PHYLOGEOGRAPHY AND POPULATION STRUCTURE OF ANTARCTIC OPHIUROIDS: EFECTS OF LIFE HISTORY, OCEANOGRAPHY AND PALEOCLIMATOLOGY Except where reference is made to the work of others, the work described in this disertation is my own or was done in collaboration with my advisory commite. This disertation does not include proprietary or clasified information. ___________________________________ Rebeca L. Hunter Certificate of Approval: _________________________ _________________________ Nanete E. Chadwick Kenneth M. Halanych, Chair Asociate Profesor Asociate Profesor Biological Sciences Biological Sciences _________________________ _________________________ Leslie R. Goertzen Scott R. Santos Asistant Profesor Asistant Profesor Biological Sciences Biological Sciences _________________________ George T. Flowers Dean Graduate School PHYLOGEOGRAPHY AND POPULATION STRUCTURE OF ANTARCTIC OPHIUROIDS: EFECTS OF LIFE HISTORY, OCEANOGRAPHY AND PALEOCLIMATOLOGY Rebeca L. Hunter A Disertation Submited to the Graduate Faculty of Auburn University in Partial Fulfilment of the Requirements for the Degre of Doctor of Philosophy Auburn, Alabama August 10 th , 2009 iii PHYLOGEOGRAPHY AND POPULATION STRUCTURE OF ANTARCTIC OPHIUROIDS: EFECTS OF LIFE HISTORY, OCEANOGRAPHY AND PALEOCLIMATOLOGY Rebeca L. Hunter Permision is granted to Auburn University to make copies of this disertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves al publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iv DISERTATION ABSTRACT PHYLOGEOGRAPHY AND POPULATION STRUCTURE OF ANTARCTIC OPHIUROIDS: EFECTS OF LIFE HISTORY, OCEANOGRAPHY AND PALEOCLIMATOLOGY Rebeca L. Hunter Doctor of Philosophy, August 10 th , 2009 (M.S., Texas A&M University, 2004) (B.S., Abilene Christian University, 2001) 192 Typed Pages Directed by Kenneth M. Halanych The Antarctic landmas and surrounding continental shelf have been isolated for over 24 milion years. Geographic and thermal isolation have resulted in a highly endemic, diverse benthic marine fauna. This fauna has been relatively wel characterized morphologicaly, but litle is known about the evolutionary history, genetic diversity and population genetic structure of Antarctic benthic marine invertebrates. Several important questions remain largely unanswered, including 1) are circumpolar species geneticaly homogeneous throughout their range, 2) are non-endemic species maintaining v connectivity betwen populations distributed in Antarctica and South America, and 3) how does early life history influence dispersal throughout Antarctica? This research examined phylogeographic paterns within four Antarctic britle stars (ophiuroids). Ophiuroid species were chosen that difered in their mode of development (i.e., presence/absence of a pelagic larval stage) and geographic distribution. A non-endemic brooding species, Astrotoma agassizi, was evaluated in order to ases connectivity across a major oceanographic barier separating Antarctic and South American populations. Thre Antarctic endemics, Ophiurolepis gelida, O. brevirima and Ophionotus victoriae, al possesing some form of pelagic larvae, were obtained from various locations around Antarctica in order to characterize their population structure and levels of gene flow throughout the Antarctic continental shelf. Analysis of intraspecific mitochondrial sequence data revealed several interesting paterns. First, al species showed evidence of restricted connectivity betwen major geographic regions in the Antarctic, subantarctic and/or South America (depending on sampling), regardles of developmental mode. Additionaly, cryptic divergence and/or speciation were recovered in al cases. Second, the brooding species, A. agassizi, displayed evidence of greater connectivity within geographic regions compared to species with pelagic larvae. These results suggest that phylogeographic paterns are not easily predicted for Antarctic britle stars, and ophiuroid diversity is underestimated in the Southern Ocean. These generalizations likely apply to other Antarctic marine invertebrates, and suggest that much more research wil be required to quantify Southern Ocean biodiversity and fully understand the contemporary and historical proceses driving evolution in this region. vi ACKNOWLEDGMENTS I would like to thank my disertation advisor, Ken Halanych, for motivating me and keeping me on task throughout this long proces. Thank you for making me go to metings, take field courses and for encouraging me to publish early. I am grateful that you alowed me to be independent, and provided just the right amount of oversight and guidance. I would also like to thank my commite members: Scott Santos, who carefully edited and improved a number of my manuscripts, and Nanete Chadwick and Les Goertzen, who provided valuable fedback and suggestions during the course of this work, as did my outside reader, Stephen Bullard. You al made the experience of geting a Ph.D. les painful than it would have been otherwise! To members of the Halanych Lab, past and present: thank you for your knowledge and expertise in the lab (Torsten Struck, Adriene Burnet, Heather Ecleston, Dan Thornhil, Liz Borda, Min Zhong, Brit Maret and Chad Smith), for help and good company in the field (Alexis Janosik, Nerida Wilson, Andy Mahon, Nicole Cox and Jon Craft), and for being good cofe buddies (Joie Cannon, Alexis Janosik, Kevin Kocot and Liz Borda). A number of individuals provided samples used in this disertation, and I am grateful for their wilingnes to collect samples on my behalf. I would like to acknowledge the NSF-EPSCOR program at Auburn University which supported me for two years during this work. Lastly, I would like to thank my husband, Ryan Hunter, who graciously put up with me during this ordeal, and whose expertise in Sigma Plot came in handy on more than one occasion! vii Style manual of journal used: Chapter 1: Molecular Ecology Chapter 2: Zootaxa Chapter 3: Journal of Heredity Chapters 4, 5 and 6: Marine Biology Computer software used: Adobe Ilustrator, Microsoft Excel, Microsoft Word, Sigma Plot, Arlequin, BEAST, Collapse, DnaSP, acClade, MegAlign, MDIV, ModelTest, MrBayes, MrodelTest, PAUP, SeqMan, TCS, TreRot vii TABLE OF CONTENTS LIST OF TABLES..................................................... xi LIST OF FIGURES.................................................... xii 1. INTRODUCTION TO DISERTATION................................. 1 1.1 INTRODUCTION................................................ 1 1.1.1 LARVAL DISPERSAL....................................... 4 1.1.2 MOLECULAR STUDIES ON ANTARCTIC FAUNA.............. 6 1.1.3 RESEARCH OBJECTIVES.................................... 7 1.2 REFERENCES.................................................. 9 2. MORPHOLOGICAL CLADISTIC ANALYSIS OF OPHIUROLEPIS ATSUMOTO, 1915 (OPHIURIDA: OPHIURIDAE) FROM THE SOUTHERN OCEAN.............................................................. 19 2.1 ABSTRACT.................................................... 19 2.2 INTRODUCTION................................................ 19 2.2.1 TAXONOMIC HISTORY..................................... 21 2.3 MATERIALS AND METHODS.................................... 24 2.3.1 DATA MATRIX............................................ 24 2.3.2 CHARACTER SCORING..................................... 25 2.3.3 PHYLOGENETIC ANALYSIS................................ 25 2.4. RESULTS...................................................... 26 2.4.1 ANALYSIS INCLUDING T. RELEGATA ND T. MADSENI........ 26 2.4.2 ANALYSIS EXCLUDING T. RELEGATA ND T. MADSENI........ 27 2.5. DISCUSION.................................................. 28 2.5.1 MORPHOLOGICAL CHARACTERS........................... 30 2.5.2 TAXONOMIC REVISION OF OPHIUROLEPIS.................. 31 2.5.3 BIOGEOGRAPHIC ONSIDERATIONS........................ 32 2.6 REFERENCES.................................................. 35 ix 3. EVALUATING CONECTIVITY IN THE BRODING BRITLE STAR ASTROTOMA AGASSIZI ACROS THE DRAKE PASAGE IN THE SOUTHERN OCEAN.................................................. 48 3.1 ABSTRACT.................................................... 48 3.2 INTRODUCTION.............................................. .. 49 3.3 MATERIALS AND METHODS.................................... 52 3.3.1 DATA COLECTION........................................ 52 3.3.2 POPULATION STRUCTURE ANALYSES...................... 53 3.3.3 PHYLOGENETIC AND GENETIC DISTANCE ANALYSES....... 55 3.4 RESULTS...................................................... 56 3.4.1 POPULATION STRUCTURE................................. 56 3.4.2 PHYLOGENETIC RELATIONSHIPS AND GENETIC DISTANCES. 59 3.5 DISCUSION................................................... 59 3.5.1 CRYPTIC SPECIES IN ASTROTOMA AGASSIZI................. 59 3.5.2 CONECTIVITY BETWEN ANTARCTICA ND SOUTH AMERICA................................................. 61 3.5.3 INTRACLADE POPULATION STRUCTURE.................... 63 3.6 REFERENCES .................................................. 67 4. PHYLOGEOGRAPHY OF THE ANTARCTIC PLANKTOTROPHIC BRITLE STAR OPHIONOTUS VICTORIAE REVEALS GENETIC STRUCTURE INCONSISTENT WITH EARLY LIFE HISTORY........................... 84 4.1 ABSTRACT.................................................... 84 4.2 INTRODUCTION............................................... 85 4.3 MATERIALS AND METHODS.................................... 88 4.3.1 DATA COLECTION........................................ 88 4.3.2 POPULATION STRUCTURE ANALYSES...................... 90 4.3.3 HISTORICAL DEMOGRAPHY AND MIGRATION ANALYSES.... 91 4.4 RESULTS...................................................... 91 4.4.1 POPULATION STRUCTURE................................. 92 4.4.2 HISTORICAL DEMOGRAPHY............................... 94 4.5 DISCUSION................................................... 95 4.5.1 INTERCLADE GENETIC RELATIONSHIPS..................... 95 4.5.2 CLADE 1 RELATIONSHIPS: THE ANTARCTIC PENINSULA..... 98 4.5.3 CLADE 1 RELATIONSHIPS: SUBANTARCTIC ISLANDS........ 100 4.5.4 CLADE 1 RELATIONSHIPS: DECEPTION ISLAND............. 102 4.6 REFERENCES.................................................. 104 x 5. GEOGRAPHICAL SUBDIVISION AND EMOGRAPHIC HISTORY IN TWO ANTARCTIC OPHIUROIDS: THE ROLE OF PLEISTOCENE GLACIAL CYCLES AND CONTEMPORARY OCEANOGRAPHY..................... 124 5.1 ABSTRACT.................................................... 124 5.2 INTRODUCTION................................................ 125 5.3 MATERIALS AND METHODS.................................... 128 5.3.1 DATA COLECTION....................................... 128 5.3.2 POPULATION STRUCTURE ANALYSES..................... 129 5.3.3 HISTORICAL DEMOGRAPHY AND IVERGENCE DATING.... 130 5.4 RESULTS...................................................... 131 5.4.1 POPULATION STRUCTURE................................. 131 5.4.2 HISTORICAL DEMOGRAPHY AND IVERGENCE............. 133 5.5 DISCUSION................................................... 134 5.5.1 O. GELIDA................................................ 135 5.5.2 O. BREVIRIMA............................................. 138 5.5.3 PHYLOGEOGRAPHY WITHIN BRANSFIELD STRAIT.......... 139 5.6 REFERENCES.................................................. 142 6. CONCLUSIONS.................................................... 161 6.1 CONCLUSIONS................................................ 161 6.2 REFERENCES.................................................. 166 APENDIX.......................................................... 