, male). Distinctive characters: Gill pouches 10–14; fused cusps 3/2; total cusps 36–46; total slime pores 71–88; dorsal nasal sinus papillae absent; gill pouches at end of the dental muscle 3; afferent branchial arteries at median ventral aorta 4; ventral aorta bifurcates at gill pouch 7; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots small prominent; ventral fin fold prominent, the distal margin pale; body color light brown with pale spotting and small blotches. Distribution: Nootka Bay, west side of Vancouver Island, British Columbia, Canada, to off Cabo Blanco, Puntarenas, Costa Rica, northeastern and eastern Pacific; occurring at depths between 16 and 966 m. Documented records in Mexican and/or Central American waters (Museum specimens): 716 specimens. CICIMAR-CI 4815 (1), 25°34'N, 113°9'W; CICIMAR-CI 4816 (1), 25°34'N, 113°9'W; CICIMAR-CI 4818 (1), 25°34'N, 113°9'W; CICIMAR-CI 4819 (1), 25°34'N, 113°9'W; CICIMAR-CI 4820 (1), 25°38'N, 113°24'W; CICIMAR-CI 4821 (1), 25°38'N, 113°24'W; CICIMAR-CI 4822 (2), 25°38'N, 113°24'W; CICIMAR-CI 4823 (1), 25°38'N, 113°24'W; CICIMAR-CI 4824 (1), 25°38'N, 113°24'W; CICIMAR-CI 4825 (1), 25°38'N, 113°24'W; CICIMAR-CI 4845 (3), 25°38'N, 113°24'W; CICIMAR-CI 4866 (1), 25°12'N, 112°51'W; LACM 32044.001 (1), 28°17.45'N, 115°09.15'W; LACM 37341.001 (9), 27°0'N, 114°0'W; NRM 39654 (9), 32°55'N, 117°0'W; SIO 62-521 (3), 168–188 mm TL, 30°13.6'N, 116°2.5'W; SIO 62-565 (29), 265–370 mm TL, 28°19'N, 115°35'W; SIO 62-91 (3), 168–209 mm TL, 28°9.8'N, 115°24.4'W; SIO 64-951 (1), 31°7.6'N, 116°35.0'W; SIO 64-953 (3), 31°8.2'N, 116°34.6'W; SIO 66-23 (2), 320–340 mm TL, 28°54.1'N, 118°12.7'W; SIO 68-408 (30), 28°19'N, 115°35.0'W; SIO 69-207 (56), 270–410 mm TL, 28°29.5'N, 112°37.0'W; SIO 69-225A (20), 31°50'N, 116°50.9'W; SIO 69-225C (10), 31°48.3'N, 116°50.2'W; SIO 69-225D (1), 31°47.6'N, 116°50.4'W; SIO 69-225E (1), 117 mm TL, 31°47.2'N, 116°50.4'W; SIO 69-228A (205), 32°5.6'N, 117°3.8'W; SIO 69-228B (6), 32°5.9'N, 117°4.4'W; SIO 69-231A (6), 32°31.3'N, 117°19.2'W; SIO 69277D (115), 31°29.2'N, 116°45.5'W; SIO 69-277E (22), 31°29'N, 116°44.9'W; SIO 69-277F (10), 31°29.2'N, 116°43.8'W; SIO 71-114 (28), 290–450 mm TL, 28°21'N, 115°43'W; SIO 71-126 (83), 195–460 mm TL, 30°22.2'N, 116°7.4'W; SIO 71-127 (40), 250–405 mm TL, 30°16.6'N, 116°9.7'W; SIO 71-164 (1), 400 mm TL, 27°12.9'N, 114°13.5'W; SIO 73373 (1), 320 mm TL, 28°49.7'N, 114°48.2'W; UCR 2705-007 (1), 351 mm TL, 9º29.83’N, 85º13.46’W; UF 234137 (5), 32°53.6'N, 117°17.1'W. Conservation status: Data deficient (Knapp et al. 2011). Major threats:

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Target of fisheries (Knapp et al. 2011). Literature: Miller & Lea (1972: 32), Jespersen (1975: 189–198), Knaggs et al. (1975: 56), Theisen (1976: 167–173), Wisner & McMillan (1990: 795–798), Fernholm (1998: 34), Mok (2001: 355–363), Mok & McMillan (2004: 737–747), Love et al. (2005: 1), Møller & Jones (2007: 64, 66), Cavalcanti & Gallo (2008; 1259, 1260, 1264, 1266), Rodríguez-Romero et al. (2008: 1768), Flores-Olivares et al. (2009: 56), Knapp et al. (2011: 404), Reyes-Bonilla et al. (2011: 199), Cruz-Mena & Angulo (2015). 16) *Eptatretus wayuu Mok, Saavedra-Diaz & Acero P., 2001. Eptatretus wayuu Mok, Saavedra-Diaz & Acero P., 2001: 1026 (original description; type locality: 12°24'N, 72°15'W, off the Guajira Peninsula, Colombia, Caribbean Sea, depth 300–306 m; holotype: INV PEC2410, 216 mm TL, sex indeterminate). Distinctive characters: Gill pouches 5; fused cusps 3/2; total cusps 41–43; total slime pores 73–75; gill pouches at end of the dental muscle 0; afferent branchial arteries at median ventral aorta 3–5; ventral aorta bifurcates at gill pouch 1–2; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots faint; ventral fin fold and caudal fin fold well developed; body color pink when fresh, dark violet in alcohol. Distribution: Off the Guajira Peninsula near Puerto Bolivar, Colombia, Caribbean Sea; occurring at depths between 300 and 306 m (Mok et al. 2001, Knapp et al. 2011, Eschmeyer 2015). Known only from the holotype and the paratype (INV PEC2411, 194 mm TL, female with eggs). Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Data deficient (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Mok et al. (2001: 1026–1033), Møller & Jones (2007: 64, 66), Fernholm & Quattrini (2008: 126), Cavalcanti & Gallo (2008; 1263, 1264), Knapp et al. (2011: 405), Polanco Fernandez & Fernholm (2014: 531). Remarks: Known only from the holotype and the paratype (INV PEC2411, 194 mm TL, female, taken with the holotype) (Mok et al. 2001; Eschmeyer 2015). 17) Myxine circifrons Garman, 1899; Pacific whitehead hagfish (En); Whiteface hagfish (En); Bruja de cara blanca (Sp). Myxine circifrons Garman, 1899: 344 (original description; type locality: 7°30'36"N, 78°39'W, Gulf of Panama, eastern Pacific, depth 1335 m; Syntypes: MCZ 28419 (2) and MCZ 91374 (1)). Distinctive characters: Gill pouches 5; fused cusps 3/2; total cusps 43–56; total slime pores 80–102; one single dorsal nasal sinus papillae; gill pouches at end of the dental muscle 0; ventral aorta bifurcates at gill pouch 2; branchial ducts on the left side

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confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right side; ventral fin fold usually well developed, caudal fin fold present, rounded; body color grayish black to reddish brown, anterior portion paler often whitish. Distribution: Eastern Pacific, from southern California, USA, to Chile; occurring at depths between 637 and 1860 m. Documented records in Mexican and/or Central American waters (Museum specimens): 244 specimens. Syntypes (3); CNPE-IBUNAM s/c (1), 419 mm TL, ¼ mi to W Aduana, Sonora (Gulf of California); SIO 0583 (1), 437 mm TL, 25°40.8'N, 109°54.4'W; SIO 59-52 (3), 197–435 mm TL, 32°11.4'N, 117°13.5'W; SIO 62-694 (10), 355–527 mm TL, 32°25.0'N, 117°26.5'W; SIO 63-19 (1), 326 mm TL, 32°25.2'N, 117°27.6'W; SIO 63-868 (9), 410–510 mm TL, 32°30.4'N, 117°25.9'W; SIO 65-192 (1), 355 mm TL, 28°51.0'N, 115°46.7'W; SIO 66-535 (5), 330–410 mm TL, 31°59.5'N, 117°6.1'W; SIO 68-59 (25), 133–510 mm TL, 23°7.3'N, 109°19.2'W; SIO 68-60 (6), 365–496 mm TL, 23°6.7'N, 109°16.5'W; SIO 68-118 (110), 125–490 mm TL, 25°36.5'N, 109°43.0'W; SIO 68-119 (25), 280–475 mm TL, 25°35.6'N, 109°45.1'W; SIO 69-227A (15), 31°35.3'N, 116°53.3'W; SIO 69-228F (8), 32°8.2'N, 117°13.0'W; SIO 69-487 (1), 493 mm TL, 32°26.2'N, 117°31.4'W; SIO 71-29 (1), 425 mm TL, 32°26.9'N, 117°28.9'W; SIO 71-116 (1), 524 mm TL, 28°25.2'N, 115°49.7'W; SIO 71-137 (1), 463 mm TL, 32°26.3'N, 117°28.4'W; SIO 72-55 (2), 335–470 mm TL, 32°29.2'N, 117°30.3'W; SIO 73-290 (5), 356– 394 mm TL, 9°48.2'N, 85°48.2'W; SIO 74-175 (2), 467–487 mm TL, 32°31.7'N, 117°34.1'W; SIO 77-116 (1), 500 mm TL, 32°45.0'N, 117°41.0'W; SIO 80-37 (1), 550 mm TL, 10°51.8'S, 78°30.0'W; SIO 8794 (1), 255 mm TL, 30°15.5'N, 116°10.7'W; UCR 2008-003 (2), 9°33'25"N, 85°22'0"W; UCR 2290-002 (2), 9º30’0"N, 85º5'39"W; UMMZ 214877 (1), 28°57'N, 116°28'W; Conservation status: Least concern (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Garman (1899: 344), Miller & Lea (1972: 32), Theisen (1976: 167–173), Hensley (1991: 1042), Wisner & McMillan (1995: 533), Pequeño (1997: 78), Chirichigno & Vélez (1998: 28), Fernholm (1998: 35), McMillan (1999: 110), Mincarone (2001: 483), Mok (2001: 355–363), Mok & Kuo (2001: 295), Mok & McMillan (2004: 741, 742), Love et al. (2005: 1), Møller et al. (2005: 379), Rubio et al. (2005: 119), Hendrickx & Hastings (2007: 274), Cavalcanti & Gallo (2008; 1259, 1261, 1264, 1266), Nakaya (2009: 46– 47), Kuo et al. (2010: 856), Knapp et al. (2011: 405), Cruz-Mena & Angulo (2015).

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18) Myxine glutinosa Linnaeus, 1758; Atlantic hagfish (En); Bruja del Atlántico. Myxine glutinosa Linnaeus, 1758: 650 (original description; type locality: Atlantic Ocean; Syntypes: NRM 89 (4), 222–275 mm TL, sex indeterminate). Distinctive characters: Gill pouches 6–7; fused cusps 2/2; total cusps 29–41; total slime pores 85–118; one single dorsal nasal sinus papillae; gill pouches at end of the dental muscle 0; ventral aorta bifurcates at gill pouch 2; branchial ducts on the left side confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right side; ventral fin fold present, thin, caudal fin fold present, thin and rounded; body color reddish brown to dark purple, with the head white and occasional pale blotches ventrally (northwestern Atlantic Ocean) or homogeneous grayish to dark brown (northeastern Atlantic Ocean). Distribution: Two populations from the north Atlantic Ocean: (1) from Murmansk, Russia, to northern Marocco; occurring at depths between 40 and 1200 m (eastern population); and (2) from Greenland to the Gulf of Mexico; occurring at depths up to 742 m (western population). Documented records in Mexican and/or Central American waters (Museum specimens): 1 specimen. SIO 86-86 (1), 392 mm TL, 24°25.0‘N, 87°38.0‘W. Conservation status: Least concern (Knapp et al. 2011). Major threats: Target of fisheries (Knapp et al. 2011). Literature: Vladykov (1973: 6), Fernholm (1981: 79), Fernholm (1998: 35), Martini et al. (1998: 516–524), Mok (1999: 61), Mok & Kuo (2001: 295), Martini & Flescher (2002: 9–16), Mok (2002: 59), Møller et al. (2005: 381), Kuo et al. (2010: 856), Knapp et al. (2011: 405). Remarks: Wisner & McMillan (1995), based on differences in size and coloration recognized M. limosa, sensu Girard (1859), as a valid species; however, this species correspond to an eastern population of M. glutinosa, sensu Linnaeus (1758). As noted by Martini et al. (1998) and Martini & Flescher (2002), in the absence of meristic and morphological supporting data, these features seem insufficient to justify dividing both populations (eastern and western) into separate species. 19) Myxine hubbsi Wisner & McMillan, 1995. Myxine hubbsi Wisner & McMillan 1995: 536 (original description; type locality: 32°38'N, 118°08°4'W, southeastern of Clemente Island, off San Diego, California, USA, northeastern Pacific, depth 2009 m; holotype: SIO 65-452, 515 mm TL, female). Distinctive characters: Gill pouches 6; fused cusps 2/2; total cusps 32–42; total slime pores 90–111; one single dorsal nasal sinus papillae; gill pouches at end of the dental muscle 0; ventral aorta bifurcates at gill pouch 1–2; branchial ducts on the left side confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right