170 xi LIST OF TABLES CHAPTER 2 Table 1: Distributions, depth ranges and catalog numbers for species of Ophiurolepis, Theodoria and Homalophiura used in this study........................42 CHAPTER 3 Table 1: Collection information for Astrotoma agassizi in South America and Antarctica. Collection station numbers correspond with Figure 1 and N refers to number of individuals sequenced for 16S and COI.....................77 Table 2: Pairwise ? ST for each Astrotoma agassizi collection station in South America and Antarctica...................................................78 Table 3: Hierarchical analysis of molecular variance (AMOVA) for South American and Antarctic populations of Astrotoma agassizi..........................79 Table 4: Genetic diversity statistics for pooled Astrotoma agassizi collection stations, N refers to number of individuals, H is the number of haplotypes, ? refers to nucleotide diversity and h is haplotype diversity........................80 CHAPTER 4 Table 1: Population summary statistics for Ophionotus victoriae (derived from the 16S+COI concatenated dataset): N refers to number of individuals, H is the number of haplotypes, ? refers to nucleotide diversity, and h is haplotype diversity. Tajima?s D and Fu?s F S refer to results of neutrality tests. Station numbers correspond with Figure 1..................................117 Table 2: Pairwise ? ST values betwen Ophionotus victoriae Antarctic and subantarctic stations. Station numbers corespond with Figure 1....................118 Table 3: Hierarchical analysis of molecular variance (AMOVA) for Antarctic and subantarctic populations of Ophionotus victoriae......................119 xii CHAPTER 5 Table 1: Collection information for Ophiurolepis gelida and O. brevirima from Antarctica. Station numbers correspond with Figure 1...................152 Table 2a: Pairwise ? ST values betwen O. gelida stations (where N ? 4) from the Antarctic Peninsula and Subantarctic Islands. Station numbers correspond with Figure 1 and Table 1............................................153 Table 2b: Pairwise ? ST values betwen O. brevirima stations (where N ? 4) from the Antarctic Peninsula and Weddel Sea...............................154 Table 3a: Results of diversity estimates and neutrality statistics for Ophiurolepis gelida and O. brevirima...............................................155 Table 3b: Results of mismatch distribution for Ophiurolepis gelida and O.brevirima..................................................156 Table 4: Time of the most recent common ancestor for O. gelida and O. brevirima lineages. Top date was obtained using a 3%/MY mutation rate, and the bottom number resulted from a slower mutation rate of 0.5%/MY. 95% CI given in parentheses....................................................157 xii LIST OF FIGURES CHAPTER 1 Figure 1: Relevant geographic localities in Antarctica and South America...........18 CHAPTER 2 Figure 1: Plate showing aboral disc view of various ophiuroid species used in this study. A. Ophiurolepis anceps, B. O. banzarei, C. O. brevirima, D. O. gelida, E. O. martensi, F. O. mordax, G. O. olstadi, H. O. scisa, I. O. tuberosa, J. Homalophiura brucei, K. H. clasta, L. H. confragosa, M. H. inornata, N. Theodoria partita, O. T. relegata, P. T. wallini........................44 Figure 2: MP strict consensus tre for data including T. relegata and T. madseni. Numbers below branches represent decay indices......................45 Figure 3: MP strict consensus tre for data excluding T. relegata and T. madseni. Numbers above branches represent bootstrap proportions and numbers below represent decay indices...........................................46 Figure 4: Morphological characters mapped onto the strict consensus MP tre for data excluding T. relegata and T. madseni. Character numbers correspond with Appendix. Bars indicate where a change has occurred, circles indicate reversals and squares indicate convergent character changes.....................47 CHAPTER 3 Figure 1: Map showing collection localities for Astrotoma agassizi from South America and Antarctica..................................................81 Figure 2: Bayesian tre of unique 16S+COI mtDNA haplotypes, with corresponding haplotype networks for each of thre phylogenetic clades. Numbers next to nodes indicate Bayesian posterior probabilities. On the Bayesian tre, haplotypes are labeled acording to station. In networks, circles are coded by station and a unique key is given for each clade. Coding does not overlap betwen clades. Haplotypes are sized acording to relative abundance and mising haplotypes are denoted by smal, closed black circles............82 xiv Figure 3: Mismatch distributions and Tajima?s D statistic for pooled Astrotoma agassizi collection stations. Significant Tajima?s D values are indicated by (P < 0.05).........................................................83 CHAPTER 4 Figure 1: Map showing collection localities for Ophionotus victoriae from the Antarctic Peninsula and subantarctic South Sandwich Islands and Bouvet Island (inset).......................................................120 Figure 2: 16S + COI parsimony networks of 134 O. victoriae individuals collected throughout the Antarctic Peninsula and subantarctic islands. Haplotype circles are coded by geographic region and sized acording to relative abundance. Mising (unsampled) haplotypes are denoted by smal black circles.......121 Figure 3: Posterior probability distributions for the number of migrants per generation (M) betwen certain O. victoriae stations/regions.....................122 Figure 4: Map showing the distribution of O. victoriae clades throughout the Antarctic Peninsula. Given that al subantarctic populations belonged to Clade 1a, these geographic localities were not included..............................123 CHAPTER 5 Figure 1: Map showing collection localities for Ophiurolepis gelida and O. brevirima from Antarctica and subantarctic South Sandwich Islands and Bouvet Island........................................................158 Figure 2: 16S parsimony networks of 118 O. gelida individuals collected throughout Antarctica and from two subantarctic islands; and 87 O. brevirima individuals collected from the Antarctic Peninsula and Weddel Sea. Haplotype circles are coded by geographic region and sized acording to relative abundance. Mising (unsampled) haplotypes are denoted by smal black circles.............159 Figure 3: Observed and expected mismatch distributions for pooled O. gelida and O. brevirima geographic regions....................................160 1 CHAPTER 1. Introduction to disertation 1.1 INTRODUCTION Understanding factors contributing to distributional paterns of organisms in the marine environment has been a focus of numerous recent studies (e.g., Palumbi et al. 1997; Lesios et al. 1998; McCartney et al. 2000; Waters et al. 2000; Muss et al. 2001). Oceanography, life history and behavior have al ben shown to afect dispersal of marine organisms, especialy invertebrates (Palumbi 1994). As a result of these studies, several bariers to dispersal have been identified based on concordant phylogeographic paterns in a wide variety of taxa. The most prominent of these include the Eastern Pacific Barier, a ~5000 km deep-water gap betwen the central and eastern Pacific Ocean, the southern tip of South Africa, due to cold water upweling, the deep-water separation of the eastern and western Atlantic (Lesios et al. 2003), and the Polar Front in the Southern Ocean (Clarke et al. 2005). While efects of oceanographic bariers and life history constraints have been evaluated for many geographic regions, one of the more distinctive biogeographical regions, the Southern Ocean, is not wel understood in this context. The Antarctic Polar Front (APF), other oceanographic features, and certain life history traits afecting dispersal of marine organisms in this part of the world require further investigation. As such, Antarctica and the surrounding Southern Ocean are ideal localities for further study of the physical and biological factors driving distributional paterns and population genetic structure in marine invertebrates. 2 Isolation of the Antarctic continent has been a driving evolutionary force for Antarctic fauna for 24-41 milion years (Lawver & Gahagan 2003; Pfuhl & McCave 2005; Scher & Martin 2006). Separation of Antarctica from South America and the ensuing onset of the Antarctic Circumpolar Current (AC) are presumed to have been primary forces driving speciation in many Southern Ocean marine taxa. AC formation is thought to have driven gradual cooling and glaciation that began ~34 mya in the Antarctic (Zachos et al. 2001). This cooling and long period of isolation have led to a diverse and abundant benthic fauna that are typicaly stenothermal, eurybathic and endemic to the Antarctic (Hempel 1985). Endemism is particularly high in certain groups such as fish (95%), amphipods (95%), pycnogonids (90%), isopods (87%), and echinoderms (73%) (Knox & Lowry 1977; Brandt 1991; Jazdzewski et al. 1991). Antarctic benthos are believed to have originated from thre sources: 1) remnant Gondwana forms, including forms originating in Antarctica, 2) forms from the surrounding deep sea, and 3) forms from South America that migrated to Antarctica (Del 1972; Hempel 1985; Watling & Thurston 1989; Dayton et al. 1994). Many present-day fauna conform to certain biogeographic paterns. The most common of these is circumpolarity, largely atributed to pasive dispersal via the AC (Fel et al. 1969). Other fauna are restricted to west Antarctica (Antarctic Peninsula, Weddel Sea and Belingshausen Sea) or the subantarctic islands (Dayton et al. 1994) (Fig. 1). Several groups have undergone extensive radiations upon isolation in Antarctica, these include pycnogonids, echinoderms, gastropods, ascidians, some isopods and notothenioid fish (Del 1972; Arntz & Galardo 1993). 3 While many Antarctic taxa exhibit high levels of endemism, others show much lower levels despite geographic and thermal isolating factors. For example, polychaete, echinoderm and mollusc conspecifics have been reported on the continental shelves of both Antarctica and South America, and a wel-recognized faunal afinity exists betwen these two regions (Del 1972; Arntz et al. 1994; Dayton et al. 1994). A lack of endemism in these species indicates some level of recent gene flow betwen populations separated by the AC and APF. Several gene flow mechanisms have been proposed, including human-mediated transport, migration of benthic adults, larval dispersal and rafting. Human-mediated transport into Antarctica is possible, though unlikely, as fouling and balast water organisms would have to be pre-adapted to the extreme environmental conditions in the Antarctic in order to survive (Thatje 2005). Migration of adults has long been asumed to occur across the Scotia Arc, a submerged ridge with a series of emergent islands including South Georgia, South Sandwich Islands and the South Orkneys. The Scotia Arc forms a ?stepping-stone? connection betwen the Antarctic Peninsula and South America (Fel et al. 1969). Dispersal of larvae or rafting adults/juveniles would most likely occur across the Drake Pasage, the portion of the AC separating Antarctica and South America representing the shortest distance betwen Antarctica and any other continent. Mechanisticaly, dispersal could occur via warm- and cold-core rings, mesoscale eddies known to transport larvae and rafting organisms into and out of Antarctica across the AC (Clarke et al. 2005). The recent findings of Antarctic kril in Chilean fjords, and South American anomuran and brachyuran crab larvae in the Antarctic Peninsula highlight the penetrability of this current (Antezana 1999; Thatje & Fuentes 2003; Clarke et al. 2005). Dispersal of rafting adults from South America to 4 subantarctic South Georgia has been shown in a brooding bivalve species (Helmuth et al. 1994). 1.1.1 Larval dispersal Reproduction in polar marine invertebrates has been the focus of much speculation and discussion in the literature for more than a century. The paradigm known as ?Thorson?s Rule?, coined by Mileikovsky (1971), resulted from few observations that led to the idea that polar and deep-sea species were almost exclusively brooders (e.g., Thomson 1878, 1885; Murray 1885; Thorson 1936). Applicability of this ?rule? is now known to be limited for many groups, especialy echinoderms and bivalves (Clarke 1992; Pearse 1994). New paradigms concerning reproductive trends in the Antarctic, based on larger amounts of data, are emerging. These data have shown that over 70% of Antarctic echinoderm species have a planktonic larval stage (Pearse 1994). Pearse (1994) summarized previous research (Bosch & Pearse 1990; Pearse et al. 1991) on echinoderm reproduction in McMurdo Sound, Ross Sea, Antarctica, and determined that 23% of echinoderms had a feding pelagic (planktotrophic) larval form, 50% had a non-feding pelagic (lecithotrophic) form and 27% had either a non-feding demersal larvae or were brooders. These prevailing modes of development can be used to infer dispersal potential, as determined by pelagic larval duration, of Antarctic benthic invertebrates. Studies on a variety of marine invertebrates have shown that species with longer pelagic larval period often show les population diferentiation than those with abbreviated larval development (Berger 1973; Crisp 1978; Janson 1987; McMilan et al. 1992; Duffy 1993; Hunt 1993; 5 Helberg 1996; Hoskin 1997; Arndt & Smith 1998). However, this relationship can vary greatly, and some marine invertebrates show high levels of population diferentiation despite a long-lived pelagic larvae (e.g., Tracey et al. 1975; Burton 1986). In addition, brooding species can be more widespread than planktotrophic species (Johanneson 1988). The enhanced dispersal ability of Antarctic marine invertebrates with pelagic larvae has been invoked to explain their ability to quickly recolonize shelf habitat recently disturbed by ice scour (Poulin et al. 2002). Historicaly, disturbances caused by expansion and contraction of the Antarctic ice sheet during Pleistocene glacial periods are thought to be responsible for the circumpolar distribution typical of many Antarctic benthic invertebrates (Thatje et al. 2005). Thatje et al. (2005) suggested that the shalow continental shelf that opened up during periods of diachronous deglaciation (Anderson et al. 2002) would be recolonized first by species with highly dispersive larvae. These species could then recolonize shelf habitat around the continent of Antarctica as it became available during interglacial periods, resulting in a circumpolar distribution (Thatje et al. 2005). Conversely, diachronous deglaciation could also drive cryptic speciation in species with low dispersal potential, such as brooders, due to isolation in shelf refugia during glacial periods (Thatje et al. 2005). This type of speciation has been shown in Antarctic brooding isopods and a brooding sea slug (Held 2003; Held & W?gele 2005; Wilson et al. 2009), as wel as a notothenioid fish (Janko et al. 2007). 6 1.1.2 Molecular studies on Antarctic fauna Studies investigating the evolutionary history of Antarctic fauna using molecular tools have emerged in recent years. These studies have focused on notothenioid fish, known for their adaptive radiation in Antarctica over the past 2-12 milion years (Bargeloni et al. 2000a, 2000b; Stankovic et al. 2002; Patarnelo et al. 2003; Janko et al. 2007), kril (Patarnelo et al. 1996; Zane et al. 1998; Bargeloni et al. 2000b), isopods (Held 2003; Held & W?gele 2005; Raupach & W?gele 2006; Lese & Held 2008), molluscs (Beaumont & Wei 1991; Alcock et al. 1997; Page & Linse 2002; Linse et al. 2007; Wilson et al. 2009), nemerteans (Thornhil et al. 2008), and pycnogonids (Mahon et al. 2008). Many studies have concentrated on the AC?s role in promoting speciation and divergence in the Southern Ocean. For example, Page and Linse (2002) concluded that bivalve sister species split after the formation of the AC, indicating that initialy it was not a barier to dispersal in this group (se also Thornhil et al. 2008). Conversely, Patarnelo et al. (1996) found evidence of vicariant speciation for Antarctic and subantarctic kril caused by formation of the AC. In Antarctica, notothenioid fish show genetic discontinuity among benthic populations distributed around the continent (Bargeloni et al. 2000b). The Antarctic kril Euphausia superba exhibits surprising diferentiation betwen populations from subantarctic South Georgia and from the Weddel Sea, given the high dispersal potential of this planktonic organism (Zane et al. 1998; Bargeloni et al. 2000b). The circumpolar crinoid Promachocrinus kerguelensis is represented by at least six cryptic lineages, despite having a pelagic larval stage in its development (Wilson et al. 2007), while the planktotrophic nemertean Parborlasia corrugatus and brooding pycnogonid Nymphon australe are geneticaly homogenenous 7 over large distances throughout the Antarctic Peninsula (Mahon et al. 2008; Thornhil et al. 2008). 1.1.3 Research objectives Despite the above examples, many Antarctic benthic groups are unstudied in terms of their evolutionary history, population connectivity and biogeography. A conspicuous example is the ophiuroid echinoderms, one of the most common and abundant Antarctic benthic groups (Fel et al. 1969). Ophiuroids represent an important ecological component of the benthic community; certain species have been reported with local densities up to 10 7 individuals/ km 2 (Fel et al. 1969). Many Antarctic ophiuroids have a circumpolar distribution, atributed to dispersal by the AC, or for deeper forms, by traversing deep ocean floors. The Antarctic Peninsula region has the highest species diversity for ophiuroids, probably because of faunal exchange with South America (Fel et al. 1969). Given that ophiuroids are an important component of the Antarctic benthic community and have received litle atention, the primary goal of this research was to evaluate genetic population structure and levels of connectivity using molecular tools. Where homogenous population genetic structure is identified, dispersal of larvae and/or adults wil be proposed. In cases where heterogeneous population genetic structure results, limited dispersal and/or physical bariers to dispersal wil be evaluated. Several ophiuroid species with diferent reproductive developmental modes wil be examined and hypotheses wil be developed based on difering dispersal capacities that correspond to expected levels of population connectivity. Pearse (1994) summarized approximate 8 persistence times for lecithotrophic and planktotrophic larval forms in the Antarctic. Pelagic lecithotrophic forms have been recorded spending 2-3 months in the plankton (based on asteroid data from Bosch & Pearse 1990), whereas pelagic planktotrophic forms are suspected to spend 5-6 months (based on echinoid and asteroid data from Pearse & Bosch 1986 and Bosch et al. 1987). Specific research objectives are: 1) Determine phylogenetic relationships betwen species in the Southern Ocean genus Ophiurolepis using morphological data 2) Evaluate population genetic structure and connectivity across the Drake Pasage for Astrotoma agassizi, a brooding Antarctic species 3) Examine population genetic structure and connectivity in Ophionotus victoriae in the Antarctic Peninsula and subantarctic islands, a planktotrophic Antarctic species 4) Determine population genetic structure and levels of connectivity for Ophiurolepis brevirima in the Antarctic Peninsula and Weddel Sea, and for Ophiurolepis gelida in the Ross Sea to Bouvet Island, both probable lecithotrophic Antarctic species 9 1.2 REFERENCES Alcock AL, Brierley AS, Thorpe JP, Rodhouse PG (1997) Restricted gene flow and evolutionary divergence betwen geographicaly separated populations of the Antarctic octopus Pareledone turqueti. Marine Biology, 129, 97?102. 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Two of the thre Ophiurolepis clades included species currently asigned to other genera, but closely alied with Ophiurolepis in the taxonomic literature. This indicates that Ophiurolepis as currently defined is not a monophyletic group. Additional forms of data, namely molecular, are needed to more definitively resolve relationships within this group. 2.2 INTRODUCTION The britle star genus Ophiurolepis Matsumoto, 1915 is one of the more speciose ophiuroid genera in the Southern Ocean (Fel et al. 1969). Nine of the fourten species are endemic to the Antarctic/subantarctic region and four species have a circumpolar 20 distribution (Table 1). A circumpolar distribution has been proposed as the general distributional patern for Antarctic ophiuroids, presumably influenced by the west-wind drift (Fel 1962; Pawson 1993). Of the five non-endemic species, O. martensi (Studer, 1885) and O. scisa (Koehler, 1908) extend from Antarctica to southernmost South America, and O. turgida Mortensen, 1936 has been found only from the southernmost South American shelf (Mortensen 1936). In addition to Antarctica, O. mordax Koehler, 1922 has been reported from the abyssal region of the Mozambique Channel (Vadon & Guile 1984; Guile & Vadon 1986). Recently, O. carinata (Studer, 1876), previously known only from the subantarctic Kerguelen and South Sandwich Islands, was collected close to methane seps off Concepci?n Bay, Chile at approximately 36?S latitude (Selanes & Krylova 2005). Litle is known about the life history of any species of Ophiurolepis. Lifespan was calculated for O. brevirima Mortensen, 1936 and O. gelida (Koehler, 1901) as 25 and 33 years, respectively, by analyzing vertebral ossicle growth bands (Dahm 1996). In his monograph on the echinoids and ophiuroids of the Antarctic/subantarctic region, Mortensen (1936) speculated about the reproductive biology of several Ophiurolepis and other closely related species. He postulated that six species (O. brevirima, O. gelida, O. tuberosa (Mortensen, 1936), Theodoria partita (Koehler, 1908), T. wallini (Mortensen, 1925), and Homalophiura inornata (Lyman, 1878)) were dioecious with some form of direct development, but were not viviparous. Egg size was reported as ~0.3 m diameter in these species. This is wel within the oocyte diameter range (0.1-1.0 m) known for brooding ophiuroids (Hendler 1991), but also within range for those with non-feding pelagic lecithotrophic larvae, the dominant larval type in Antarctic echinoderms (Pearse 21 1994). Two exceptions noted by Mortensen (1936) were O. martensi, asumed to be viviparous based on the presence of five to six juveniles per bursa in some specimens, and O. carinata, thought to develop via the typical planktotrophic ophiopluteus larvae due to the smal size and high number of eggs. Several Ophiurolepis species are wel known because of the dominant role they play in the Antarctic benthic community. For example, O. brevirima, O. gelida and O. martensi are among the dominant asterozoan species in Weddel Sea benthic asemblages (Piepenburg et al. 1997). Ophiurolepis brevirima and O. gelida are considered ?permanent species? (CixDi% > 5; where Ci is a measure of frequency of occurrence and Di is a measure of numerical dominance) throughout the South Shetland Islands of the Antarctic Peninsula (Manj?n-Cabeza & Ramos 2003). Ophiurolepis martensi is the most numerous ophiuroid around Marion Island, present in abundances of up to 232 m -2 (Beckley & Branch 1992). In contrast, other Ophiurolepis species are known from only a few localities where they have been collected in low numbers. Both O. accomodata Koehler, 1922 and O. granulifera Bernasconi and D?Agostino, 1973 were described from thre or fewer specimens and have been reported only from one or two localities (Koehler 1922; Madsen 1955; Bernasconi & D?Agostino 1973). One species, O. turgida, has been described from a single specimen (Mortensen 1936). 