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side; ventral fin fold thin, caudal fin fold rounded; body color light to dark purplish black, rarely with pale blotches, head usually pale anteriorly. Distribution: Eastern Pacific, from southern California, USA, to Chile; occurring at depths between 1100 and 2440 m. Documented records in Mexican and/or Central American waters (Museum specimens): 92 specimens. CAS 77360 (3 P), 315–395 mm TL, 31°00'N, 118°06'W; MOVI 27508-27509 (2), 215–410 mm TL, 31°02.7'N, 116°59.25'W; SIO 59-363 (3), 322–410 mm TL, 31°0.0'N, 118°6.0'W; SIO 59-366 (2), 225– 435 mm TL, 31°2.7'N, 116°59.3'W; SIO 68-60 (36 P), 120–484 mm TL, 23°6.7'N, 109°16.5'W; SIO 68-61 (2 P), 350–400 mm TL, 23°5.7'N, 109°12.5'W; SIO 68-676 (16), 258–486 mm TL, 23°37.0'N, 118°7.5'W; SIO 71-116 (1), 415 mm TL, 28°25.2'N, 115°49.7'W; SIO 72-55 (1), 310 mm TL, 32°29.2'N, 117°30.3'W; SIO 72-176 (8), 326–473 mm TL, 32°29.2'N, 117°30.3'W; SIO 73-293 (16 P), 230–480 mm TL, 9°23.5'N, 85°6.4'W; USNM 325214 (2 P), 440–450 mm TL, 32°39'N, 118°11'W. Conservation status: Least concern (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Wisner & McMillan (1995: 530–550), Pequeño (1997: 78), Fernholm (1998: 35), Mok (2001: 355–363), Mok & Kuo (2001: 295), Mok (2002: 59), Mok & McMillan (2004: 737–747), Love et al. (2005: 1), Møller et al. (2005: 381), Cavalcanti & Gallo (2008; 1259–1266), Knapp et al. (2011: 405). 20) Myxine mccoskeri Wisner & McMillan, 1995. Myxine mccoskeri Wisner & McMillan, 1995: 534 (original description; type locality: 9°39'N, 78°60'W, off San Blas Archipelago, Panama, Caribbean Sea, depth 530–560 m; holotype: SIO 70-363, 201 mm TL, female). Distinctive characters: Gill pouches 5; fused cusps 3/2; total cusps 36– 48; total slime pores 77–92; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 0; ventral aorta bifurcates at gill pouch 1; branchial ducts on the left side confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right side; ventral fin fold vestigial to well developed, caudal fin fold present, rounded; body color variably light to dark brown, with the head, barbells and ventral aspects anterior to gill apertures white or lighter than body. Distribution: Off Panama, Colombia, and Venezuela, Southern Caribbean Sea; occurring at depths between 439 and 1174 m. Documented records in Mexican and/or Central American waters (Museum specimens): 3 specimens. Holotype; SIO 70-363 (2), 117–170 mm TL, taken with the holotype (Panama). Conservation status: Least concern (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries

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(Knapp et al. 2011). Literature: Wisner & McMillan (1995: 530–550), Fernholm (1998: 35), Mok (2001: 355–363), Mok & McMillan (2004: 737–747), Cavalcanti & Gallo (2008; 1259, 1261), Knapp et al. (2011: 405). 21) *Myxine mcmillanae Hensley, 1991. Myxine mcmillanae Hensley, 1991: 1040 (original description; type locality: 17°51'48"N, 66°58'54"W, off southern Puerto Rico, Caribbean Sea, depth 925 m; holotype: USNM 308405, 390 mm TL, sex indeterminate). Distinctive characters: Gill pouches 6, rarely 7; fused cusps 2/2; total cusps 42–48; total slime pores 101–113; one single dorsal nasal sinus papillae; gill pouches at end of the dental muscle 1–3; ventral aorta bifurcates at gill pouch 2; branchial ducts on the left side confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right side; ventral fin fold prominent, caudal fin fold rounded; body color bluish gray to brown with the head anterior to first slime pore white. Distribution: Northeastern Gulf of Mexico and Caribbean Sea, Atlantic Ocean; occurring at depths between 603 and 1500 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats: Potentially impacted by trawling and other fisheries (Knapp et al. 2011). Literature: Hensley (1991: 1040– 1043), Wisner & McMillan (1995: 539), Fernholm (1998: 35), McEachran & Fechhelm (1998: 34), Grana-Raffucci (1999: 9), Mincarone (2001: 481), Mok (2001: 355–363), Mok & Kuo (2001: 295), Mok (2002: 59), Mok & McMillan (2004: 741, 744), Cavalcanti & Gallo (2008; 1261), McEachran (2009:1256), Knapp et al. (2011: 405). 22) *Myxine robinsorum Wisner & McMillan, 1995. Myxine robinsi Wisner & McMillan 1995: 534 (original description; type locality: 11°37'N, 60°50'W, off northern Trinidad & Tobago, Caribbean Sea, depth 783–1281 m; holotype: SIO 90-149, 475 mm TL, female). Distinctive characters: Gill pouches 5; fused cusps 3/2; total cusps 56–58; total slime pores 94–104; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 1; ventral aorta bifurcates at gill pouch 1–2; branchial ducts on the left side confluent with the pharyngocutaneous duct, forming a gill aperture larger than that of right side; ventral fin fold well developed, caudal fin fold present, rounded; body color light to medium brown, with the head whitish. Distribution: Southern Caribbean Sea; occurring at depths between 783 and 1768 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Least concern (Knapp et al. 2011). Major threats:

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None known (Knapp et al. 2011). Literature: Wisner & McMillan (1995: 530–550), Fernholm (1998: 35), Mok (2001: 355–363), Knapp et al. (2011: 405). Remarks: The species name “robinsi” was corrected to “robinsorum” by Fernholm (1998) according to ICZN (1985), article 32c(ii). 23) *Rubicundus lakeside (Mincarone & McCosker, 2004). Eptatretus lakeside Mincarone & McCosker, 2004: 163 (original description; type locality: 00°17'30"S, 91°39'36"W, off Cabo Douglas, northwestern Fernandina Island, Galápagos Islands, eastern Pacific, depth 762 m; holotype: CAS 201880, 201 mm TL, female). Distinctive characters: Gill pouches 5; fused cusps 3/3; total cusps 36; total slime pores 88; dorsal nasal sinus papillae paired; gill pouches at end of the dental muscle 0; afferent branchial arteries at median ventral aorta 3; ventral aorta bifurcates at gill pouch 2; gill apertures linear; last gill apertures and pharyngocutaneous duct fused; eyespots weak; ventral fin fold conspicuous, caudal fin fold quite thin and rounded; body color reddish. Distribution: Galápagos Islands, Ecuador, eastern Pacific; occurring at depths about 762 m. Documented records in Mexican and/or Central American waters (Museum specimens): None known. Conservation status: Data deficient (Knapp et al. 2011). Major threats: None known (Knapp et al. 2011). Literature: Mincarone & McCosker (2004: 162– 168), Cavalcanti & Gallo (2008; 1263), Fernholm & Quattrini (2008: 131), McCosker & Rosenblatt (2010: 187), Knapp et al. (2011: 404), Fernholm et al. (2013: 10). Remarks: Known only from the holotype (Mincarone & McCosker 2004, Eschmeyer 2015).

Concluding remarks Knowledge about the biology of hagfishes is lacking in several areas of the world due to their cryptic nature and the lack of research and sampling. For a better understanding, their distribution, population trends, habitat and ecology need to be studied in much greater detail. In this chapter the readers will find revised and updated taxonomic and distributional information about the hagfishes of Mexico and Central America (Pacific and Caribbean coasts). It includes all the nominal species of the family Myxinidae known to be present in the area as well as contiguous waters (Galápagos Islands, West Indies, and the Colombian Pacific and Caribbean coasts). A total of 3 genera and 23 species are listed; a brief diagnosis for each one and an identification key are included.

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This work constitutes a comprehensive review of hagfishes of the region, presented in an accessible form to researchers interested on hagfish systematics, biodiversity and distribution, in order to encourage future works at the regional scale.

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Myxinidae) from the North Atlantic Ocean. Copeia 2005, 374-385. Nakaya K. 2009. Myxinidae. In Nakaya K., Yabe M., Imamura H., Romero M.C., Yoshida M. (eds.): Deep-sea fishes of Peru. Pp. 46–47. Tokyo: Japan Deep Sea Trawlers Association. Nelson J.S. 2006. Fishes of the World. 4th ed. Hoboken, New Jersey: John Wiley & Sons. Page L.M., Espinosa-Pérez H., Findley L.D., Gilbert C.R., Lea R.N., Mandrak N.E., Mayden R.L. & Nelson J.S. 2013. Common and scientific names of fishes from the United States, Canada, and Mexico. Seventh Edition. Maryland: American Fisheries Society. Palacios-Salgado D.S., Burnes-Romo L.A., Tavera J.J. & Ramíez-Valdez A. 2012. Endemic fishes of the Cortez biogeographic province (Eastern Pacific Ocean). Acta Ichthyologica et Piscatoria 42, 153-164. Pequeño G. 1997. Fishes of Chile. Systematic list reviewed and commented: addendum. Revista de Biología Marina y Oceanografía 32, 77-94 (In Spanish). Polanco Fernandez A. & Fernholm B. 2014. A New species of hagfish (Myxinidae: Eptatretus) from the Colombian Caribbean. Copeia 2014, 530-533. Reyes-Bonilla H., Bedolla-Guzmán Y.R., Calderón-Aguilera L.E., AyalaBocos A., Ramírez-Valdez A., González-Romero S., OlivaresBañuelos N.C., Sánchez-Alcántara I. & Walther-Mendoza M. 2011. Checklist and biogeography of fishes from Guadalupe Island, western Mexico. California Cooperative Oceanic Fisheries Investigations Reports 51, 195-209 Rodríguez-Romero J., Palacios-Salgado D.S., López-Martínez J., Hernández-Vázquez S. & Ponce-Díaz G. 2008. Taxonomic composition and zoogeographical relationships of demersal fishes of the western coast of Baja California Sur, Mexico. Revista de Biología Tropical 56, 1765-1783 (In Spanish). Rubio E.A., Pedraza M.J. & Zapata L.A. 2005. First record of Myxine circifrons Garman 1899 (Agnatha: Myxinidae) on the Pacific coast of Colombia. Gayana 69, 118-121. Sabaj Perez M.H. 2014. Standard symbolic codes for institutional resource collections in herpetology and ichthyology: an Online Reference (Version 5.0, 22 September 2014). Accessed on the internet at http://www.asih.org/sites/default/files/documents/resources/symbolic_ codes_for_collections_v5.0_sabajperez_2014.pdf. on 15 June 2015. INVEMAR. 2015. Marine Biodiversity Information System of Colombia (Version 4 October 2011). Accessed on the internet at http: www.invemar.org.co/siam/sibm on 15 June 2015.

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Smith K.L. & Hessler R.R. 1974. Respiration of benthopelagic fishes: in situ measurements at 1230 meters. Science 184, 72-73. Theisen B. 1976. The olfactory system in the pacific hagfishes Eptatretus stoutii, Eptatretus deani, and Myxine circifrons. Acta Zoologica 57, 167-173. Vladykov V.D. 1973. Myxinidae. In Hureau J.-C. & Monod T. (eds.): Check-list of the Fishes of the North-eastern Atlantic and of the Mediterranean. Pp. 6. Paris: CLOFNAM, UNESCO. Wisner R.L. & McMillan C.B. 1988. A new species of hagfish, genus Eptatretus (Cyclostomata, Myxinidae), from the Pacific Ocean near Valparaiso, Chile, with new data on E. bischoffii and E. polytrema. San Diego Society of Natural History 21, 14, 227-244. Wisner R.L. & McMillan C.B. 1990. Three new species of hagfishes, genus Eptatretus (Cyclostomata, Myxinidae), from the Pacific Coast of North America, with new data on E. deani and E. stoutii. Fishery Bulletin 88, 787-804. Wisner R.L. & McMillan C.B. 1995. Review of new world hagfishes of the genus Myxine (Agnatha, Myxinidae) with descriptions of nine new species. Fishery Bulletin 93, 530-550.

Table 5-1 (next page). Morphometric data of the Mexican and Central American species of hagfishes; measurements expressed as percentages of TL; data from McMillan & Wisner (1984), Hensley (1985), Wisner & McMillan (1988, 1990, 1995), Fernholm (1998), McMillan (1999), Mok et al. (2001), Mincarone & Sampaio (2004), Møller & Jones (2007), Fernandez & Fernholm (2014) and Cruz-Mena & Angulo (2015). Measurements unavailable are represented with an en-dash (–). POL: Preocular length; PBL: Pre-branchial length; BRL: Branchial length; TRL: Trunk length; TAL: Tail length; TAD: Tail depth; BDF: Body depth with finfold; BOD: Body depth without finfold.

331–385

130–523

207–592

142–380

147–470

283–320

350–450

223–395

410–815

129–481

338–590

179–468

194–216

125–650

117–501

209–522

E. caribbeaus

E. deani

E. fritzi

E. grouseri

E. mcconnaugheyi

E. mccoskeri

E. mendozai

E. minor

E. multidens

E. sinus

E. springeri

E. stoutii

E. wayuu

M. circifrons

M. glutinosa

M. hubbsi

220.3

328–360

E. ancon

E. bobwisneri

584

TL (mm)

E. aceroi

Species

–

–

–

–

4.0–7.7

–

4.1–8.2

–

3.1–6.2

5.8–6.6

–

4.5–7.4

–

4.8–8.2

4.2–8.9

5.6–6.1

–

–

–

POL

25–28

23–30

29–30

33.1–33.0

18.7–25.3

21.5–26.8

19.7–27.8

18.4–22.2

20.1–25.9

22.2–25.2

24–26

14.7–18.1

20–24

17.6–24.5

14.4–20.4

21.4–23.6

19.4–22.9

37.3

22.7

PBL

–

–

–

3.0–3.5

11.5–14.2

2.5–5.6

9.2–17.2

5.4–8.1

5.1–7.2

4.7–6.6

9.3–10.1

15.5–21.6

6.3–8.1

11.5–15.8

12.7–18.2

5.8–7.8

10.0–11.1

1.95

4.5

BRL

58–65

54–62

55–58

47.8–50.2

47.0–53.5

52.9–61.1

45.0–54.0

53.3–57.1

50.6–55.9

51.0–54.5

49–50

48.2–55.7

54–57

46.2–55.6

48.0–55.5

50.4–56.0

50.3–2.8

47.6

63.5

TRL

Measurements

12.0

9.4

TAL

10–12

12–17

10–15

10.9–14.9

10.4–17.8

13.4–16.8

10.2–17.4

16.8–19.1

13.9–18.3

16.2–19.3

15.6–17.7

12.2–16.1

14.6–17.5

13.2–18.1

12.6–19.2

16.5–19.6

16.7–17.1

Hagfishes of Mexico and Central America

3–5

3–6

5–6

10.1–11.0

4.5–8.3

6.4–9.3

4.8–9.0

8.6–10.4

5.3–11.6

8.0–9.4

8.7–10.2

6.0–8.8

6.3–7.9

5.8–9.2

5.2–10.3

7.5–10.9

7.6–7.8

7.4

3.1

TAD

4–6

4–9

6–8

8.6–9.4

5.0–9.7

6.6–9.9

4.9–10.4

7.9–11.6

7.1–11.4

9.1–11.6

9.4–10.6

6.1–9.8

4.2–8.8

–

4.7–10.5

7.7–9.4

8.5–9.2

7.6

3.2

BDF

4–5

3–8

4–7

7.7–8.7

4.1–9.0

6.2–9.7

4.6–10.1

7.7–11.0

7.1–10.8

8.0–10.9

–

5.5–9.0

–

5.3–10.2

4.5–10.5

7.4–10.6

–

6.7

2.4

BOD

123

165–286

M. robinsorum –

–

–

–

4.7

29–32

26–31

27–34

6.2

–

–

–

Chapter Five

50.9

57–60

56–61

52–56

18.2

11–14

11–14

13–17

6.0

4–5

4–6

4–7

7.2

5–7

4–8

5–9

6.4

4–5

4–7

4–7

10–12

7

E. caribbeaus

E. fritzi

8

E. bobwisneri

10–12

6

E. ancon

E. deani

5

GPO

E. aceroi

Species

3

3

3

3

3

2–3

MAR

2

2

3

2

2

2

MPR

7–10

6–10

11–13

9

13

13

UAR

7–9

7–9

10–11

8

12

12

UPR

38–46

37–46

54–58

44

60

58

TCU

9

26

44

PSP

10–15

4–10

13–15

Character

9–12

9–13

6–7

7

0

6

BSP

40–49

39–49

47–52

46–47

45

107

TSP

8–15

9–15

11–13

13–14

9

17

CSP

74–85

67–80

79–85

76

80

174

TTP

Table 5-2. Meristic data of the Mexican and Central American species of hagfishes; data from McMillan & Wisner (1984), Hensley (1985), Wisner & McMillan (1988, 1990, 1995), Fernholm (1998), McMillan (1999), Mok et al. (2001), Mincarone & McCosker (2004), Mincarone & Sampaio (2004), Møller & Jones (2007), Fernandez & Fernholm (2014) and Cruz-Mena & Angulo (2015). GPO: Gill pouches; MAR: Multicusp in anterior row; MPR: Multicusp in posterior row; UAR: Unicusp in anterior row; UPR: Unicusp in posterior row; TCU: Total cusps; PSP: Prebranchial slime pores; BSP: Branchial slime pores; TSP: Trunk slime pores; CSP: Tail slime pores; TTP: Total slime pores.

275

271–473

M. mcmillanae

R. lakeside

165–286

M. mccoskeri

124

E. multidens

5

5

6–7

6

5

6–7

5

5

E. wayuu

M. circifrons

M. glutinosa

M. hubbsi

M. mccoskeri

M. mcmillanae

M. robinsorum

R. lakeside

6

10–14

E. stoutii

E. springeri

9–12

6

E. minor

E. sinus

6

5–6

E. mendozai

8

12–14

E. mcconnaugheyi

E. mccoskeri

5–6

E. grouseri

3

3

2

3

2

2

3

3

3

3

3

3

3

3

3

3

3

3

2

2

2

2

2

2

2

2

2

2

3

3

3

3

2

2

6

11–13

8–10

6–9

5–9

4–8

7–13

7–8

6–10

c.a. 11

6–9

10–12

8–11

11–13

9–10

7–9

9

6

11–12

8–10

7–10

5–8

5–8

8–12

8–9

6–10

c.a. 9

6–10

9–12

8–10

10–12

9–10

7–9

8

36

56–58

42–48

36–48

32–42

29–41

43–56

43

36–46

48–52

34–46

50–58

46–54

56–61

48–51

38–45

44

15

25–32

26–35

22–27

18–28

20–38

17–33

24

10–16

15–19

10–17

14–16

15–18

13–15

14–15

6–11

12–13

Hagfishes of Mexico and Central America

4

–

–

–

–

–

–

2

9–14

3–6

8–11

5–7

4–6

5–6

7

11–16

4–5

50

59–61

60–76

43–53

57–73

50–69

48–62

38–40

39–51

44–57

36–49

52–55

41–48

45–48

40–42

39–50

44–46

19

7–11

9–12

9–14

8–14

8–16

7–13

9

8–14

11–14

7–14

17–20

11–14

12–15

10–12

8–13

14–15

88

94–104

101–113

77–92

90–111

85–118

80–102

73–75

71–88

77–92

66–82

88–93

74–82

77–82

72–74

67–84

76–77

125

CHAPTER SIX THE STRUCTURE AND TAXONOMY OF THE GILL PORE IN LAMPREYS OF THE GENUS ENTOSPHENUS RICHARD BEAMISH

Introduction Lamprey species have been distinguished using morphological differences of metamorphosed individuals that rely heavily on the teeth on the oral disc and the tongue (Vladykov & Follett 1967, Hubbs & Potter 1971, Renaud 2011). The recent use of molecular methods to analyze the genetic composition of lampreys has clarified relationships (Mateus et al. 2013a) as well as added another level of complexity to their taxonomy (Docker 2009, Potter et al. 2015). Molecular methods have also shown that there is greater species diversity than previously recognized (Reid et al. 2011, Boguski et al. 2012, Mateus et al. 2013b). The structure in the gill pore is a new morphological character that can distinguish species as well as identify taxonomic diversity (Beamish 2010). The number, arrangement and structure of papillae along the posterior rim of the gill pore differ among genera and many species (Beamish 2010). I have examined 37 different species, including holotypes and paratypes from 10 collections around the world and find that gill pore papillae provide taxonomic information consistent with the structures currently in use (Beamish, unpublished data). Although the paper by Beamish (2010) focused on introducing the character, there was information that related to the taxonomy and biology of particular species. Lampetra ayresii from areas throughout their distribution were examined. Some specimens at the southern limit of the species’ distribution from the Sacramento River, California had fused papillae, forming rope-like structures that differed from all other samples. It is possible that this structure distinguishes populations that are older, perhaps indicating that

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127

the oldest L. ayresii population is from this area as well as showing that the population may have a genetic stability to maintain the unique structure of the papillae. In a second example, the gill pore structure of specimens of L. pacifica were examined from collections in the Canadian Museum of Nature, and at Oregon State University that included the holotype and paratypes (Beamish 2010). The greatly reduced number of papillae that were also larger than in L. richardsoni and in a single row, readily distinguished L. pacifica from L. richardsoni. Reid et al. (2011) reexamined the morphological data Vladykov (1973) used to describe L. pacifica as well as doing a genetic analysis using cytochrome b (cyt b). They concluded that L. pacifica is a distinct species as Vladykov (1973) proposed and the species is now listed in the recent Common and Scientific Names of Fishes from the United States, Canada and Mexico (Page et al. 2013). In this paper, I show how the gill pore structure can be used to distinguish the species within the genus Entosphenus as well as identify some of the diversity that as yet is not recognized taxonomically. Entosphenus was considered to be a subgenus of Lampetra by Hubbs & Potter (1971), but the results in Docker et al. (1999) and the analyses by Gill et al. (2003) showed that Entosphenus is monophyletic and a separate genus that is recognized in Renaud (2011) and in the recent names of fishes published by the American Fisheries Society (Page et al. 2013). The genus is characterized by tricuspid lateral circumoral teeth and an infraoral lamina with five cusps. The anadromous parasitic species from which a number of species are derived is E. tridentatus or the Pacific lamprey. The Pacific lamprey is distributed from Mexico, in Baja California through to Norton Sound Alaska (Renaud 2011). It is found in Northern Japan (Renaud 2011), but there are no records from any river along the eastern coast of Russia (Orlov, personal communication). There are three freshwater parasitic species derivatives and three nonparasitic derivatives, although one nonparasitic species, E. hubbsi, has recently been placed in the genus Lampetra by some authors (Docker et al. 1999, Lang et al. 2009, Page et al. 2013). There are also a number of populations that have individuals of questionable taxonomic status. The different species and life histories characterize changes that have occurred in all lamprey over their evolutionary history, possibly providing cues to success of all lampreys over their 360 million year history (Gess et al. 2006). In this study, the gill pore structure is compared to determine if it provides a character that is useful in the taxonomy of species in the genus Entosphenus. The gill pore structure can also be used to recognize populations of a species that have distinct structure. This is relevant because lampreys are now recognized as a part of the community of fishes

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that can be protected by government legislation. Renaud (1997), Jelks et al. (2008) and Potter et al. (2014) list the lamprey taxa that receive conservation status in North America. In British Columbia, a variety of L. richardsoni (L. richardsoni var. marifuga) is protected by the Species at Risk Act (Renaud et al. 2009), as well as E. macrostomus (Renaud et al. 2009, Potter et al. 2014). In the United States, a petition in January 2003 to list E. tridentatus and E. hubbsi as threatened or endangered failed, but drew attention to the need to know more about the particular population of the species (Siskiyou Regional Education Project 2003; see http://www.fws.gov/policy/library/ 2004/04-28167.pdf). Better information about particular populations is important as there is evidence of populations of Entosphenus that differ from taxons currently recognized as species (Hubbs 1925, Coots 1955, Moyle 2002, Moyle et al 2009). The information on the gill pore structure of species and populations within the genus Entosphenus adds to the relatively few characters that are available for classification (Gill et al. 2003) as well as providing a method to detect populations that may ultimately be recognized taxonomically.

Methods Methods used to count and describe the marginal papillae and the central processes are described in Beamish (2010). A standard binocular microscope is needed to provide the necessary magnification. The papillae are best observed in mature specimens that have been preserved to avoid a mucous build up on the skin surface, around the branchial pore area of the right side. The most anterior pore was number one, with counts from pore number three on the left side being preferred. However, Beamish (2010) showed that pores two to six could be used. Counts were made from specimens with well-developed papillae unless specified. The identification of museum specimens was accepted and specimens in my own collection were identified using the characters that were used to determine the holotype and paratype (Renaud 2011). Metamorphosed specimens were collected using an electroshocker with some specimens kept alive in freshwater tanks at the Pacific Biological Station. Some nonparasitic species were preserved after they matured and others were preserved from the holding tanks in the winter following metamorphosis in the river. Parasitic species were fed and some kept to the spring of the following year, but not until sexual maturity. Gill pore structure of L. pacifica is included for comparison with the structure in E. hubbsi. The material used in this study is in Table 6-1.