2.2.1 Taxonomic history Ophiurolepis was erected by Matsumoto (1915) to acommodate a single species that had been independently described by two taxonomists and placed in diferent genera. In 1876, Studer described Ophiolepis carinata from Kerguelen Island while two years 22 later Lyman (1878) described Ophioglypha deshayesi from Kerguelen Island. The two names were synonymized and transfered to Ophiurolepis, with O. carinata as the type specimen. Matsumoto (1915) diagnosed Ophiurolepis as having a disk with large rounded plates surrounded by belts of smaler plates, wel-developed dorsal arm plates, triangular ventral arm plates, one minute arm spine and thre tentacle scales, both teth and oral papilae present, and with the second oral tentacle pores opening outside the mouth. Within Ophiurolepis several species have been shufled among closely related genera owing to morphological similarities with congeners and a lack of sufficient synapomorphies characterizing some genera. For example, O. martensi, originaly described as Ophioglypha martensi (Studer, 1885), was transfered to Ophiozona (Bel 1902), then back to Ophioglypha (Koehler 1911), and then to Homalophiura and subsequently Amphiophiura (Clark 1915), prior to being asigned to Ophiurolepis (Koehler 1922). Two other species, O. gelida and O. scisa, were at one time placed in Homalophiura, and O. anceps (Koehler, 1908) was provisionaly placed in Amphiophiura (Clark 1915). Additionaly, Fel (1961) excluded two taxa from Ophiurolepis on the basis of thre external skeletal characters shared by O. wallini and O. partita, thereby restricting the definition of Ophiurolepis. A new genus, Theodoria Fel 1961, was established for these two species and one other species, Amphiophiura relegata (Koehler, 1922). The genus Homalophiura is closely related to Ophiurolepis and includes some species that might be referable to Ophiurolepis. The type species for this genus, H. inornata, has been suggested to belong within Ophiurolepis. Several workers (Hertz 23 1926; Mortensen 1936; Bartsch 1982) have stated that Homalophiura and Ophiurolepis are not easily distinguished and that Homalophiura probably cannot be maintained, despite the fact that Clark (1915) claimed Homalophiura was a ?wel-characterized and homogeneous group?. Clark (1915) diagnosed Homalophiura as having large plates mingled with smaler plates on the disk, with a reduced arm comb and smal tentacle pores, few minute arm spines, and second oral tentacle pores opening outside the mouth. In the taxonomic literature (Madsen 1951, 1967; Tommasi 1968; Bernasconi & D?Agostino 1971, 1973, 1977), the application of H. inornata has been inconsistent. Paterson (1985) proposed reconciling the genus by suggesting reasignments for the nineten currently recognized species in Homalophiura. Homalophiura inornata, H. brucei (Koehler, 1908) and H. confragosa (Lyman, 1878) were reasigned to Ophiurolepis; however, no formal revision of the genus has since been made. Litle information exists on phylogenetic relationships within Ophiurolepis. Mortensen (1936) stated that O. gelida and O. brevirima were closely related, based on overal morphological similarity. However, no phylogenetic relationships have been suggested for the group apart from subjective asesments due to morphological similarity. Given uncertain relationships within Ophiurolepis and uncertain afinity with several Theodoria and Homalophiura species, a formal asesment of phylogenetic relationships is needed. Using morphological characters in a cladistic framework, this study explores the evolutionary relationships among these important Southern Ocean species and wil provide phylogenetic hypotheses that can be tested with additional data. Gaining insight into the evolutionary history of Ophiurolepis in Antarctica wil alow 24 further understanding of the relevant biogeographic proceses and speciation mechanisms that have contributed to biodiversity in this area. 2.3 MATERIALS AND METHODS 2.3.1 Data matrix In order to test phylogenetic relationships of species of Ophiurolepis, a data matrix of 36 morphological characters and 24 ingroup species was constructed in MacClade 4.0 (Maddison & Maddison 2000) (Appendix). The folowing species were included (Table 1 and Fig. 1): the fourten species currently recognized in Ophiurolepis, Theodoria wallini, T. partita, T. relegata, T. madseni Tommasi, 1976, Homalophiura inornata, H. brucei, H. confragosa, H. intorta (Lyman, 1878), H. euryplax Clark, 1939 and H. clasta (Clark, 1911). Theodoria species were included to determine the relationship of Theodoria to Ophiurolepis, since two of these species were at one time placed in Ophiurolepis (T. wallini and T. partita). Several Homalophiura species were included to help elucidate the relationship of Homalophiura and Ophiurolepis. Homalophiura inornata, H. brucei and H. confragosa were suggested by Paterson (1985) to belong in Ophiurolepis while H. euryplax and H. intorta were alied with Ophiura and H. clasta was suggested to belong in Homophiura (Paterson 1985). Additionaly, a second analysis was done excluding Theodoria relegata and T. madseni, since these two species have reportedly never been considered as having a close afinity to Ophiurolepis, but are congeneric with two species that were once placed in Ophiurolepis. Two species from the same subfamily (Ophiurinae) as Ophiurolepis, Ophiuroglypha carinata (Koehler, 1901) and Ophiuroglypha lymani (Ljungman, 1870), were used as outgroups. 25 2.3.2 Character scoring Of the 36 characters used in analyses, 34 were ossicular, one was soft tisue and one was ecological. Characters were scored using published descriptions (Studer 1876; Lyman 1878; Koehler 1902, 1922; Clark 1911; Matsumoto 1915; Mortensen 1925, 1936; Madsen 1955, 1967; Fel 1961; Cherbonnier 1962; Bernasconi & D?Agostino 1973; Bartsch 1982; Paterson 1985) and verified using museum specimens when possible. Samples were obtained from the Smithsonian Institution National Museum of Natural History and the South Australian Museum, and collected during a 2004 Antarctic cruise. Twenty-six characters were scored as binary and 10 were scored as multistate. Character descriptions and justification of scoring are given in the Appendix and select characters are photographicaly ilustrated. 2.3.3 Phylogenetic analysis Data were analyzed in PAUP 4.0b10 (Swofford 2002) using maximum parsimony (MP) based reconstructions. Al characters were treated as unordered and equaly weighted. For the analysis including T. relegata and T. madseni, a heuristic search with 1000 random addition replicates and tre bisection reconnection (TBR) branch-swapping was performed. For the analysis excluding T. relegata and T. madseni, a branch and bound search was done. Character-state optimization used the acelerated transformation (ACTRAN) option in PAUP. To test the robustnes of the MP tres, 1000 bootstrap replicates were done and decay indices (Bremer 1988) were calculated in TreRot.v2 (Sorenson 1999). Character transformation was evaluated in MacClade 4.0. 26 2.4 RESULTS Analysis of morphological data yielded 817 most parsimonious tres (strict consensus shown in Fig. 2) with a tre length (TL) = 78, consistency index (CI) = 0.60 and retention index (RI) = 0.77. Bootstrapping proved to be too computationaly intensive for searches including T. relegata and T. madseni, therefore only decay indices were calculated. Searches excluding T. relegata and T. madseni yielded six most parsimonious tres (strict consensus shown in Fig. 3) with TL = 71, CI = 0.65 and RI = 0.79. 2.4.1 Analysis including T. relegata and T. madseni The most parsimonious tres from searches including T. relegata and T. madseni required seven additional steps and were supported by lower CI and RI values when compared to searches excluding these taxa. The strict consensus of these tres is not wel resolved (Fig. 2), however, two clades, denoted A and C, are present. Resolution among al other species in the tre is lacking. These are denoted as clade B in order to make comparisons with the analysis that excluded T. relegata and T. madseni. This lack of resolution is probably due to the confounding efect of adding these two taxa, which share similarities to the other two Theodoria species (T. wallini and T. partita), which in turn share similarities to Ophiurolepis species. Characters shared betwen the four Theodoria species are primarily those used in diagnosing the genus: deeply excavate jaws and conspicuous basal tentacle pores. 27 2.4.2 Analysis excluding T. relegata and T. madseni Owing to the confounding efect of T. relegata and T. madseni, I focus instead on relationships recovered when these two taxa were removed. Morphological data suggest that two, possibly thre, major clades (Fig. 3) are present within Ophiurolepis. Two of these clades, clades A and C, are present also in the analysis that includes T. relegata and T. madseni, whereas Clade B collapses when these two taxa are included. Clade A, a ten species clade, contained only species currently recognized as Ophiurolepis. Clade B contained thre Ophiurolepis species as wel as Theodoria wallini, T. partita and Homalophiura brucei. Homalophiura brucei was previously suggested to have a greater afinity with Ophiurolepis than with Homalophiura (Paterson 1985). Within clade B, T. partita and T. wallini were supported as sister taxa. The third clade, clade C, was basal to A and B and included thre Homalophiura species (H. clasta, H. confragosa and H. inornata) and O. scisa. As previously stated, H. inornata and H. confragosa have been suggested as belonging within Ophiurolepis, while H. clasta was alied with Homophiura (Paterson 1985). Ophiurolepis scisa had at one time been placed within Homalophiura (Clark 1915), reflecting a previously recognized afinity betwen this species and some members of Homalophiura. Homalophiura intorta and H. euryplax were basal to al Homalophiura and Ophiurolepis species. This was not surprising given that Paterson (1985) grouped these two species with Ophiura in his revision of the Homalophiura, and not Ophiurolepis. Both bootstrap proportions and decay indices indicated low support for relationships within and betwen clades A-C, but moderate support for grouping these clades to the exclusion of H. euryplax and H. intorta. 28 2.5 DISCUSION This first atempt to elucidate phylogenetic relationships within Ophiurolepis has revealed two major clades, and tentatively a third. Many species within Ophiurolepis are distinguished by only a few morphological characters, some of which vary widely within these species. This condition semingly creates a continuum among certain species that are dificult to separate from one another based on morphological character data. For example, O. olstadi Madsen, 1955 was noted by Madsen (1955) as resembling O. gelida in ?overal appearance?, but distinguished by a more thickened skeleton and short genital slits. It could be that skeleton thicknes is a variable morphological character and that this species is actualy a form of O. gelida with short genital slits. Another species, O. brevirima, difers from O. olstadi only in having broader jaws and adoral plates and in having fewer arm spines (Madsen 1955). These distinctions may represent morphological variability in these characters and not species boundaries. Therefore, the lack of strong support for relationships within Ophiurolepis is not surprising. Twenty-thre morphological character states difered betwen Ophiurolepis clades A/B and the outgroup Ophiuroglypha (Fig. 4), suggesting that considerable morphological evolution has occurred betwen these two genera. In stark contrast, only two synapomorphies separate clades A/B from clade C, composed primarily of Homalophiura species. Clades A/B are distinguished by their disc elevation (character 1) while members of clade C posses fragmented dorsal arm plates (character 31). However, this later synapomorphy has convergently arisen in both O. tuberosa and T. partita of clade B, casting doubt on its phylogenetic utility. 