The Gill Pore in Lampreys of the Genus Entosphenus

129

Table 6-1. Material used. The number (n) of specimens used is in brackets. Species

E. tridentatus (n = 13)

E. macrostomus (n = 30)

E. similis (n = 4)

E. minimus (n = 15)

Collection number UW14612 UW29385 CAS 85223 CAS 208642 CAS 210309 Private collection Private collection Private collection Private collection Private collection Private collection Private collection Private collection Private collection OS02902 CMNF1 19990029.1 CMNFI 19990027.1 Private collection USNM 35319 USNM 210967 OS 002875 OS 015884 CAS 28706 CMNFI-19860728.1

Location Columbia River, Oregon Lake Aleknagik, Alaska Trinity River, California Trinity River, California Copco Lake, California Russia, collected off the coast of Kamchatka Mid-North Central Pacific Ocean, attached to a Pacific salmon North Pacific Ocean off the Aleutian Islands Copper River, British Columbia Sweltzer Creek, British Columbia Keogh River, British Columbia Sakinaw Lake, British anadromous form Merced River, California

Columbia

Mesachie Lake, British Columbia Upper Klamath paratype Oregon, paratype

Lake,

Oregon, paratype Sprague River, Oregon Miller Lake, holotype Miller Lake, paratype Miller Lake, paratype Sycan River 1997 Miller Lake, paratype Miller Lake 1951

Oregon

Chapter Six

130 Undescribed Entosphenus A (n = 4) E. folletti (n = 4)

E. lethophagus (n = 10)

E. hubbsi (n = 19)

L. pacifica (n = 5)

Private collection

San Joaquin River, California

NMC 86 0805 CMNFI 86-0806 CMNFI 19751549.1 CMNFI 198607333.1 USNM 232567 CAS 13391 Private collection CAS 35988 CAS 35987 OS 004945 Private collection CMNFI 19710769.1 CMNFI 19710769A.1 OS 004945

California, paratype Fall Creek, California, paratype California, holotype Oregon Oregon Hat Creek Hat Creek, California California, paratype California, holotype California, paratype California Clackamus River, Oregon Clackamus River, Oregon Willamette River, Oregon

Results Entosphenus tridentatus E. tridentatus has a large number of gill pore papillae making it difficult to make accurate counts without some dissection of the gill pore to display the papillae (Fig. 6-1). In Beamish (2010), 10 specimens from around the North Pacific Ocean from off the coast of Russia to the Merced River in California were examined and the results are reported here. The number of marginal papillae ranged from 60 to 73 with an average of 67 (Table 6-2). Importantly, the number, appearance and arrangement were virtually identical in all specimens. The gill pore papillae were pencil shaped dorsally with a broader base that was pigmented (Fig. 6-2). Papillae formed a broad band and along the posterior margin of the pore. In the denser areas, papillae could form three or even four short rows (Fig. 6-2). There was no evidence of fused papilla. The central process was large and the largest of all lamprey species examined (R.J. Beamish, unpublished data). It was about four times as long and three times as thick

The Gill Pore in Lampreys of the Genus Entosphenus

131

as the marginal papilla. The central process was weakly pigmented with tissue around the base of the process also being pigmented. In the collection at the California Academy of Sciences, there were two small (162 mm, 172 mm) E. tridentatus that were collected from Copco Lake in April 1980 (CAS 210309). Both were feeding and immature. Each had a small number of very small marginal papillae that were almost in a single row along the posterior rim of the pore. The central process was large relative to the marginal papillae. The papillae were not easily counted, but there were about 15 in one specimen and 16 in the second specimen.

Figure 6-1. Photographs of the gill pore cups of E. tridentatus from: (A) off the coast of Kamchatka, Russia, and (B) the Merced River, California.

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132

Figure 6-2. Representation of the typical gill pore structure of E. tridentatus. (The central process and marginal papillae are labeled in Fig. 6-1.)

Table 6-2. The number of gill pore papillae from 10 Pacific lamprey specimens collected throughout the subarctic Pacific Ocean (from Beamish 2010). Location Russia North Central Pacific Ocean North Pacific Ocean off the Aleutian Islands Lake Aleknagik, Alaska Copper River, British Columbia Sweltzer Creek, British Columbia Keogh River, British Columbia Sakinaw Lake, British Columbia Columbia River, Oregon Merced River, California Average (SD)

Number of marginal papillae 68 60 70 73 64 68 68 62 72 64 67 (4.3)

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133

Entosphenus macrostomus Entosphenus macrostomus was described as a new species in 1982 (Beamish 1982). A study by Taylor et al. (2012) using DNA supported the species designation. In the study by Beamish (2010), gill pore papillae were examined from 30 specimens from the only known population in Mesachie and Cowichan lakes. The 30 specimens were from the original collection that was used to describe the species and remain in my own collection. Other specimens from the same sample are catalogued as holotype and paratypes in the Canadian Museum of Nature as: Holotype– CMNFI 1981-1219.1, Paratypes–CMNFI 1981-1220.1, 1221.1, 1222.1.

Figure 6-3. (A) Photograph of pore number three from E. macrostomus from Mesachie Lake, British Columbia, showing the large central process and numerous marginal papillae. (B) Representation of the typical gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) of E. macrostomus.

The number of papillae in gill pore cups number 1 to 7 ranged from 33 to 79. The numbers in pores one and seven were the smallest, but there were no significant differences in the counts in pores two to six. Counts in pore number three had the lowest standard deviation and when possible it was used as the representative structure for each specimen. There was no significant relationship between the number of papillae in pore three and length of the specimens or the sex of the specimen (Beamish 2010). Thus, the structure of the central process and the marginal papillae in pore three was considered to be representative of the species (Fig. 6-3A). The

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number of papillae in the 30 specimens ranged from 46 to 72 and averaged 59. The papillae varied in size, but were tubular and not pigmented (Fig. 6-3B). Papillae were larger than in E. tridentatus and those deeper in the pore could be longer than at the margin. All marginal papillae were in a continuous band that could be about three or four papillae deep into the pore, but not arranged in rows (Fig. 6-3B). The central process was large, prominent, but shorter than in E. tridentatus and rarely pigmented at the base. Entosphenus similis Entosphenus similis was first described by Vladykov & Kott (1979). It is a freshwater, parasitic derivative of E. tridentatus that has been reported from the Klamath River Basin and the Klamath River in California. Vladykov & Kott (1979) distinguished E. similis from E. tridentatus by E. similis having a larger disc, smaller eye, fewer myomeres and more robust teeth in the anterior field of the disc. However, the most diagnostic difference was the greatly reduced number of the velar tentacles, with E. similis having an average of 8 and no wing-like appendages. Docker et al. (1999) reported that DNA-based comparisons for E. similis from the Merced River in California were not distinguishable from E. tridentatus, whereas E. similis from Klamath Basin of Oregon was distinguishable from E. tridentatus using DNA.

Figure 6-4. Representation of the typical gill pore structure of E. similis (the central process and marginal papillae are labeled in Fig. 6-1.)

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Four specimens of E. similis, including three paratypes were examined from two museum collections and one specimen from my own collection. There was a moderate number (28-40, average 36) of unpigmented, smaller, pencil-shaped papillae that were arranged in a continuous band that had imperfect rows (Fig. 6-4). Papillae were similar in size with a larger concentration of papillae towards the dorsal and ventral areas along the posterior of the pore. The central papilla was prominent, large, about two times the size of the marginal papillae and unpigmented (Fig. 6-4). Entosphenus minimus E. minimus was described by Bond & Kan (1973) from material collected from Miller Lake, Oregon in 1950 and 1952. The species was originally described as parasitic, cannibalistic, and a scavenger (Bond & Kan 1973). Later, these authors speculated that some E. minimus may not feed. The length range of 72 mm to 120 mm for the spawning lamprey indicated that feeding and scavenging was minimal. The species was believed to have been eradicated in the late 1950s as a result of a fish control program to kill the lamprey that were considered to be harming introduced trout (Lorion et al. 2000). Lorion et al. (2000), however, reported that the species was not extinct and occurred in a disjunct distribution in Miller Creek, Jack Creek and the upper sections of the Williamson and Sycan rivers. These authors concluded that the new and previously collected specimens were most similar to the nonparasitic E. lethophagus but that E. minimus was smaller, had a larger disc length, larger prebranchial length and a larger eye. Lorion et al. (2000) speculated that E. minimus might have evolved when a nonparasitic population produced a parasitic species which could indicate that E. minimus might have had a non-feeding ancestor. Importantly, they noted that the recently discovered E. minimus differed slightly from the first described specimens. Gill pore papillae were examined from 15 specimens in 4 collections that included the holotype and paratypes. One specimen was from the recently described population. The central process was prominent but much smaller relative to E. tridentatus (Fig. 6-5A). The base was about two times the width of the marginal papillae and the length was slightly longer than the marginal papillae. Marginal papillae were reduced in number and varied among specimens with a range of 9 to 16 (average = 12; Fig. 6-5A) for the samples from the extinct populations and 9 to 22 (average = 15) for the specimen from the recently identified population (Fig. 6-5C). Papillae were commonly fused into flap-like structures, with

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two small bumps toward the ends of the dorsal edge of the papillae along the margin (Fig. 6-5B). Marginal papillae may be paired or irregularly arranged in an approximate band along the posterior margin. There was no consistent space that separated the dorsal and ventral sections of the marginal papilla, indicating that the papillae generally formed a continuous band (Fig. 6-5A). The holotype had 15 marginal papillae with some papillae fused into a flap-like papilla dorsally and one ventrally.

Figure 6-5. Representations of the gill pore structure in the populations of E. minimus: (A) no fusion and some pairing of papillae; (B) some fusion; (C) Representation of the gill pore structure from the recently identified population of E. minimus (the central process and marginal papillae are labeled in Fig. 6-1.)

Undescribed Entosphenus A On November 2-3, 1990, metamorphosed lamprey were collected in the San Joaquin River below the bridge in Friant, California. Four specimens were subsequently found to have a unique arrangement of gill pore papillae that have not been found in any specimens in the genus Entosphenus. The specimens had marginal papillae extending into the middle of the pore from the posterior margin (Fig. 6-6). Papillae in this area and on the extension were larger than the papillae that may be in pairs and became progressively smaller dorsally and ventrally. There was variation in the

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positioning of the papillae among specimens (Fig. 6-6) but the extension into the middle of the pore was consistent. The number of marginal papillae ranged up to about 20 when they could be distinguished; however, smaller papillae were difficult to distinguish in some specimens. The most common structure was the prominent papillae in the mid-pore region and some pairing of papillae dorsally and ventrally. Furthermore, some papillae were fused to form bars and the number of papillae were reduced in some specimens. The central papilla was small, but prominent, about one and one half times the size of the larger marginal papillae (Fig. 6-6). Two specimens were preserved shortly after being collected on November 16, 1990, and two were kept alive in fresh water at the Pacific Biological Station in Nanaimo, British Columbia, Canada. Both survived until they were preserved on May 15, 1991 and on July 23, 1991 without becoming sexually mature. Both specimens were in a tank with other lamprey that fed on prey, but it was not determined if these two specimens fed. However, the prominent, sharp and cornified teeth clearly indicated that the four specimens were parasitic. In addition to the gill pore structure, the specimens were also noteworthy because of a large eye (Fig. 6-7, Table 6-3). The velar tentacles of the four specimens all had a single tentacle in the middle with wings of smaller tentacles on each side. The single tentacle in the middle was longer or about the same length as the larger tentacle in the wings. The total number ranged in number from 8 to 13 and averaged 10. The number of the velar tentacles readily distinguished this lamprey from E. tridentatus or E. macrostoma. The undescribed specimens were most similar to E. minimus, but had a larger eye and very different gill pore papillae. The sample was small and there was enough variability in the four specimens to indicate that further study is needed before the appropriate classification is understood. What is clear is that the structure of the gill pore papillae is unique within the genus. There was a second group of specimens that were more abundant in the collection and had an even larger eye but gill pore papillae were arranged in a straight continuous band and were very small. The gill pore papillae for this unnamed group need to be examined from mature specimens in order to be certain of the structure.

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Figure 6-6. Representations of typical gill pore structure of the undescribed Entosphenus A showing the accumulation of marginal papillae in the mid-pore area (the central process and marginal papillae are labeled in Fig. 6-1.)

Figure 6-7. Photograph of undescribed Entosphenus A showing the exceptionally large eye (scale bar is in mm). See Table 6-3 for morphometric comparisons.

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Table 6-3. Summary of selected morphometric measurements (% of total length) reported in Beamish (1982) and Lorin et al. (2000). Character Total length (range) Branchial length (range) Prebranchial length (range) Eye diameter (range) Disc length (range) N Velar tentacles 1

E. similis E. similis Lorin et al. Beamish 2000 1982 168.3 226 (124.7-229.0) (188-269) 10.2 9.9 (9.2-11.3) (8.5-11.8) 15.1 14.3 (12.8-16.2) (13.0-15.6) 2.3 2.0 (1.8-2.7) (1.4-2.4) 8.9 9.3 (7.7-9.5) (7.8-10.4) 19 22 N = 7-9 and wings1

E. minimus E. minimus Lorin et al. Beamish 2000 1982 109.0 8.5 (79.9-145) (7.2-12.9) 10.1 9.2 (8.9-12.2) (7.9-10.8) 13.5 14.8 (11.0-17.0) (13.4-16.4) 2.6 2.4 (2.1-3.3) (2.1-3.1) 6.7 6.2 (5.0-8.6) (5.0-7.4) 58 45 N = 5-9 and wings1

Undescribed Entosphenus A 150.5 (141-158) 9.5 (9.1-10.1) 13.1 (12.2-15) 3.2 (3.1-3.5) 6.2 (5.4-7.3) 4 N = 8-13 and wings

from Renaud (2011)

Entosphenus folletti Entosphenus folletti was first described by Vladykov & Kott (1976a). It was separated from E. tridentatus and E. minimus by its non-functional intestine and it differed from the nonparasitic E. lethophagus in the same river system, mainly because of the larger disc in E. folletti. Other differences were an additional cusp on the middle two inner lateral teeth and a longer, median velar tentacle, but the major diagnostic characters were the life history type and the larger disc. E. folletti is thought to be restricted to the Lost River sub basin, the Lower Klamath River and Goose Lake sub basin of the Sacrament River drainage (Vladykov & Kott 1976a). There were four specimens examined, all from the Canadian Museum of Nature, which included the holotype and three paratypes. The marginal papillae were about one half as numerous as found in E. tridentatus and the 34 in the holotype were considered to be moderately abundant relative to E. tridentatus. The small to medium sized papillae were in a continuous band along the posterior margin (Fig. 6-8). Papillae were slightly clumped dorsally and arranged in two or three imperfect rows (Fig. 6-8). In two specimens, a few papillae were fused to form spade-shaped papillae. The central process was about four times the size of the marginal papillae (Fig. 6-8). The base was about four times the width of the base of the marginal papilla. Two of the paratypes had 42 and 43 papillae and a third specimen had 34 papillae. However, the paratype from Lost River near the outlet of Clear Lake (CMNFI 1975-08051) had a smaller, dome-shaped central process and between 13 and 18 marginal papillae that were mostly in a single row with three fused papillae (Fig. 6-8). It is possible that the four

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specimens represented the range of variation for E. folletti, but it is also possible that individuals similar to the paratype from Lost River that had the much smaller number of papillae also had other differences of taxonomic relevance.