29 The question remains whether Homalophiura is a valid genus, or if H. inornata should be transfered to Ophiurolepis, invalidating the genus. The present analysis indicates that thre Homalophiura species (H. inornata, H. confragosa and H. clasta), one of which is the type species, are scarcely distinguishable from other Ophiurolepis species. One Homalophiura species, H. brucei, even fals within an Ophiurolepis clade. Also of importance is that an Ophiurolepis species, O. scisa, was recovered as sister to H. inornata, the type species. Therefore, it sems practical to consider clade C as a basal Ophiurolepis clade, sister to the two derived clades A and B. This placement has been proposed by others (Hertz 1926; Mortensen 1936; Bartsch 1982), most explicitly by Paterson (1985). Conversely, H. intorta and H. euryplax are distinguished by a greater number of synapomorphies and separate from the ingroup species with moderate support. This indicates, as previously recognized (Paterson 1985), that at least these two Homalophiura species do not belong within Ophiurolepis. The two derived Ophiurolepis clades are diferentiated by the gonad character (character 34) of clade B, and by the characteristic thickening of the disc plates (character 2) in clade A (Fig. 4). It was not surprising that Theodoria partita and T. wallini fel within Ophiurolepis. In the original description, Fel (1961) stated that Theodoria was most closely related to Ophiurolepis, difering only by deeply excavate jaws, thre conspicuous tentacle pores on the proximal arm joints, and relatively smaler and often fragmented oral shields. However, four Ophiurolepis species are known to have fragmented oral shields, as does H. inornata. Furthermore, the size of the oral shields appears to be comparable among the two genera when scaled for disc size (personal observation). Therefore, only two diagnostic morphological characters separate T. partita 30 and T. wallini from Ophiurolepis species. These characters could be easily explained if T. partita and T. wallini were derived from within clade B. Potentialy, T. relegata and T. madseni also are derived taxa within Ophiurolepis, but more data are needed to unambiguously determine the relationship of these two species to other Ophiurolepis and Theodoria species. However, it is likely also that these two taxa are alied with T. partita and T. wallini only on the basis of the characters discussed above. Some of these characters, such as size and fragmentation of the oral shields, have been shown to be convergent and may not reflect any real phylogenetic relationship among these two taxonomic groups. 2.5.1 Morphological characters The utility of morphological characters used in this study was examined by analyzing their patern on the resulting phylogeny. Convergence or reversals were evident for fiften characters, most of which are diagnostic characters used in ophiuroid taxonomy. For example, the length of the genital slit often is used to separate closely related species, such as O. gelida and O. brevirima. However, this study has shown that this character may not be useful in asesing phylogenetic relationships among these ophiuroid species, due to repeated convergent evolution. Similarly, fragmentation of the oral shields has arisen independently in several lineages. The convergent nature of many characters used currently in ophiuroid taxonomy sems to limit their efectivenes in reconstructing phylogenies, especialy of closely related species. This is reflected in the paucity of morphological cladistic studies that exist for ophiuroids at al taxonomic levels (e.g., Smith et al. 1995). 31 2.5.2 Taxonomic revision of Ophiurolepis Although a systematic revision of Ophiurolepis, Homalophiura and Theodoria is beyond the scope of this study, preliminary recommendations may facilitate a more formal revision in the future. Morphological evidence points to the inclusion of H. inornata, H. clasta, H. confragosa and H. brucei in Ophiurolepis. Following this recommendation, Ophiurolepis species would be diagnosable by thre synapomorphies: rudimentary arm combs, distalmost arm spine separated from the other arm spine and tentacle scales by a gap, and crescent-shaped genital plates. Remaining Homalophiura species would consequently be invalidated due to re-asignment of the type species, H. inornata. Further examination of these species, including two used in this study, H. intorta and H. euryplax, is necesary to determine whether re-asignments into existing genera are appropriate or if a new genus/genera is needed to acommodate these species. For the genus Theodoria, T. partita and T. wallini share one synapomorphy, jaws excavate on the midline, that separates them from other Ophiurolepis species. However, these two species also share two character reversals, evenly spaced arm spines across the distal margin of the lateral arm plate and conspicuous basal tentacle pores. Theodoria relegata exhibits these character states, but the position of this species remains unclear from the phylogenetic analysis conducted herein. Therefore, given that T. partita and T. wallini are distinct within Ophiurolepis, it is recommended that a more thorough investigation of these two taxa, with T. relegata and T. madseni, be caried out. 32 2.5.3 Biogeographic considerations With some understanding of the phylogenetic relationships within Ophiurolepis, it is possible to evaluate hypotheses on the biogeographic history of this group. The topology depicted in Figure 3 suggests that clades A and B arose from a geographicaly widespread ancestor, based on the wide distribution of its sister group, clade C. Subsequently, part of this ancestral stock occurring around Antarctica could have then become isolated as Antarctica separated from Australia and then South America (~41 mya), and become further isolated by the onset of the Antarctic Circumpolar Current in the late Eocene (Scher & Martin 2006). Given that the greatest diversity of the genus is in the Antarctic and that al but one species occur there, it is likely that speciation occurred after isolation, resulting in the present day clades A and B. Some species then subsequently spread into southern South America and in the case of O. mordax and O. carinata, spread to ocean basins based on the supposed ability of deeper water forms to traverse ocean floors (Fel et al. 1969). This sems feasible given that O. mordax has been collected as deep as 2500 m. In fact, this particular species could be a rare example of ?polar emergence?, in which cold stenothermal forms are found in the same Antarctic water layer, the Antarctic Bottom Water, but at diferent latitudes (Vadon & Guile 1984). Faunal exchange betwen South America and the Antarctic Peninsula and South Georgia region has long been presumed and is reflected in the twenty-five ophiuroid species shared betwen these two areas (Fel et al. 1969). Faunal afinities betwen these regions have been determined for a number of Antarctic taxa (se Bargeloni et al. 2000a; Bargeloni et al. 2000b; Page & Linse 2002; Stankovic et al. 2002). Therefore, it is 33 highly probable that several Ophiurolepis species independently established populations in southern South America. One of these species, O. turgida, may have even become extinct in the Antarctic, now being endemic to southern South America. Why is Ophiurolepis so speciose in the Antarctic? One possibility is reflected in the smal degre of morphological diference betwen many Ophiurolepis species and could be the result of ?taxonomic spliting?. This efect can lead to many taxonomic species within a group that do not represent biological species. Future molecular and ecological data could begin to answer these questions that remain unresolved at the morphological level. Second, at least seven species, O. brevirima, O. gelida, O. martensi, O. tuberosa, T. partita, T. wallini and H. inornata are probably brooders, while for the rest the reproductive mode is unknown (Mortensen 1936; Hendler 1991). Thus, it is possible that speciation in this group was enhanced or even driven by the lowered dispersal capabilities of brooding species, especialy if brooding was the ancestral state. This patern has been discussed for Antarctic asteroids (Pearse & Bosch 1993) and noted for Antarctic brooding echinoids, which are more speciose than their relatives who undergo planktotrophic development (Pearse et al. 1991). Examination of morphological traits in Ophiurolepis has shed some light on phylogenetic relationships within this genus. However, since these and other ophiuroid species appear to be plagued by convergent morphology, other types of data, namely molecular, are necesary to resolve relationships among these ecologicaly important and abundant animals. 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Sorenson, M.D. (1999) TreRot, version 2. Boston University, Boston, MA. Stankovic, A., Spalik, K., Kamler, E., Borsuk, P. & Weglenski, P. (2002) Recent origin of sub-Antarctic notothenioids. Polar Biology, 25, 203?205. Studer, T. (1876) Uber Echinodermen aus dem antarktischen Mere und zwei neue Seigel von den Papua-Inseln, gesamelt auf der Reise S.M.S. Gazele um die Erde. Monatsberichte der K?niglichen Preussische Akademie des Wisenschaften zu Berlin, 1876, 452?465. 41 Studer, T. (1885) Die Sesterne S?d-Georgiens, nach der Ausbeute der Deutschen Polarstation in 1882 und 1883. Jahrb. der Wisenschaften Anstalten zu Hamburg, 2, 141?166. Swofford, D.L. (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Asociates, Sunderland, MA. Tommasi, L.R. (1968) Os ofiur?ides existentes nas cole?oes do museu de Buenos Aires coletados do la Plata at? 42? latitude sul. Pap?is Avulsos de Zoologica Sao Paulo, 21, 115?124. Tommasi, L.R. (1976) Ophiuroidea collected in the Peru-Chile Trench by the USNS Eltanin during cruise II. Pap?is Avulsos de Zoologica Sao Paulo, 29, 281?318. Vadon, C. & Guile, A. (1984) The family Ophiuridae in the bathyal zone of the Indian Ocean: Origin and biogeography. In: Kegan, B.F. & Balkema, A.. (Eds.), Procedings of the Fifth International Echinoderm Conference, 24?29 September, Galway, Ireland. Rotterdam, Balkema, pp. 645?652. 42 Table 1 Distributions, depth ranges and catalog numbers for species of Ophiurolepis, Theodoria and Homalophiura used in this study. Taxon Distribution Depth Range USNM Catalog No. Reference Ophiurolepis acomodata Marion Is. 130 m Koehler 192 anceps Ros Sea, Wedel Sea 700-2580 m 1014081 Koehler 1908; USNM 1 banzarei east Antarctica 190-30 m SAM 2 : K1206-1208, K1210 Madsen 1967 brevirima circumpolar, South Georgia 200-750 m E43649, E43679 Lyman 1878; Mortensen 1936; Fel 1961; Madsen 1967; Bernasconi & D?Agostino 1975; USNM carinata Chile, South Sandwich Is., Kerguelen Is. 50-760 m Studer 1876; Lyman 1878; Mortensen 1936; Selanes & Krylova 205 gelida circumpolar Antarctic/subantarctic 40-650 m E43606, E43634 Koehler 1902, 192, 1923; Mortensen 1936; Clark 1951; Fel 1961; Madsen 1967 granulifera Peterman Is., Petrel Base 250-40 m Bernasconi & D?Agostino 1973 martensi circumpolar Antarctic/subantarctic, Cape Horn 20-310 m 1078489, E4507, E10583, E52048 Koehler 192, 1923; Mortensen 1936; Clark 1951; Fel 1961; Madsen 1967; Rowe & Clark 1975; USNM mordax east Antarctica, South Sandwich Is., Mozambique Chanel 220-250 m 1078474 Koehler 192; Vadon & Guile 1984; USNM olstadi Antarctic Peninsula, Ros Sea 60 m E52417, E52834 Madsen 195; USNM scisa Wedel Sea, Falkland Is. 650-2560 m E46838 Koehler 1908; USN tuberosa Antarctic Peninsula, Ros Sea 200-750 m E43785, E43789 Mortensen 1936; Madsen 195; Fel 1961; USNM tumescens circumpolar 200-2450 m Koehler 192; Madsen 195, 1967; Bernasconi & D?Agostino 1975 turgida Falkland Is. 340 m Mortensen 1936 Theodoria madseni Laguna Grande Peru 3883-404 m E11375 Tomasi 1976 partita Antarctic Peninsula, Ros Sea, South Georgia 130-3250 m E44698, E46807 Mortensen 1936; USNM 43 relegata circumpolar 110-50 m E43592, E44602 Fel 1961; USNM walini circumpolar 130-640 m E43786, E44701 Mortensen 1925, 1936; Madsen 1967 Homalophiura brucei west Antarctica 3000-440 m 1019242, 1019630 Koehler 1908; USNM clasta Japan 930-1680 m 25547 Clark 191 confragosa north Atlantic, Patagonia?Buenos Aires 100-10 m 9848, 26267 Lyman 1878; USNM euryplax Gulf of Aden, Oman Sea, Maldives 1270 m Clark 1939; Vadon & Guile 1984 inornata cosmopolitan 50-380 m E52508 Lyman 1878; Koehler 1904, 1906, 1914, 192; Clark 1915; Mortensen 1936; Madsen 1967; Bernasconi & D?Agostino 1971, 197; USNM intorta Marion Is. 90-140 m Lyman 1878 1 refers to distribution records of catalogued specimens from the Smithsonian Institution National Museum of Natural History (USNM) 2 South Australian Museum 44 F Figure 1 Plate showing aboral disc view of various ophiuroid species used in this study. A. Ophiurolepis anceps, B. O. banzarei, C. O. brevirima, D. O. gelida, E. O. martensi, F. O. mordax, G. O. olstadi, H. O. scisa, I. O. tuberosa, J. Homalophiura brucei, K. H. clasta, L. H. confragosa, M. H. inornata, N. Theodoria partita, O. T. relegata, P. T. wallini. 