Figure 6-8. (A) Representation of typical gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) of E. folletti. (B) Representation of the gill pore structure of the paratype (CMNFI 1975-08051) showing the different arrangement of the marginal papillae and a smaller central process.

Entosphenus lethophagus E. lethophagus was initially described by Hubbs (1971). It was the first species to be recognized as a nonparasitic derivative of E. tridentatus. Specimens were from the Pit River system of northeastern California and the Klamath River system in south-central Oregon. The holotype and paratypes were from the Fall River, a tributary of the Pit River in California. In the description of the new species Hubbs (1971) noted the local variability of lampreys in the area where E. lethophagus was found that he believed did not warrant species or subspecies recognition. In addition to being nonparasitic, E. lethophagus had a larger disc and fewer velar tentacles than E. tridentatus. Interestingly, Lorion et al. (2000) found that their description of E. minimus identified sufficient similarities with L. lethophagus for them to consider that they were sister species derived from a common ancestor that was not E. tridentatus.

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The description of the gill pore papillae was based on four museum specimens. An additional description was provided for specimens from Hat Creek that were identified as E. lethophagus, but had a different gill pore structure. The marginal papillae were small in size and number (n = 23, 25, 25, 25). Papillae were tube- or spade-shaped (Fig. 6-9A). The papillae formed a band along the posterior margin with some spaced randomly and some in pairs or threes (Fig. 6-9A). The central process was prominent and large relative to the marginal papillae, approximately three times the size of the marginal papillae (Fig. 6-9A).

Figure 6-9. (A) Representation of typical gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) of E. lethophagus. (B) Representation of the typical structure of specimens with fused papillae and a smaller central process.

Six specimens from Hat Creek were examined from two collections. Distinct papillae were rarely found. Instead papillae were fused into bars or a rope-like structure (Fig. 6-9B). In some specimens there was a separation of the bar into smaller segments. The central process was thick and small, and different than in those specimens represented in Fig. 6-9A. It is apparent that the structure found in the Hat Creek sample is not similar to the type specimens. The relevance of the differences in

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structures requires additional study that should include DNA-based genetic analysis. Entosphenus hubbsi E. hubbsi was identified by Vladykov & Kott (1976b) with an additional description in 1984 (Vladykov & Kott 1984). This is a nonparasitic species that they considered to be more closely related to Entosphenus tridentatus. However, Docker et al. (1999) reported that E. hubbsi was genetically more similar to L. ayresii and L. richardsoni than it was to other Entosphenus species. The recent American Fisheries Society book on the Common and Scientific Names of Fishes from the United States, Canada, and Mexico (Page et al. 2013) uses the genus Lampetra but Renaud (2011) and Potter et al. (2014) retain the genus Entosphenus. The species was first described from the Friant-Kern Canal in California, and is now known from a number of locations with a center of abundance in the San Joaquin River drainage (Brown & Moyle 1993). I have specimens in my collection from the San Joaquin River below the bridge in the Friant and the Merced River at the Merced Hatchery. I examined the holotype and three paratypes from one collection (Table 6-1) as well as 15 specimens from my own collection. The gill pore papillae are unique within the genus Entosphenus. The papillae were located on two fleshy pads with a distinct separation between the pads that were part of the posterior wall of the gill pore (Fig. 6-10). The pads and the papillae were almost “hand-like,” with the papillae resembling fingers. The number of papillae at the posterior edge of each pad varied from two to seven, but most often were four or five, resulting in a total number ranging from six to 13. In mature E. hubbsi, the papillae were large and extended through the gill pore (Fig. 6-10). The central process was small and variable in structure from small dome shaped to small and pencil-like (Fig. 6-10). The five samples from the San Joaquin River collected and preserved in November 1990, were large averaging 167 mm (156 mm-173 mm).

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Figure 6-10. (A) Representation and (B) photograph of a typical gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) of E. hubbsi.

Lampetra pacifica Five specimens were examined from two collections including the holotype and four paratypes. The central process was very small and smaller than in E. hubbsi (Fig. 6-11). The number of marginal papillae was reduced, ranging from eight to 12. Marginal papillae were in a single row or distinct dorsal and ventral fleshy pads with a clear separation between the pads at the mid posterior margin of the pore. In some specimens, a single papilla was positioned outside of the fleshy pad in the mid-posterior area (Fig. 6-11).

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Figure 6-11. Representation of typical gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) of L. pacifica.

Discussion The differences in the gill pore papillae in the species of the genus Entosphenus provide species-specific information as well as population differences which may be of taxonomic significance. There is a trend in the derivatives of E. tridentatus to a reduction in the number of marginal papillae, but less in the size of the central process (Fig. 6-12, Table 6-4). As the number of the marginal papillae is reduced, there also is a trend for the papillae to fuse. The functions of the papillae and the central process remain to be determined, but because the papillae increase in size as animals mature prior to spawning, it is probable that they have at least some sensory function. A more confined distribution in the freshwater parasitic and nonparasitic derivatives may change the need for the structures, resulting in the observed changes.

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Fig. 6-12. Relationship of gill pore structure (the central process and marginal papillae are labeled in Fig. 6-1) among species in the genus Entosphenus. Nonparasitic species are shown below the dashed line. Lines indicate the possible evolutionary relationships among species. Structures within the gill pore of E. hubbsi do not appear to be closely related to other species in the genus.

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Table 6-4. Summary of the gill pore structures. Species E. tridentatus

E. macrostomus

E. similis

E. minimus

Undescribed Entosphenus A

E. folletti

E. lethophagus

E. hubbsi

L. pacifica

Central process Large, 4 times the length of the marginal papillae, pigmented at base Large, 3 times the length of the marginal papillae, not pigmented at base Prominent, about 2 times the length of the marginal papillae

Short, stubby, about 1 to 1 ½ times the length of the marginal papillae Short, stubby , about 1 to 1 ½ times the length of the marginal papillae, wide base

Prominent, about 4 times the length of the marginal papillae, wide base Prominent, about 3 times the length of the marginal papillae Not prominent, smaller in length than the marginal papillae

Not prominent, about one fifth the length of the marginal papillae, and smaller than in E. hubbsi

Marginal papillae Large number (~70), pencil shaped, pigmented at base, in a broad band Large number (~60), larger than E. tridentatus, not pigmented, in a broad band Moderate number (~35) in a continuous band that form imperfect rows with some clumping dorsally and ventrally Small number (9-22) in an irregular band with frequent fusions Small number (~20) that form a narrow band along the posterior edge of the pore where the papillae extend into the mid pore area and increase in size Moderate number (34-43) in imperfect rows with some clumping dorsally and ventrally Small in size and number (23-25) Small number (6-13) of large marginal papillae that are located on the edge of two separate fleshy pads that extend out from the gill pore Small number (8-12) of large marginal papillae in a single row on a dorsal and ventral fleshy pad

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E. tridentatus has the most papillae (average of 67) that are pigmented at the base, and the longest and most prominent central process of the species in the genus. The pigmentation of the papilla base virtually only occurs in E. tridentatus. Beamish (2010) showed that gill pore structure is similar throughout the distribution of E. tridentatus although the sample size was minimal. Despite the small sample size, the consistency of the structure is evidence of the importance of the papillae in the anadromous life history. E. macrostomus which probably is the most recently derived species (Beamish 1982), has slightly fewer papillae than E. tridentatus (average of 59, Table 6-4), but the papillae are larger and the central process remains large. There was virtually no pigmentation of papillae or the central process. E. similis has even fewer marginal papillae, about one half of the number for E. tridentatus, and a smaller central process. E. minimus has the smallest number of papillae of which some may be fused. Samples in this study from the extinct population averaged only 12 marginal papillae. There were slightly more in the single sample of the recently discovered population (average of 15). The central process was also greatly reduced in size. The gill pore structure, including the fused papillae, readily distinguished E. minimus from E. similis. The gill pore structure of the two nonparasitic derivatives is similar, but E. lethophagus has a smaller number of papillae of about 23-25 compared to 34-43 for E. folletti. One paratype of E. folletti from Lost River (CMNFI-1975-08051) had between 13 and 18 marginal papillae that were mostly in a single row with several papillae fused into bar-shaped structures. As only a small number of specimens were examined, the variability may occur within the species, but it is also possible that there are other differences of taxonomic importance in the Lost River population. There also was a major difference in the structure of the marginal papillae from a population of E. lethophagus in Hat Creek. In a number of specimens (Fig. 6-9B), papillae were fused into rope-like structures that could be one continuous bar or a mixture of bars and an occasional separate papilla. In both the Hat Creek population and the Lost River population, a DNA-based genetic analysis may help resolve the taxonomy. It is apparent from the gill pore structure that: E. macrostomus and E. similis are derivatives of E. tridentatus with the smaller number of papillae in E. similis suggesting that it evolved earlier than E. macrostomus (Fig. 612). It is likely that E. minimus evolved from an E. similis-like ancestor and not directly from E. tridentatus. The very small number and irregular shape of the papillae suggest that the separation occurred well in the past. The undescribed Entosphenus A would also have a similis-like ancestor. It is possible that E. folletti and E. lethophagus evolved from an E. similis-

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like ancestor as there needed to be a reduction in gill pore papillae as well as the ability to osmoregulate in fresh water. Lorion al. (2000) proposed that E. minimus may originate atavistically from E. lethophagus and not from an E. tridentatus-like ancestor. We show in Beamish et al. (2016) that a population of the non-parasitic L. richardsoni can produce a parasitic life history type. However, the gill pore structure of E. lethophagus is very different from E. minimus and more similar to an E. similis-like ancestor which I interpret to indicate that an E. similis-like species was the ancestor of both E. folletti and E. lethophagus. A benefit of studying gill pore structure is that there are clear examples of major differences at what might be considered the population level. One example is included in this paper, but there are several more examples from specimens in my collection and in museum collections. In this paper, the undescribed Entosphenus A had a gill pore structure that was unique to Entosphenus with a concentration of small papillae in the middle of the pore and small number of smaller papillae dorsally and ventrally. The central process was also small. The four specimens in my collection all had a very large eye (Fig. 6-7, Table 6-3). The taxonomic significance of these specimens remains to be determined, but the specimens can readily be distinguished by the bunched marginal papillae at the center of the distal edge of the pore. The specimens from Copco Lake were another example of where further study is needed to determine the taxonomic significance of the gill pore structure. Papillae can be difficult to count in immature specimens of parasitic species and it needs to be confirmed that the mature individuals have a similar number of papillae. However, despite being immature, the specimens were of sufficient size, that I suspect that the small number in the two specimens is representative of the population. Importantly, it is the gill pore structure that readily allows specimens to be selected for study. An extreme example of the changed gill pore structure occurs in E. hubbsi. Marginal papillae in E. hubbsi were reduced in number (ranging 613) and at the margins of two fleshy pads with a distinct space between the pads. The central process was small and almost reduced to a small swelling in some pores. A characteristic feature of the papillae was that they extended through the pore and were easily visible without magnification. E. hubbsi was originally placed in the genus Entosphenus primarily because of the dentition, including the presence of posterials (Vladykov & Kott 1976b). Using mitochondrial DNA, Docker et al (1999) reported that L. hubbsi was a southern relict species of L. ayresii. However, the gill pore papillae structure had no similarity to the structure of L. ayresii (Beamish 2010). Of importance is the similarity in the

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structure to L. pacifica. Beamish (2010) reported that the reduced number of papillae in L. pacifica readily distinguished the lamprey from L. richardsoni. In this study, I show that the few papillae were also on a fleshy pad almost identical to E. hubbsi which was not found in any specimens from other species in the genus Entosphenus. This similarity and the similar low myomere number may indicate a past close relationship between L. pacifica and E. hubbsi. Boguski et al. (2012) used DNA from the mitochondrial cytochrome b gene to demonstrate that L. pacifica and E. hubbsi formed distinct clades that had been isolated for a long time. The similarity in the gill pore structure between these two species also supports the closer relationship of E. hubbsi to Lampetra as proposed by Docker et al. (1999), but I suspect that we still have much more to learn about the derivation of E. hubbsi. Gill pore papillae provide evidence of taxonomic relationships, but they may not be a diagnostic character in some species. Further studies that include understanding more about the function of the papillae may show that for some species or populations, the variability is too great for the character to be of taxonomic importance. There also is a restriction in the use of gill pore papillae to maturing specimens of some species, particularly anadromous parasitic species. I found that gill pore structure is particularly useful in the taxonomy of nonparasitic lampreys. I have looked at a number of populations of L. richardsoni and found specific differences in gill pore structure. In one case where DNA samples were available, it was found using the methods in Beamish et al. (2016), specimens originally distinguished by their gill pore structure were possibly a distinct species. The existence of greater variation in lamprey populations has recently been recognized in several studies. Boguski et al. (2012) reported the possibility of four new species of Lampetra based on DNA from ammocoetes. Mateus et al. (2013b) identified three new cryptic species of Lampetra planeri using mitochondrial DNA markers. Despite looking at only a small number of specimens, the gill pore structure shows a similar diversity in the genus Entosphenus, indicating that gill pore structure is another character that will contribute to the understanding of the taxonomic relationships among lampreys.