45 Figure 2 MP strict consensus tre for data including T. relegata and T. madseni. Numbers below branches represent decay indices. 46 Figure 3 MP strict consensus tre for data excluding T. relegata and T. madseni. Numbers above branches represent bootstrap proportions and numbers below represent decay indices. 47 Figure 4 Morphological characters mapped onto the strict consensus MP tre for data excluding T. relegata and T. madseni. Character numbers correspond with Appendix. Bars indicate where a change has occurred, circles indicate reversals and squares indicate convergent character changes. 48 CHAPTER 3: Evaluating connectivity in the brooding britle star Astrotoma agassizi across the Drake Pasage in the Southern Ocean 3.1 ABSTRACT Studies examining population genetic structure and genetic diversity of benthic marine invertebrates in the Southern Ocean have emerged in recent years. However, many taxonomic groups remain largely unstudied, echinoderms being one conspicuous example. The britle star Astrotoma agassizi is distributed widely throughout Antarctica and southern South America. This species is a brooding echinoderm and therefore may have limited dispersal capacity. In order to determine the efect of hypothesized isolating bariers in the Southern Ocean, such as depth, geographic distance and the Polar Front, two mtDNA markers were used to compare populations from the South American and Antarctic continental shelves. Astrotoma agassizi was shown to be geneticaly discontinuous across the Polar Front. In fact, populations previously asumed to be panmictic instead represent thre separate lineages that lack morphological distinction. However, within lineages, genetic continuity was displayed across a large geographic range (> 500 km). Therefore, despite lacking a pelagic larval stage, A. agassizi can disperse across substantial geographic distance within continental shelf regions. These results indicate that geographic distance alone may not be a barier to dispersal, but rather 49 the combined efects of distance, depth and the Polar Front act to prevent gene flow betwen A. agassizi populations in the Southern Ocean. 3.2 INTRODUCTION Oceanographic current paterns and life history traits, such as reproductive strategy, have been shown to directly afect dispersal of organisms in the marine environment (reviewed in Palumbi 1994), thereby profoundly influencing distributional paterns in the world?s oceans. These factors and their influence on population genetic structure of marine organisms have been studied across a wide range of taxa, alowing generalizations to be established on which hypotheses can be based. For example, studies have shown that marine invertebrate species with longer pelagic larval duration often show les population diferentiation than those with abbreviated larval development (Berger 1973; Crisp 1978; Janson 1987; McMilan et al. 1992; Duffy 1993; Hunt 1993; Helberg 1996; Hoskin 1997; Arndt and Smith 1998). However, this relationship can vary greatly, with some species showing high levels of diferentiation despite long-lived pelagic larvae (e.g., Tracey et al. 1975; Burton 1986; Hare and Avise 1996). Efects of oceanographic bariers and life-history constraints have been evaluated across many geographic regions, however one particularly distinctive biogeographic region, the Southern Ocean, is not wel understood in this context. The roles of the Polar Front, smal-scale gyres and life history traits that afect dispersal of marine organisms in the Southern Ocean are only beginning to be understood. Isolation of the Antarctic continent is hypothesized to have been a driving evolutionary force for Antarctic fauna. Separation of Antarctica from South America and 50 the ensuing onset of the Antarctic Circumpolar Curent (AC), dated to betwen 24?41 mya (Lawver and Gahagan 2003; Pfuhl and McCave 2005; Scher and Martin 2006), are presumed to have been primary forces promoting speciation in Southern Ocean taxa (Patarnelo et al. 1996; Page and Linse 2002; Clarke et al. 2005). The Polar Front, the region of the AC marked by a 3-4?C temperature change and high-flow velocity (Eastman 1993), is a particularly strong physical barier (Clarke et al. 2005). AC formation, coupled with decreasing atmospheric CO 2 concentration (DeConto and Pollard 2003), is thought to have driven the gradual cooling and glaciation that began ~34 mya in the Antarctic (Zachos et al. 2001). This cooling and long period of isolation have led to a diverse and abundant benthic fauna that is typicaly stenothermal, eurybathic and endemic to Antarctica (Ekman 1953; Hempel 1985). Endemism is particularly high in certain groups including fish (95%), amphipods (95%), pycnogonids (90%), isopods (87%), and certain echinoderm clases (73%) (Knox and Lowry 1977; Brandt 1991; Jazdzewski et al. 1991). While many Antarctic benthic organisms exhibit high levels of endemism, others show much lower levels despite apparent geographic and thermal isolation. For example, polychaete, echinoderm and molusc conspecifics have been reported on both Antarctic and South American continental shelves, and a wel-recognized faunal afinity exists betwen these two geographic regions (Del 1972; Arntz et al. 1994; Dayton et al. 1994). Lack of endemism in these species suggests some level of recent or ongoing gene flow betwen populations separated by the AC. Several gene flow mechanisms have been proposed, including migration of benthic adults, larval dispersal and rafting. Migration of adults is thought to occur along the Scotia Arc, a submerged ridge with a series of 51 emergent islands that form a ?stepping-stone? connection betwen the Antarctic Peninsula and South America (Fel et al. 1969). Dispersal of larvae or rafting adults/juveniles would most likely occur across the Drake Pasage, the portion of the AC separating Antarctica and South America representing the shortest distance betwen Antarctica and any other continent. Mechanisticaly, dispersal could occur across the AC via warm- and cold-core rings (Clarke et al. 2005), mesoscale eddies known to transport larvae and rafting organisms (Robinson 1983; Scheltema 1986). Studies investigating the evolutionary history of Antarctic fauna using molecular tools have emerged in recent years. These studies have focused primarily on groups such as notothenioid fish (Bargeloni et al. 2000a, 2000b; Stankovic et al. 2002), kril (Patarnelo et al. 1996; Bargeloni et al. 2000b), and molluscs (Brierley et al. 1993; Alcock et al. 1997; Page and Linse 2002), and concentrate on the AC?s role in promoting speciation and divergence in the Southern Ocean. Many Antarctic benthic organisms remain unstudied in terms of their evolutionary history, population connectivity and biogeography, and only a few studies exist evaluating population connectivity of Southern Ocean species within South America (e.g., Brierley et al. 1993; Shaw et al. 2004). A conspicuous example of an unstudied taxon is the Ophiuroidea, abundant and ecologicaly important components of the Antarctic benthic community. Astrotoma agassizi is one of thirten ophiuroid species shared betwen Antarctica and South America (Fel et al. 1969). This species has a circumpolar Antarctic/subantarctic distribution and occurs throughout the southern part of South America, in depths of 80?1200 m (Bartsch 1982). Astrotoma agassizi broods its embryos (Bernasconi 1965; De La Serna De Estaban 1966; Bartsch 1982; IS Smirnov, pers. 52 comm.) and therefore lacks a dispersive larval stage. Astrotoma agassizi is recognized as a morphologicaly uniform species throughout Antarctica and South America. However, given potential for significant population genetic structure owing to presumed limited dispersal capacity, we wanted to determine whether morphological uniformity corresponds also with genetic uniformity in this geographicaly and bathymetricaly widespread species. Two mtDNA gene fragments were employed to evaluate the efects of geographic distance, depth and the Polar Front on population genetic structure and connectivity in this conspicuous Southern Ocean species. 3.3 MATERIALS AND METHODS 3.3.1 Data collection Astrotoma agassizi samples were collected during two cruises to the southern tip of South America and Antarctic Peninsula aboard the R/V Laurence M. Gould. The first cruise took place from 23 November ? 22 December 2004 and the second from 12 May ? 13 June 2006. In total, 207 individuals were collected from eleven stations in South American waters and 30 individuals were collected from six Antarctic stations (Fig. 1 and Table 1). Benthic samples were collected with an epibenthic sled, Blake trawl, or rock dredge. Samples intended for DNA analysis were either frozen upon collection at -80?C or preserved in ~85% ethanol. DNA was extracted using the DNeasy ? Tisue Kit (QIAGEN) following manufacturer?s protocol. Two mitochondrial gene fragments, 16S rDNA (16S) and cytochrome oxidase subunit I (COI), were amplified using standard PCR protocols. 16SarL (5?-CGCTGTTATCAAACAT-3?) and 16SbrH 53 (5?-CGTCTGACTCAGATCACGT-3?) (Palumbi et al. 1991) amplify a ~500 bp fragment from the middle of 16S. For COI, primers were designed based on a COI alignment spanning the diversity of extant echinoderms. The novel primers CO2_23AF (5?-MCARCTWGWTWCAGA-3?) and CO2_577R (5?- TCSGARCATGSCATARA-3?) amplify a ~550 bp fragment from the 5? end of the gene. Double-stranded PCR products were purified using either a gel-freze method or Montage? PCR Filter Units (Milipore). Purified PCR products were bi-directionaly sequenced using a CEQ8000 Genetic Analysis System (Beckman Coulter). Al A. agassizi haplotypes were deposited in GenBank and correspond to acesion numbers EF565745?EF565820. 3.3.2 Population structure analyses Sequences were edited in SeqMan (DNA* LASERGENE) and aligned with Clustal W (Thompson et al. 1994) in MegAlign (DNA* LASERGENE). Alignments were examined visualy in MACLADE v4.0 (Maddison and Maddison 2000) and COI sequences were translated to ensure stop codons were not present. 16S and COI alignments are available in TreBASE (acesion number M3740). Repetitive sequences were collapsed into representative haplotypes in COLAPSE v1.2 (http:/darwin.uvigo.es/). Preliminary analyses of 16S and COI indicated they were congruent and were subsequently combined for select analyses. Parsimony networks were constructed using mtDNA haplotypes in TCS v1.18 (Clement et al. 2000), with a 95% connection limit betwen haplotypes. Gaps were treated as mising data. To determine the number of genetic populations present across 54 the sampled range of A. agassizi, pairwise ? ST were computed betwen al collection stations in ARLEQUIN v3.1 (Excoffier et al. 2005). Acordingly, collection stations where pairwise comparisons were not significantly diferent from zero were pooled in subsequent analyses. ARLEQUIN was used to perform an analysis of molecular variance (AMOVA) on mtDNA sequences to ases how haplotypic variation is partitioned geographicaly. For the AMOVA, variance was partitioned into thre hierarchical components: within collection stations (? ST ), among collection stations within a clade (? SC ), and among clades (? CT ), where clades were determined by phylogenetic analysis (se below). For both pairwise ? ST and AMOVA, a 16S+COI concatenated dataset was used with 10,000 permutations and the Tamura-Nei model (Tamura and Nei 1993) with among site rate variation. Nucleotide (?) and haplotype (h) diversities were calculated in DNASP v4.1 (Rozas et al. 2003). Isolation by distance among collection stations was tested using a Mantel test (Mantel 1967) in ARLEQUIN with 1000 permutations. For the Mantel test, 16S+COI pairwise ? ST values were used for genetic distances, and the linear distance betwen collection stations was log 10 transformed and used for geographic distances. Tajima?s D (Tajima 1989) test statistic was calculated in DNASP to evaluate the asumption of selective neutrality of mtDNA sequences. Mismatch analyses were done in DNASP by comparing the observed versus expected distribution of pairwise nucleotide diferences betwen 16S and COI haplotypes, to determine if population expansion had occurred in the history of A. agassizi. Population expansion was also evaluated by the neutrality test, because values significantly diferent from zero can be indicative not only of selection, but also of demographic paterns such as past population expansion (Aris- 55 Brosou and Excoffier 1996). With the exception of the haplotype network analysis, population-level analyses were performed only with collection stations where four or more individuals were sampled. In order to evaluate levels of migration betwen pairs of populations, an MCMC approach was taken as implemented in the program MDIV (Nielsen and Wakely 2001; http:/cbsuapps.tc.cornel.edu/mdiv.aspx). Initial runs were done with al population pairs (where N ? 6) to obtain an upper limit of the scaled migration rate (M max ) for subsequent runs. Thre independent runs with diferent random number seds were completed for each comparison and results averaged. For these analyses, the finite-sites model (HKY) was used, with Markov chain length = 5 ? 10 6 , 10% burn-in and M max = 10, 25, 50 or 100. The migration rate per generation was determined by the M value with highest posterior probability. 3.3.3 Phylogenetic and genetic distance analyses Phylogenetic relationships among mtDNA haplotypes were estimated using Bayesian methods in MRBAYES v3.1 (Huelsenbeck and Ronquist 2001). For Bayesian analysis, 16S and COI were treated as unlinked partitions and MRMODELTEST v2.2 (Nylander 2004) was used to determine the best-fit model for each partition under the Akaike information criterion (AIC). For 16S, the HKY+I+? model was selected, while for COI the GTR+? model was chosen. Conditions for analysis were uniform prior distribution of parameters and two sets of four simultaneous chains run for 1 ? 10 6 generations with tres sampled every 100 generations. Stationarity was evaluated by examining log-likelihood values per generation. Burn-in tres were discarded before 56 computing a 50% majority-rule consensus tre with nodal support given by the posterior probability of each recovered clade. Resulting topologies were rooted with the outgroup species Astrohamma tuberculatum. Genetic distances were calculated using 16S+COI combined data in PAUP* v4.0 (Swofford 2002) in order to evaluate levels of divergence within A. agassizi. MODELTEST v3.7 (Posada and Crandal 1998) was used to determine the best-fit model of sequence evolution under the AIC for the corrected genetic distances. The transversional (TVM) model with gama shape parameter (? = 0.778) and proportion of invariable sites (0.7696) was selected. 3.4 RESULTS In total, 118 individuals were sequenced for 16S (490 bp) and COI (493 bp), resulting in a 983 bp concatenated dataset. The combined dataset included 64 mitochondrial haplotypes, representing 25 individuals from Antarctica and 93 from South America. No insertions, deletions or stop codons were observed among the 118 individuals for COI. For 16S, the inclusion of a few gaps were required for alignment. 3.4.1 Population structure Parsimony network analysis resulted in thre haplotype networks whether mtDNA gene fragments were analyzed separately (data not shown) or combined (Fig. 2). For the combined data, network 1 ( = clade 1) included 38 haplotypes from 58 individuals from South America, network 2 ( = clade 2) was comprised of 14 haplotypes 57 from 35 individuals from South America, and network 3 ( = clade 3) included 12 haplotypes from 25 individuals from Antarctica. Pairwise ? ST (Table 2) indicated that some collection stations within South America were geneticaly indistinct from one another, as were al collection stations within Antarctica. South American stations 1, 3 and 8 from clade 1 were pooled and stations 4 and 14 from clade 2 were pooled. In Antarctica, al collection stations (where N > 4) were pooled (stations 47, 82 and 85). Within clade 1, ? ST values were significant for every pairwise comparison that included station 5, suggesting that this station is geneticaly isolated from other clade 1 stations. As expected, betwen-clade ? ST values approached 1.0 due to the thre clades being fixed for alternate mtDNA haplotypes. AMOVA results (Table 3) further confirmed genetic isolation betwen clades as the greatest proportion of variance (84%, P = 0.0001) was atributable to that betwen clades. The second largest variance proportion (11%, P < 0.0001) was that within collection stations while only 5% (P < 0.0001) was atributable to that betwen collection stations within a clade. Corroborating parsimony network results, nucleotide (?) and haplotype (h) diversity values indicated that clade 1 was more geneticaly diverse than clades 2 or 3 (Table 4; 16S and COI analyzed separately). These later clades exhibited much lower levels of nucleotide and haplotype diversities, with the exception of COI haplotype diversity, which was slightly higher in clades 2 and 3 compared to clade 1. Results of the Mantel test, performed only on collection stations within clades 1 and 3 given that clade 2 contained only two geographic localities, did not support isolation by distance for stations within clade 1 (r = ?0.03; P = 0.49) or clade 3 (r = 0.02; P = 0.51). 58 Tajima?s D was negative but non-significant for 16S, whereas thre populations (St. 5; St. 9; Sts. 4, 14) had significantly negative values for COI (Fig. 3), indicative of past population expansion (Aris-Brosou and Excoffier 1996). The shape of the 16S and COI mismatch distributions were unimodal for clade 2 and 3 (Fig. 3), suggesting past population expansion. Clade 1 distributions were primarily ragged and multimodal (Fig. 3), suggesting stable population size (Harpending et al. 1998). However, stations 5 and 9 COI mismatch distributions were characterized by a high frequency peak corresponding to low pairwise diferences and a secondary low frequency peak corresponding to higher number of pairwise diferences, potentialy explaining the significantly negative Tajima?s D value for these two stations. Migration analyses revealed stations 47 and 82 (clade 3) from Antarctica to be experiencing the highest levels of gene flow. These two collection stations are the most geographicaly distant populations sampled in Antarctica (with sufficient numbers to perform analyses), therefore it is presumed that geographicaly intermediate stations are experiencing equivalent if not higher levels of gene flow. For stations 47 and 82, the posterior probability distribution plateaued beyond M = 30 migrants per generation, with its highest value atained at M = 66. Although lower than clade 3, relatively high levels of gene flow were also estimated for clade 2. The best estimate of the number of migrants per generation betwen stations 4 and 14 was M = 9.5. For clade 1, migration rates betwen station 5 and both stations 1 and 3 (the furthest and closest stations to station 5, respectively) suggested litle to no gene flow, as highest posterior probability values corresponded with les than 1 migrant per generation (M = 0.30, 0.32). Lack of gene flow is similarly reflected in significant pairwise ? ST values for station 5. Betwen other clade 59 1 populations, migration rates were also low, albeit slightly higher than station 5 comparisons. Migration estimates betwen stations 1, 3 and 9 were around 1 migrant per generation (0.62?1.7). 3.4.2 Phylogenetic relationships and genetic distances Phylogenetic analysis of mtDNA haplotypes revealed thre distinct lineages within Astrotoma agassizi, two in South America and one in Antarctica (Fig. 2). These thre clades correspond to the thre networks recovered in the parsimony network analysis. The two South American clades (clades 1 and 2) were recovered as sister clades with a posterior probability of 0.94, while the Antarctic clade (clade 3) was supported as sister to the South American clades with a posterior probability of 0.99. Intraclade genetic distances were low, averaging from 0.34% (clade 3) to 1.13% (clade 1), while interclade distances were substantialy higher, 4.8% betwen clades 1 and 2, 5.1% betwen clades 2 and 3, and 6.8% betwen clades 1 and 3. 3.5 DISCUSION 3.5.1 Cryptic species in Astrotoma agassizi Astrotoma agassizi is not a geneticaly contiguous, panmictic species, but rather characterized by substantial levels of cryptic diversity. Parsimony network based approaches to recognizing species boundaries have been advocated in recent years (Le and ?Foighil 2004; Tarjuelo et al. 2004; Uthicke et al. 2004; Addison and Hart 2005; Barati et al. 2005; Jolly et al. 2005; Hart et al. 2006), where multiple haplotype networks have been interpreted as multiple species. Acording to these criteria, A. agassizi as 60 currently defined constitutes at least thre putative species. Thre networks were recovered at the 95% connection limit, and each network can tentatively be infered as corresponding to a separate species. Phylogenetic analysis, pairwise ? ST and AMOVA provide additional support for the existence of thre distinct lineages. Further, mtDNA genetic distances betwen the thre clades ranged from 4.8%?6.8%, and distances of this magnitude (5%?7%) are typicaly found betwen echinoderm species easily distinguished by phenotypic or behavioral diferences (Foltz 1997; Hart et al. 1997; Lesios et al. 2001; O?Loughlin et al. 2003; Uthicke and Benzie 2003; Waters and Roy 2003; Uthicke et al. 2004; Waters et al. 2004; Hart and Podolsky 2005). Cryptic speciation has been documented extensively in the marine environment and reported for the Southern Ocean as wel (Beaumont and Wei 1991; Held 2003; Held and W?gele 2005; Raupach and W?gele 2006; Wilson et al. 2007). Held (2003) discussed several criteria for establishing cryptic species. First, species in question should have a bimodal distribution of pairwise genetic distances with no intermediate values. Second, genetic divergence must be found at a level known to exist betwen sister species that are closely related to the species in question. Third, this level of divergence should be found among sympatric populations, although this last criterion was suggested to be les crucial. Astrotoma agassizi fulfils criterion one and likely criterion two. Although no studies exist examining sister species closely related to A. agassizi, genetic divergences among other echinoderms are consistent with the hypothesis of separate species. While geneticaly divergent sympatric populations were not sampled for this study, degre of spatial continuity betwen collection sites is unknown and divergent populations were found at sites separated by les than 80 km. Furthermore, examination of diagnostic 61 morphological characters did not reveal fixed diferences betwen clades. Namely, genital slit length, arm spine number, shape of teth, and disc and arm granulation were uniform among voucher specimens from each collection station. Taken together, these data provide compeling evidence that Astrotoma agassizi is a complex of at least thre cryptic species in the Southern Ocean. 