Summary There are papillae in the gill pores of metamorphosed lamprey that are positioned along the posterior margin of the pore and a more central, rigid structure called the central process. The number, arrangement and shape of

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these structures differ among many of the known species of lampreys. In the genus Entosphenus, there are either six or seven recognized species. Gill pore structures are similar between E. tridentatus and E. macrostomus. E. similis has about one half the number of marginal papillae found in E. tridentatus and E. macrostomus, but a smaller central process. E. minimus has even fewer marginal papillae that may be fused and a much reduced central process. The gill pore structure in E. folletti is similar to E. similis. E. lethophagus retained a large central process, but the number of marginal papillae is reduced and more paired than in all other species in the genus. The gill pore structure of E. hubbsi gill is unlike any other species in the genus Entosphenus with a very small central process and marginal papillae that are positioned on two fleshy pad-like appendages instead of along the posterior margin of the pore. This structure is similar to the gill pore structure of Lampetra pacifica, supporting the interpretation that E. hubbsi belongs in the genus Lampetra. Major differences in gill pore structure occur in some populations of Entosphenus that may indicate that they are new species. There is recent recognition that there is more variability of taxonomic significance within lamprey populations than is reflected in the current number of species and gill pore papillae are a character that will help identify potentially different species and populations of significance.

Acknowledgements Chrys Neville collected many of the specimens in the late 1980s and early 1990s. Curators of museums were always helpful. Tyler Stitt prepared some figures and Lana Fitzpatrick assisted in the preparation of the manuscript and provided interpretative drawings. Joy Wade reviewed an earlier draft.

References Beamish R.J. 1982. Lampetra macrostoma, a new species of freshwater parasitic lamprey from the west coast of Canada. Canadian Journal of Fisheries and Aquatic Sciences 37, 1906-1923. Beamish R.J. 2010. The use of gill pore papillae in the taxonomy of lampreys. Copeia 2010, 618-628. Beamish R.J., Withler R., Wade J. & Beacham T. 2016. A nonparasitic lamprey produces a parasitic life history type: the Morrison Creek enigma. In Orlov A.M. & Beamish R.J. (eds.): Jawless Fishes of the World. Vol. 1. Pp. 191-230. Cambridge: Cambridge Scholars Publishing.

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Boguski D.A. Reid S.B., Goodman D.H. & Docker M.F. 2012. Genetic diversity, endemism and phylogeny of lampreys within the genus Lampetra sensu stricto (Petromyzontiformes: Petromyzontidae) in western North America. Journal of Fish Biology 81, 1891-1914. Bond C.E. & Kan T.T. 1973. Lampetra (Entosphenus) minima n. sp., a dwarfed parasitic lamprey from Oregon. Copeia 1973, 568-574. Brown L.R. and Moyle P.B. 1993. Distribution, ecology and status of the fishes of the San Joaquin River drainage, California. California Fish and Game 79, 96-113. Coots M. 1955. The Pacific lamprey, Entosphenus tridentatus, above Copco Dam, Siskiyou County, California. California Fish and Game 41, 118-119. Docker M.F. 2009. A review of the evolution of nonparasitism in lampreys and an update of the paired species concept. In Brown L.R., Chase S.D., Mesa M.G., Beamish R.J. & Boyle P.B. (eds.): Biology, Management, and Conservation of Lampreys in North America. Pp. 71-114. Bethesda: American Fisheries Society. Docker M.F., Youson J.H., Beamish R.J. & Devlin R.H. 1999. Phylogeny of the lamprey genus Lampetra inferred from mitochondrial cytochrome b and ND3 gene sequences. Canadian Journal of Fisheries and Aquatic Sciences 56, 2340-2349. Gess R.W., Coates M.I. & Rubidge B.S. 2006. A lamprey from the Devonian period of South Africa. Nature 443, 981-984. Gill H.S., Renaud C.B., Chapleau F., Mayden R.L. & Potter I.C. 2003. Phylogeny of living parasitic lampreys (Petromyzontiformes) based on morphological data. Copeia 2003, 687-703. Hubbs C.L. 1925. The life cycle and growth of lampreys. Papers of the Michigan Academy of Science Arts and Letters 4, 587-603. Hubbs C.L. 1971. Lampetra (Entosphenus) lethophaga, a new species, the nonparasitic derivative of the Pacific lamprey. Transactions of the San Diego Society of Natural History 16, 125-164. Hubbs C.L. & I.C. Potter. 1971. Distribution, phylogeny and taxonomy. In Hardisty M.W. & Potter I.C. (eds.): The Biology of Lampreys. Volume 1. Pp. 1-65. London: Academic Press. Jelks H.L., Walsh S.J., Burkhead N.M., Contreras-Balderas S., Diaz-Pardo E., Hendrickson D.A., Lyons J., Mandrak N.E., McCormick F., Nelson J.S., Platania S.P., Porter B.A., Renaud C.B., Schmitter-soto J.J., Taylor E.B. & Warren M.L., Jr. 2008. Conservation status of imperiled North American freshwater and diadromous fishes. Fisheries 33, 372407.

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Lang N.J., Roe K.J., Renaud C.B., Gill H.S., Potter I.C., Freyhof J., Naseka A.M., Cochran P., Espinoza Perez H., Habit E.M., Kuhajda B.R., Neely D.A., Reshetnikov Y.S., Salnikove V.B., Stoumboudi M.T. & Mayden R.L. 2009. Novel relationships among lamprey (Petromyzontiformes) revealed by a taxonomically comprehensive molecular data set. In Brown L.R., Chase S.D., Mesa M.G., Beamish R.J. & Boyle P.B. (eds.): Biology, Management, and Conservation of Lampreys in North America. Pp. 41-56. Bethesda: American Fisheries Society. Lorion C.M., Markle D.F., Reid S.B. & Docker M.F. 2000. Redescription of the presumed-extinct Miller Lake lamprey, Lampetra minima. Copeia 2000, 1019-1028. Mateus C.S., Stange M., Berner D., Roesti M., Quintella B.R., Alves M.J., Almeida P.R. & Salzburger W. 2013a. Strong genome-wide divergence between sympatric European river and brook lampreys. Current Biology 23, R649-R650. Mateus C.S., Alves M.J., Quintella B.R. & Almeida P.R. 2013b. Three new cryptic species of the lamprey genus Lampetra Bonnaterre. 1788 (Petromyzontiformes: Petromyzontidae) from the Iberian Peninsula. Contributions in Zoology 82, 37-53. Moyle P.B. 2002. Inland Fishes of California: revised and expanded. Berkeley: University of California Press. Moyle P.B., Brown L.R., Chase S.D. & Quiñones R.M. 2009. Status and conservation of lampreys in California. In Brown L.R., Chase S.D., Mesa M.G., Beamish R.J. & Boyle P.B. (eds.): Biology, Management, and Conservation of Lampreys in North America. Pp. 279-292. Bethesda: American Fisheries Society. Page L.M., Espinosa-Pérez H., Findley L.T. Gilbert C.R., Lea R.N., Mandrak N.E., Mayden R.L. & Nelson J.S. 2013. Common and Scientific Names of Fishes from the United States, Canada, and Mexico, 7th edition. Bethesda: American Fisheries Society. Potter I.C., Gill H.S. & Renaud C.B. 2014. Petromyzontidae: Lampreys. In Warren M.L., Jr. & Burr B.M. (eds.): Freshwater Fishes of North America. Volume 1. Petromyzontidae to Catostomidae. Pp. 105-139. Baltimore: Johns Hopkins University Press. Potter I.C., Gill H.S., Renaud C.B. & Haoucher D. 2015. The taxonomy, phylogeny, and distribution of lampreys. In Docker M.F. (ed.): Lampreys: Biology, Conservation and Control. Volume 1. Pp. 35-73. Dordrecht: Springer Science+Business Media. Reid S.B., Boguski D.A., Goodman D.H. & Docker M.F. 2011. Validity of Lampetra pacifica (Petromyzontiformes: Petromyzontidae) a brook

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lamprey described from the lower Columbia River basin. Zootaxa 3091, 42-50 Renaud C.B. 1997. Conservation status of Northern Hemisphere lampreys (Petromyzontidae). Journal of Applied Ichthyology 13, 143-148. Renaud C.B. 2011. Lampreys of the world. An annotated and illustrated catalogue of lamprey species known to date. FAO: Rome. Renaud C.B., Docker M.F. & Mandrak N.F. 2009. Taxonomy, distribution, and conservation of lampreys in Canada. In Brown L.R., Chase S.D., Mesa M.G., Beamish R.J. & Boyle P.B. (eds.): Biology, Management, and Conservation of Lampreys in North America. Pp. 293-310. Bethesda: American Fisheries Society. Siskiyou Regional Education Project. 2003. A petition for rules to list: Pacific lamprey (Lampetra tridentata), river lamprey (Lampetra ayresi), western brook lamprey (Lampetra richardsoni), and kern brook lamprey (Lampetra hubbsi) as threatened under the Endangered Species Act. Submitted to U.S. Fish and Wildlife Service on 23 January 2003, 70 pp. Taylor E.B., Harris L.N., Spice E.K. & Docker M.F. 2012. Microsatellite DNA analysis of parapatric lamprey (Entosphenus spp.) populations: implications for evolution, taxonomy, and conservation of a Canadian endemic. Canadian Journal of Zoology 90, 291-303. Vladykov V.D. 1973. Lampetra pacifica, a new nonparasitic species of lamprey (Petromyzonidae) from Oregon and California. Journal of the Fisheries Research Board of Canada 30, 205-213. Vladykov V.D. & Follett W.I. 1967. The teeth of lampreys (Petromyzonidae): their terminology and use in a key to the Holarctic genera. Journal of the Fisheries Research Board of Canada 24, 10671075. Vladykov V.D. & Kott E. 1976a. A second nonparasitic species of Entosphenus Gill, 1862 (Petromyzonidae) from Klamath River system, California. Canadian Journal of Zoology 54, 974-989 Vladykov, V.D., & E. Kott. 1976b. A new nonparasitic species of lamprey of the genus Entosphenus Gill, 1862, (Petromyzonidae) from south central California. Bulletin of the Southern California Academy of Science 75, 60-67 Vladykov, V.D. & Kott E. 1979. Satellite species among the holarctic lampreys (Petromyzonidae) Canadian Journal of Zoology 57, 860-867. Vladykov, V.D., & Kott E. 1984. A second record for California and additional morphological information on Entosphenus hubbsi Vladykov and Kott, 1976 (Petromyzonidae). California Fish and Game 70, 121-127.

CHAPTER SEVEN REVIEW OF WESTERN TRANSCAUCASIAN BROOK LAMPREY, LETHENTERON NINAE NASEKA, TUNIYEV & RENAUD, 2009 (PETROMYZONTIDAE) SAKO TUNIYEV, ALEXANDER NASEKA AND CLAUDE RENAUD

Introduction Lampreys constitute a small group of about 40í45 species depending, in part, on the methodological approach used (Docker et al. 2015) and the availability of reliable data to infer the boundaries between species. For example, Renaud (2011) recognized 40 species, Potter et al. (2015) recognized 41 species, and Maitland et al. (2015) list 44 species recognizing three recently-described brook lamprey species from Portugal (Mateus et al. 2013) as distinct from Lampetra planeri (Bloch 1784). All three publications treated Eudontomyzon vladykovi Oliva & Zanandrea, 1959, considered a distinct species by some authors (e.g., Kottelat & Freyhof 2007), as a synonym of Eudontomyzon mariae (Berg 1931). Also, a number of putative lamprey species, either extant (e.g., Yamazaki et al. 2003, 2006; Boguski et al. 2012) or extinct (e.g., Kottelat et al. 2005), have not yet been formally described. If one accepts the more liberal taxonomy of Kottelat and Freyhof (2007) and Maitland et al. (2015), 21 of the 45 species, belonging to six genera, are distributed in Eurasia. Interestingly, only seven species are parasitic while the majority, 14, are nonparasitic. In the Black Sea-Sea of Azov basin, the following nonparasitic species occur: Danubian brook lamprey E. vladykovi (Danube), Ukrainian brook lamprey E. mariae (Dniester, Dnieper in the Black Sea basin to Don and Kuban' in the Sea of Azov basin), Western Transcaucasian brook lamprey Lethenteron ninae Naseka, Tuniyev &

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Renaud, 2009 (Psezuapse, Shakhe, Mzymta, Psou, Bzipi (Bzyb’), and Mokvi (Mokva) rivers in Russia and Georgia), and Turkish brook lamprey Lampetra lanceolata Kux & Steiner, 1972 (øyidere and økizdere streams west of Rize, north-eastern Turkey, and a stream in Lake Sapanca basin in northwestern Turkey) (e.g., Lang et al. 2009; Naseka et al. 2009; Renaud 2011; Freyhof 2014b). Morphological and evolutionary affinities between several parasitic and nonparasitic species have been suggested for a long time (e.g., Hubbs 1925); these species were termed “paired species” (Zanandrea 1959). Vladykov & Kott (1979) introduced the more general term “stem-satellite species” because they identified, based on morphological criteria, several cases in which more than one brook lamprey (satellite) species had apparently been derived from a single parasitic (stem) species; several socalled relict species (nonparasitic lampreys that occur at the southern limits of distribution of the Northern Hemisphere lampreys) could not be unambiguously paired with extant parasitic species (e.g., Docker 2009; Potter et al. 2015). Based on morphological criteria, among the four nonparasitic lampreys distributed in the Black Sea basin, E. mariae (including E. vladykovi when synonymized with the latter species) is paired with the Carpathian lamprey Eudontomyzon danfordi Regan, 1911, L. lanceolata is, probably, a derivative of the European river lamprey Lampetra fluviatilis (Linnaeus, 1758) though the two species do not occur sympatrically, while the affinity of L. ninae is unclear (e.g., Potter et al. 2015). The Black Sea nonparasitic species were traditionally assigned to three genera (or subgenera of Lampetra according to some authors, such as Bailey (1980)) representing distinct morphological clades (Gill et al. 2003; Monette & Renaud 2005; Renaud 2011). Diagnostic morphological characters in adults are limited to few character sets among which the pattern and morphology of teeth in the oral disc is the most informative. Eudontomyzon Regan, 1911 is characterised by numerous labial teeth commonly present on all fields of the oral disc; in Lampetra Bonnaterre, 1788, the labial teeth are present only in the anterior field while exolaterals and posterials are absent; and in Lethenteron Creaser & Hubbs, 1922, anterials are present, exolaterals are usually absent but if present, only one or two occur per lateral field, exceptionally a complete row, and a single row of posterials is present, either complete or incomplete (Gill et al. 2003; Renaud 2011). Additionally, European species of Eudontomyzon typically have unicuspid upper endolateral teeth and bicuspid middle endolateral teeth, European species of Lampetra (including the Po brook lamprey L. zanandreai Vladykov, 1955) typically possess bicuspid upper

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endolateral teeth and tricuspid middle endolateral teeth, while Eurasian Lethenteron commonly have both the upper and middle endolaterals bicuspid. Molecular methods have been used to help resolve the phylogeny and taxonomy of lampreys of the genera Eudontomyzon, Lethenteron, and Lampetra at different taxonomic levels (e.g., Docker et al. 1999; Yamazaki et al. 2006; Lang et al. 2009; Li 2014; White 2014). For the Black Sea brook lampreys, especially important were data from the mitochondrial cytochrome b gene by Lang et al. (2009), who showed that some nonparasitic species formed clades with parasitic species which, from their morphology, had been allocated by taxonomists to different genera. Thus, L. zanandreai placed in the genus Lethenteron because its endolaterals are usually bicuspid and because posterials are commonly present (e.g., Bianco 1986; Renaud 2011), was returned to Lampetra and Asian Pacific E. morii was shown to have no affinity with true European Eudontomyzon. Additionally, based on unpublished results deposited in GenBank (GQ206176 from the Chakhtsutsyr Stream, misidentified as L. lanceolata), L. ninae was transferred to Lampetra (Freyhof 2014a). These data (Lang et al. 2009) suggested that some morphological characters may be homoplasious within lampreys contrary to what had been previously hypothesised. Recently, Li (2014), based on phylogenetic trees using the cytochrome b gene, supported the hypothesis that L. zanandreai and L. ninae had derived from a Lampetra fluviatilis-type ancestor. Her cytochrome b gene analyses did not include any L. lanceolata specimen, but using the nuclear TAP2 gene intron L. ninae and L. lanceolata were not reciprocally monophyletic. However, the resolution of the TAP2 trees was not high enough to resolve the specieslevel relationships since L. fluviatilis and L. zanandreai were not retrieved as monophyletic either. In summary, the molecular data (Lang et al. 2009; Li 2014) have considerably changed traditional taxonomic concepts in three major aspects as far as Eudontomyzon, Lampetra, and Lethenteron are concerned, namely: 1. Lampetra from the Pacific drainage of North America and Lampetra aepyptera (Abbott, 1860) should each be separated from Lampetra from the Atlantic drainage of Eurasia as distinct genera; 2. Lampetra from the Atlantic drainage of Eurasia includes Lampetra zanandreai; 3. Lethenteron includes Eudontomyzon morii. With regard to morphology, it supports the hypothesis (Kottelat & Freyhof 2009) that those types of dentition, which were traditionally accepted for distinguishing genera, might have evolved convergently in different clades and may be useful in diagnosing species rather than in defining lineages.

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As shown above, although both L. ninae and L. lanceolata have close molecular phylogenetic affinities with L. fluviatilis (Lang et al. 2009; Li 2014), it is still unclear if the latter species represents their immediate "stem-species". Sequences of the mitochondrial COI gene (1,072 bp) in two larvae of L. ninae from the Mzymta River showed that these shared some haplotypes with Arctic lamprey Lethenteron camtschaticum (Tilesius, 1811) (Artamonova et al. 2011). The authors proposed three hypotheses to explain their observation and synonymisation of L. ninae with L. camtschaticum. The first hypothesis, which they called the least probable, was that L. camtschaticum in the rivers of the Caucasian Black Sea coast is a relict of the ancient Tethys Sea; the second was that the Arctic lamprey dispersed into the Black Sea basin during a glacial period; and the third suggested a human-mediated introduction of L. camtschaticum into the Black Sea basin. Synonymization of L. ninae and L. camtschaticum has already been accepted in subsequent publications by Makhrov et al. (2013) and Parin et al. (2014). However, Li (2014) showed that the mean genetic (Kimura 2-parameter) distance (cytochrome b gene) between L. ninae and Asian Lethenteron species was at least 7.79%, while between L. ninae and L. fluviatilis it was much less, 3.91%, and between L. ninae and L. zanandreai only 1.63%. Also, with regards to their third hypothesis, Artamonova et al. (2011) provided no explanation for the presence of the nonparasitic species among geographically isolated river drainages across a wide area during a short period of time since the supposed lamprey introduction(s) in the last century. It is known that nonparasitic lampreys possess very low dispersal ability within and between river basins (Schreiber & Engelhorn 1998; Mateus et al. 2011). Artamonova et al. (2011) also ignored the fact that a resident brook lamprey has been reported in the eastern Black Sea area long before any fish introductions started (De Filippi 1865, p. 360, under the name Petromyzon sp.; Barach 1939, p. 60, and Barach 1941, p. 71, under the name Lampetra mariae). The allocation by Naseka et al. (2009) of L. ninae to the genus Lethenteron and of L. lanceolata to the genus Lampetra was based principally on the usual presence of a row of posterials, albeit incomplete, in the former and its absence in the latter; the same reasoning that had caused the transfer of Lampetra zanandreai to the genus Lethenteron mentioned above. While the parasitic stem species of L. ninae has not been identified, that of L. lanceolata is inferred to be L. fluviatilis (Potter et al. 2015). This contrasts with the treatment by some authors (e.g., Artamonova et al. 2011; Makhrov et al. 2013) of L. camtschaticum, widely distributed across Eurasia, comprising both an anadromous parasitic form

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and two allopatric nonparasitic resident forms; the latter (L. reissneri (Dybowski, 1869) and L. kessleri (Anikin, 1905)) having been described as distinct species. Molecular data (Li 2014) also revealed that L. camtschaticum camtschaticum, L. camtschaticum septentrionalis (Berg, 1931), L. reissneri, and L. kessleri were not reciprocally monophyletic because their relationships had not been resolved in the cytochrome b tree. In contrast, although two putative species from Japan, Lethenteron sp. S and Lethenteron sp. N are morphologically indistinguishable from each other, they were shown to be genetically distinct using both allozyme and mitochondrial sequence data (Yamazaki & Goto 1998; Yamazaki et al. 2006; Li 2014). Apparently, more genes, and in particular nuclear genes, should be used to help resolve the basis for these differences between morphological and molecular phylogenies, and taxonomic changes need not be made hastily (Potter et al. 2015).

Historical data on the distribution of the eastern Black Sea lampreys Geographic localities mentioned below can be found in Fig. 7-1. A lamprey was first recorded in Western Transcaucasia (an area south of the Greater Caucasus Range from Novorossiysk to the उoruh River (Chorokh, Chorokhi), which includes river drainages of the Black Sea in Russia, Georgia, and the Abkhazia region) under the name Petromyzon sp. by De Filippi (1865) who collected "many individuals at the larval stage in a stream near Batumi. Overall, they resemble the common small lamprey of Europe (Lampetra planeri (Bloch, 1784)) but differ only by their larger size" (p. 360, translated from Italian). Berg (1911) referred to De Filippi (1865) and Yashchenko (1895); the latter author, in a catalogue of the St. Petersburg University zoological collection, listed (p. 99) a lamprey larva identified as "Petromyzon ponticus ?" from "Novorossiysk". Since that time, the locality of Novorossiysk (a town on the north-eastern or Caucasian coast of the Black Sea in Russia) had been commonly given whenever distribution of the Transcaucasian lamprey was discussed (e.g. Berg 1931, 1948). However, Yashchenko's specimen is absent from the collection in St Petersburg University at present (our data), and we know of no extant lamprey specimens collected near Novorossiysk. Most probably, the label "Novorossiysk" refers not to the locality where the specimen was sampled but to the collection from which this specimen was obtained í i.e., Novorossiyskiy University located in the city of Odessa, north-western Black Sea coast, where the donator of the specimen, Ernst von Ballion (1816í1901), an entomologist, worked at the time (Kottelat et

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al. 2005). The former Novorossiya Province (with Odessa as its capital; now in Ukraine) is an area of distribution of E. mariae. Berg (1931), when he described the species E. mariae, did not have any specimens of lamprey from Western Transcaucasia but extended its range to include this area.

Figure 7-1. Geographic distribution of brook lampreys in eastern and south Black Sea basin. Lethenteron ninae (solid circles): Psezuapse (1), Shakhe (2), Mzymta (3), Psou (4), Bzipi (5), Mchishta (6), Gumista (7), Kelasuri (8), Kodori (9), Mokvi (10); unidentifiable (no diagnostic characters known) species (square): Lake Bebesiri (11), Inguri (12), Khobi (13), Tsivi (Rioni tributary, 14), Lake Paliastomi (15), Supsa (16), Kintrishi (17), Chakvistskali with Chelta tributary (18), Chorokhi (20); Eudontomyzon species as described by Kokotshashwili (1942) (solid square): Makhindzhauris-tskali (19); Lampetra lanceolata (solid triangle): øyidere (21), økizdere (22), stream running to Lake Sapanca (23). Question marks indicate unverified taxonomic identification.

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Barach (1939, 1941) reported a lamprey under the name E. mariae from Lake Bebesyr (Bebesiri) in the Abkhazia region and Lake Paliastomi in Georgia based on information from local people only and gave no description of any original material. Later, Barach (1960) mentioned a lamprey from Mchishta River and Sharashidze (1960) recorded one individual 160 mm long under the name Lampetra planeri from a tributary of the Kodori (both localities are in the Abkhazia region) but, again, the authors provided no information on whether adults or larvae were found. Sharashidze (1960) also provided a list of other rivers from where this brook lamprey was known (Khobi, Inguri, Kelasuri, Gumista). The first description of a lamprey specimen (a spent adult female) was made by Kokotshashwili (1942) who collected, on the 4th of March 1935, two specimens (male and female) in the Makhindzhauris-tskali (Makhindzhauri) River north of the town of Batumi in Georgia. He provided a drawing of the oral disc of the lamprey, which clearly shows the diagnostic characters of Eudontomyzon (a broad supraoral lamina and the presence of exolaterals and posterials), as was already mentioned by Naseka et al. (2009). It is important to note that his drawing does not seem to be either a reproduction or a modified re-drawing of Berg's specimen of E. mariae from the Don River drainage (e.g., Berg 1931, 1932) although Kokotshashwili (1942) identified his specimen as E. mariae. Elanidze & Demetrashvili (1973) and Shervashidze (1980) briefly listed rivers in Georgia (including the Abkhazia region) where a lamprey identified as Lampetra mariae is distributed, namely, Chorokhi, Chakvistskali (Chakvi) with a tributary at the village of Chaisubani, Khobi, Inguri, Kodori, Makhindzhauri, Bzipi, Gumista, and Kelasuri; neither examined material nor developmental stage was mentioned. Elanidze (1983) reported the finding of two larvae and one adult specimen identified as L. mariae from the middle reaches of the Inguri River in Georgia í this was the second known report of an adult lamprey from Western Transcaucasia. However, the description provided does not allow determination of the specimen even to genus. Elanidze (1983) also reported three larvae from the middle Kodori River (the Abkhazia region) identified as L. mariae but "different in external appearance and some characters from the specimens from the Inguri River" (p. 19, translated from Russian) and numerous larvae in the Chelta River at Chaisubani, one adult (no description was given) in the mouth of the Chorokhi River and two specimens (no indication as to larvae or adults) in the Chakvis-tskali River, as well as the probable occurrence of a lamprey in the Supsa River, Georgia. Lampetra lanceolata Kux & Steiner, 1972 was described from the øyidere stream in the Black Sea basin near Rize, Turkey, and Bogutskaya & Naseka (2004)

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concluded that this species rather than E. mariae is the one distributed in Russian Western Transcaucasia. The first vouchered records of a lamprey from the Black Sea coast in the Russian Federation were those of B. Tuniyev (1999)íhe collected lamprey larvae in four rivers near the towns of Sochi and Adler: Psezuapse, Shakhe, Mzymta, and Psou. Drogan (2002) collected larvae from the same rivers but no adults were found. The first adult specimen of a lamprey from Russian Western Transcaucasia (and only the third report of an adult in 150 years of ichthyological investigation from the entire Western Transcaucasia) was collected by S. Tuniyev on the 20 September 2006 in the Mzymta River near the village of Kazachiy Brod. Since then, 1 adult and 12 larvae reared in aquaria to the postmetamorphic stage have been collected and were the basis for the original description of L. ninae together with larvae collected in the Shakhe, Mzymta, Psou (Chakhtsutsyr Stream), Bzyb', and Mokva rivers (Naseka et al. 2009). To summarise, a lamprey originally described as L. ninae is known from rivers of the Black Sea coast in Russia and north-western Georgia (Abkhazia region), from west to east: Psezuapse, Shakhe, Mzymta, Psou (Chakhtsutsyr Stream), Bzyb’, and Mokva rivers (Tuniyev 1999; Drogan 2002; Bogutskaya & Naseka 2004; Tuniyev 2005, 2006, 2008; Naseka et al. 2009). Within this range, a lamprey identified as L. mariae is also known from the Mchishta River, which is located east of the Bzyb' (Barach 1960), and, south-eastwards, in Gumista, Kelasuri, and Kodori rivers (Elanidze & Demetrashvili 1973; Elanidze 1983) (Fig. 7-1). Further southwards, a lamprey is known (De Filippi 1865; Kokotshashwili 1942; Barach 1960; Elanidze 1983; Ninua & Japoshvili 2008) from Lake Bebesyr, the Inguri (Enguri) River, a tributary of the Rioni River (the Tsivi River), Lake Paliastomi (south of the Rioni River delta), the Supsa River (probably), the Kintrishi River (our data, only larvae), the Chakvis-tskali River, the Makhindzhauri River, the Chelta River and rivulets near Batumi, the Khobi River, and in the Chorokh River at the border between Georgia and Turkey. However, identification of this lamprey still needs to be established because only four adults have been recorded from the entire area and only one (the Makhindzhauri female specimen) of these was described and it refers to a Eudontomyzon species. Further along the Black Sea coast, the species L. lanceolata is distributed in the øyidere and økizdere, streams flowing into the Black Sea west of Rize, north-eastern Turkey, andí800 km westwards from these localitiesíin a stream running to Lake Sapanca in north-western Turkey (Kux & Steiner 1972; Lang et al. 2009; Freyhof 2014b; Li 2014). Thus, our knowledge of the brook lamprey(s) along the Black Sea coast from Psezuapse River (Russia) to the øyidere River and Lake

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Sapanca (Turkey) (Fig. 7-1) is rather fragmentary. Two morphologically distinct "relict" nonparasitic species (Lethenteron-like and Lampetra-like) are respectively distributed in the north and in the south of the area and separated from each other by a poorly known form morphologically close to Eudontomyzon. Therefore, we believe that an increase in our knowledge of morphological variability within the known range of the species formally described as L. ninae will help to clarify the taxonomy of the Eastern Black Sea lampreys, the limits of their ranges, and their phylogenetic relationships.

Material and Methods All measurements on specimens were taken within a short period of time to avoid problems due to differential shrinkage of body sections, which occur during initial fixation in 4í5% formalin followed by preservation in 70% ethanol. For methods for description of body measurements and counting of the teeth, trunk myomeres, and oral fimbriae and evaluation of the extent of pigmentation coverage and the defined areas for those various characters in ammocoetes see Naseka et al. (2009). Note, however, that we refer here to the bulb of the tongue precursor as the middle prong. Also, the following characters not recorded by Naseka et al. (2009) were added here: urogenital papilla length, cloacal slit length, myomeres to origin of first dorsal fin and to insertion of first dorsal fin, oral papillae, and pigmentation in the area between upper lip and cheek, the predorsal and the lower lip. Velar wings are defined as one or more tentacles that are folded onto the dorsal surface of the velar apparatus and should not be confused with the tentacles that lie on the ventral surface of the velar apparatus (see Renaud 2011, fig. 5). For discussion on terms used to describe the stages of a lamprey life cycle í ammocoetes (=larvae), metamorphosing individuals (=transformers), and adults (=metamorphosed individuals) í see Docker et al. (2015). Statistic calculations were done using the Excel application for Microsoft Office 2003. Correlation coefficient (Pearson's r) values from 0.50 to 0.75 and from -0.50 to -0.75 indicate moderate to good correlation, and r values from 0.75 to 1 and from -0.75 to -1 indicate very good to excellent correlation between variables (Dawson & Trapp 2004). Abbreviations: CMNFI, Canadian Museum of Nature Fish Collection, Ottawa; NMW, Naturhistorisches Museum Wien, Vienna; SNP, Sochi National Park; TGU, Tomsk State University; ZIN [formerly, abbreviation ZISP was used as in Naseka et al. (2009)], Zoological Institute of Russian

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Academy of Sciences, St Petersburg; and ZMB, Museum für Naturkunde, Berlin. TL, total length; b, bicuspid tooth; u, unicuspid tooth. For intraspecific comparisons, specimens described by Naseka et al. (2009) are used (holotype ZIN 54431, paratypes ZIN 54432, 54433, 54434, 54435, 54436; CMNFI 2008-0059, non types SNP 23, 65) supplemented by the following newly collected specimens (99 ammocoetes, 1 recently metamorphosed individual, and 14 adults): SNP 253 (15 ammocoetes TL 95.3í144.3 mm, 3 adult males TL 115í153 mm; Mzymta River near Galitsino, Sochi District, Russia, 17 Oct. 2009, coll. A.N. Pen’kovskiy). SNP 258a (27 ammocoetes TL 55.3í135.5 mm; Mzymta River near Galitsino, Sochi District, Russia, 17 Oct. 2009, coll. A.N. Pen’kovskiy). SNP 258b (20 ammocoetes TL 95.4í160.1 mm, 2 adult males and 1 adult female, TL 139.0í155 mm; same data as 258a, coll. S.B. Tuniyev). SNP 286 (8 ammocoetes TL 81í122 mm; Shakhe River, Sochi District, Russia, 2009, coll. S.B. Tuniyev). SNP 377 (1 recently metamorphosed individual TL 172.5 mm, 4 adult males and 1 adult female, TL 116í140 mm; Mokva River, Abkhazia region, Georgia, Oct. 2014, coll. S.B. Tuniyev). SNP 378 (1 adult male and 2 adult females, TL 134.8í139.1 mm; Psiya River near Khartsyz, tributary of Shakhe River, Sochi District, Russia, 23 Sept. 2014, coll. S.B. Tuniyev). SNP 379 (3 ammocoetes TL 139.3í162.2 mm; Shakhe River at Malaya Kienka, Sochi District, Russia, Oct. 2014, coll. S.B. Tuniyev). SNP 380 (9 ammocoetes TL 115.6í162.0 mm; Shakhe River at Kirov, Sochi District, Russia, 17 Feb. 2014, coll. S.B. Tuniyev). SNP uncat. (8 ammocoetes TL 90.5í126.6 mm; Shakhe River at Kirov, Sochi District, Russia, 17 Feb. 2014, coll. S.B. Tuniyev). NMW 98630 (9 ammocoetes TL 129.4í157.8 mm; Mzymta River, Sochi District, Russia, 09 Nov. 2009, coll. S.B. Tuniyev). These specimens (except for the Mokva sample) were collected in large oxbow lakes formed in the Shakhe and Mzymta river systems due to recent constructions that changed the direction of watercourses. The comparative material only included the genus Lethenteron, mostly types of nominal taxa to avoid any misidentification; other data are principally taken from Renaud (2011) and Renaud & Naseka (2015).

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Comparative description of the 2009–2014 samples versus the holotype and paratypes Ammocoetes. Morphometric and meristic characters are given in Tables 7-1 and 7-2. The maximum size of ammocoetes was 162.2 mm TL; this means that ammocoetes can reach at least the latter size. Trunk myomeres 57í61 with a modal range of 58í60, similar to the counts found in the type series; myomeres to origin of first dorsal fin (32)33í35 and myomeres to insertion of first dorsal fin 45í49 with a modal range 46í48 (Table 7-2). The middle prong of the tongue precursor is clearly triangular, with a wide base and a pointed apex bearing few cirrhi (examined in 45 specimens) (Fig. 7-2, centerfold, page xiv). The pigmentation coverage is absent on the tongue precursor middle prong in all examined specimens (Fig. 7-2, centerfold, page xiv, Table 7-2). Pigmentation of the areas lateral to the elastic ridge is rarely slight (2) and sometimes moderate (10, Fig. 7-2, centerfold, page xiv), but usually strong (19, 61% of specimens) (Table 7-2). The character, thus, is variable but with a clear mode of strong pigmentation coverage. Although Naseka et al. (2009) reported only slight coverage in the areas lateral to elastic ridge, re-examination of the type series revealed that these areas had the following condition: + (1), ++ (3), +++ (5), in line with the present observations. Table 7-1. Measurements in ammocoetes of Lethenteron ninae. Numbers in parentheses following the catalogue numbers represent the sample size. Character Total length, mm Prebranchial length Prenostril length Branchial length Interbranchial opening length Trunk length Tail length Cloacal slit length

SNP 379 (3), SNP 380 (3), SNP 253 (8), SNP 258 (10), NMW 98630 (9), n=33 range mean SD 97.1í162.2 132.3 18.98 % TL 6.2í7.8

6.9

0.47

2.0í2.9 10.8í12.6

2.4 11.8

0.21 0.45

1.1í1.8

1.4

0.15

49.0í54.3 26.0í30.5

51.9 27.8

1.33 1.07

0.9í1.6

1.3

0.19

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Table 7-2. Pigmentation coverage and myomeres in ammocoetes of Lethenteron ninae. Degree of pigmentation coverage is absent (-), slight (+), moderate (++), and strong (+++). Character

SNP 379 (3), SNP 380 (3), SNP 253 (8), SNP 258 (10), NMW 98630 (9), n=33

Middle prong of tongue í (33) precursor pigmentation Pigmentation of areas + (2), ++ (10), +++ (19), undetermined (2) lateral to elastic ridge Upper lip pigmentation + (1), ++ (31), +++ (1) Lower lip pigmentation í (2), + (30), ++ (1) Area between upper lip and ++ (1), +++ (32) cheek pigmentation Cheek pigmentation +++ (33) Subocular pigmentation + (3), ++ (5), +++ (25) Upper prebranchial ++ (4), +++ (29) pigmentation Lower prebranchial + (7), ++ (12), +++ (14) pigmentation Upper branchial ++ (3), +++ (30) pigmentation Lower branchial í (17), + (16) pigmentation Ventral branchial í (9), + (22), ++ (2) pigmentation Predorsal pigmentation +++ (33) Caudal fin pigmentation + (12), ++ (20), +++ (1) Lateral line neuromasts unpigmented (33) Trunk myomeres 57 (2), 58 (15), 59 (4), 60 (9), 61 (3) Myomeres to origin of first 32 (1), 33 (9), 34 (15), 35 (8) dorsal fin Myomeres to insertion of 45 (4), 46 (14), 47 (7), 48 (6), 49 (2) first dorsal fin Body colouration (in live and freshly preserved specimens) is without mottling. External pigmentation is generally well developed (Fig. 7-3A, B, centerfold, page xv). In most specimens the pigmentation coverage of the area between the upper lip and cheek, cheek, subocular, upper prebranchial, upper branchial, and predorsal (anterior to first dorsal fin) areas is 75% or more (Table 7-2). Naseka et al. (2009) did not record the

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pigmentation between the upper lip and cheek or the predorsal, but their evaluation of the cheek, upper prebranchial and upper branchial pigmentations agree with the present observations, while for the subocular, pigmentation coverage was equally moderate or strong. Pigmentation coverage of other areas is usually either slight (1% to 0.05), but the average length of the L. richardsoni was significantly larger (t test P