3.5.2 Connectivity betwen Antarctica and South America Data from this study indicate that A. agassizi in Antarctica is geneticaly distinct and geographicaly isolated from A. agassizi in South America. Gene flow is not occurring betwen Antarctic and South American populations. The Drake Pasage separating Antarctica and South America spans approximately 900 km and reaches ~4500 m depths in some places (Whitworth et al. 1982), potentialy beyond the range of dispersal or migration for A. agassizi. However, genetic homogeneity was found across distances greater than 500 km within the Antarctic continental shelf and across distances greater than 300 km within the South American continental shelf. Therefore, it is likely that the Polar Front and/or deep-water pasages, but not sheer geographic distance, act as bariers to gene flow betwen Antarctica and South America. A similar patern has been found in Antarctic demersal fish where genetic homogeneity is maintained within continental shelves but breaks down in populations separated by distances greater than 1000 km or deep-water troughs (Shaw et al. 2004). Restricted gene flow betwen shelf areas separated by great depths (1000?1750 m) has also been shown for the Antarctic octopus Pareledone turqueti (Alcock 1997). 62 Studies investigating the role of the Polar Front in structuring Southern Ocean populations have shown it to be a barier even for organisms with high dispersal potential, such as kril (Patarnelo et al. 1996). However, other studies have shown the Polar Front to be penetrable. For example, bivalve sister species were infered to have diverged after formation of the Polar Front, indicating that at one time individuals were able to disperse across this water mas (Page and Linse 2002). Using an approximate echinoderm tDNA divergence rate of 3.1%?3.5%/my (Lesios et al. 1999; McCartney et al. 2000), separation of Antarctic and South American populations can be dated roughly at 1.4?1.6 mya, wel after Polar Front formation 24?41 mya. These dates suggest that Antarctic and South American populations split wel after the environmental factors that isolate Antarctica were established. In the case of A. agassizi, the Polar Front may have prevented high levels of gene flow, but historicaly was not an absolute barier. Even though these dates are crude estimates, the conclusion that dispersal occurred across the Polar Front at least once is a robust conclusion even if we asume a vastly slower echinoderm tDNA molecular clock. Restricted gene flow was also evident within the South American continental shelf. The two South American clades had lower interclade genetic distances when compared to the Antarctic clade. However, genetic distances were similar for clade 1?2 and clade 2?3 comparisons, indicating that the South American clades split soon after diverging from the common ancestor of al thre clades. Comparisons of physical characteristics betwen collection stations belonging to the two South American clades revealed no obvious diferences in depth distribution or faunal asemblages. Interestingly, samples from station 5 (clade 1), which were significantly diferentiated from al other 63 stations, were collected from substantialy deeper depths (850 m) than any other South American samples. Conversely, station 47 in Antarctica was 900 m and showed no significant diferentiation with any other Antarctic collection locality. Bathymetry may be an isolating force for South American populations of A. agassizi but not Antarctic populations; not surprising given Antarctic fauna are typicaly eurybathic (Hempel 1985). 3.5.3 Intraclade population structure The Mantel test showed no evidence for increasing genetic diferentiation with geographic distance. Within Antarctica, high levels of gene flow resulting in mtDNA homogeneity were demonstrated across a 518 km range throughout the Antarctic Peninsula, an unexpected result given the brooding nature of this species. Genetic homogeneity spanning large geographic distances has been reported for brooding marine invertebrates (Sponer and Roy 2002; Richards et al. 2007). Given A. agassizi lacks a pelagic larval stage, this species must rely on dispersal of benthic adults or juveniles to maintain population connectivity. Pasive transport of rafting adults has been suggested as a means of dispersal for britle stars (Sponer and Roy 2002). Astrotoma agassizi is known to tightly wrap its coiled arms around gorgonians and hydrocorals (Bartsch 1982) and has been recorded climbing up rocks, sponges, bryozoans and other sesile organisms projecting off the seafloor (Dearborn et al. 1986; Ferari and Dearborn 1989). The epifaunal propensity of this species could provide numerous opportunities for pasive transport of dislodged organisms. Furthermore, A. agassizi has been reported to occur in dense aggregations clinging to octocorals (Dearborn et al. 1986; Ferari and Dearborn 1989), increasing the likelihood that individuals could be dislodged and caried by ocean 64 currents to other geographic localities. Juveniles and smal adults probably have a greater chance for pasive dispersal due to the large size atained by full-grown adults (? 60 m disc diameter; Mortensen 1936). Migration of adults/juveniles along the Antarctic continental shelf could also maintain connectivity across these distances. Benthic migration sems les plausible, however, because A. agassizi is iregularly distributed throughout Antarctica, occurring primarily in localy abundant patches (Dearborn et al. 1986). Therefore, connectivity via movement of adults/juveniles along the continental shelf is les likely and dispersal probably occurs by occasional uprooting of smal adults or juveniles atached to sesile substrate and pasively dispersed by ocean currents. Pasive transport betwen A. agassizi patches throughout the Antarctic Peninsula could occur from Bransfield Strait (Fig. 1) to the southern peninsula, and vice versa. A surface current circulates counterclockwise around the Antarctic coast (Philpot 1985) and could promote dispersal from Bransfield Strait southward. Conversely, water from the Belingshausen Sea in the southern peninsula flows north into the Bransfield Strait (Wilson et al. 1999) and could alow for northern transport. In South America, significant genetic diferentiation was absent within clade 1 across distances as great as 320 km, despite low levels of ongoing gene flow. Conversely, high migration was shown for clade 2, however, clade 2 populations are separated by only 131 km. The fact that clade 1 and 2 individuals are separated by 72?484 km suggests that dispersal ability may not be the primary factor driving isolation betwen these clades. Instead, present-day population structure in South America may be explained by alopatric divergence of clade 1 and 2. For example, station 14 of clade 2, 65 situated at the easternmost margin of the sampled range of A. agassizi, or a similar unsampled population, could have at one time been sufficiently peripheral for alopatric divergence to occur, resulting in two South American clades. Subsequently, this once isolated population could have undergone population expansion resulting in a wider distribution. Past population expansion is supported for clade 2 based on the negative Tajima?s D value, unimodal mismatch distribution and shape of the haplotype network. The demographic history of clade 1, the more widely sampled and geneticaly diverse clade, was characterized by stable population size for some populations whereas others showed signatures of past population expansion. Interestingly, within South America there were two instances of divergent haplotypes co-occurring at the same collection station. Station 5 was comprised exclusively of clade 1 haplotypes with the exception of a single individual possesing a clade 2 haplotype. This occurrence could be the result of a rare migration event betwen stations 5 and 14 (where the majority of that clade 2 haplotype were sampled), or additional sampling could reveal clade 1 and 2 to be existing sympatricaly at this locale. Similarly, station 7b, where only two individuals were sampled, was characterized by a single clade 1 haplotype and a single clade 2 haplotype. In summary, Astrotoma agassizi is characterized by unexpected levels of genetic diversity and represents a complex of cryptic species. Populations of A. agassizi separated by the Polar Front are geneticaly isolated and belong to separate lineages. Paradoxicaly, this ?species? was also found to have unexpected levels of genetic continuity for a brooding invertebrate over large geographic distances within continental shelf regions. Similar levels of genetic diversity and divergence likely exist within many 66 other Southern Ocean benthic invertebrates. Additional work is needed to further document biodiversity in this isolated biogeographic region in order to more fully understand the dynamic physical proceses and extreme environmental conditions driving this diversity. 67 3.6 REFERENCES Addison JA, Hart MW, 2005. 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Collection station numbers correspond with Figure 1 and N refers to number of individuals sequenced for 16S and COI. Geographic Colection region station N Latitude Longitude Depth South America 1 12 S 53?16' W 6?23' 96 m 3 6 S 53?47' 61?48' 403 m 4 15 S 53?47' W 60?42' 170 m 5 15 S 53?47' 59?3' 854 m 6 2 S 54?49' W 60?16' 10 m 7a 1 S 54?27' 63?53' 108 m 7b 2 S 54?21' W 60?60' 125 m 8 4 S 54?23' 61?53' 274 m 9 16 S 54?28' W 62?12' 321 m 14 18 S 54?41' 59?24' 207 m 18 2 S 54?41' W 63?14' 254 m Antarctica 31 1 S 6?37' W 68?19' 261 m 47 8 S 62?51' 59?27' 90 m 78 1 S 65?37' W 67?47' 217 m 82 11 S 65?40' 68?02' 278 m 85 4 S 64?41' W 65?56' 368 m 78 Table 2 Pairwise ? ST for each Astrotoma agassizi collection station in South America and Antarctica. St. 1 St. 3 St. 5 St. 8 St. 9 St. 4 St. 14 St. 47 St. 82 St. 85 Geographic region Clade Colection station South America 1 St. 1 ? 1 St. 3 0.142 ? 1 St. 5 0.347 0.497 ? 1 St. 8 -0.043 0.212 0.326 ? 1 St. 9 0.264 0.029 0.561 0.303 ? 2 St. 4 0.851 0.908 0.827 0.873 0.905 ? 2 St. 14 0.873 0.930 0.851 0.902 0.920 -0.010 ? Antarctica 3 St. 47 0.869 0.936 0.870 0.891 0.927 0.926 0.94 ? 3 St. 82 0.82 0.943 0.82 0.909 0.932 0.930 0.946 -0.038 ? 3 St. 85 0.849 0.93 0.85 0.861 0.923 0.926 0.947 -0.153 -0.073 ? Bold indicates P < 0.05 79 Table 3 Hierarchical analysis of molecular variance (AMOVA) for South American and Antarctic populations of Astrotoma agassizi. Source of variation % variation ? statistic P value Among clades 83.65 ? CT =0.84 0.001 Among colection stations 5.36 ? SC =0.3 * within a clade Within colection stations 10.9 ? ST =0.89 * *P < 0.001 80 Table 4 Genetic diversity statistics for pooled Astrotoma agassizi collection stations, N refers to number of individuals, H is the number of haplotypes, ? refers to nucleotide diversity and h is haplotype diversity. Geographic Colection N H ? h region Clade station 16S COI 16S COI 16S COI South America 1 Sts. 1, 3, 8 22 13 6 0.054 ? 0.006 0.013 ? 0.024 0.93 ? 0.04 0.59 ? 0.12 St. 5 14 7 8 0.03 ? 0.008 0.04 ? 0.018 0.82 ? 0.08 0.7 ? 0.12 St. 9 16 6 8 0.030 ? 0.008 0.050 ? 0.023 0.7 ? 0.08 0.70 ? 0.13 2 Sts. 4, 14 33 3 12 0.003 ? 0.002 0.037 ? 0.006 0.12 ? 0.08 0.82 ? 0.05 Antarctica 3 Sts. 47, 82, 85 23 5 10 0.009 ? 0.004 0.037 ? 0.005 0.32 ? 0.12 0.8 ? 0.04 81 Figure 1 Map showing collection localities for Astrotoma agassizi from South America and Antarctica. 82 Figure 2 Bayesian tre of unique 16S+COI mtDNA haplotypes, with corresponding haplotype networks for each of thre phylogenetic clades. Numbers next to nodes indicate Bayesian posterior probabilities. On the Bayesian tre, haplotypes are labeled acording to station. In networks, circles are coded by station and a unique key is given for each clade. Coding does not overlap betwen clades. Haplotypes are sized acording to relative abundance and mising haplotypes are denoted by smal, closed black circles. 83 Figure 3 Mismatch distributions and Tajima?s D statistic for pooled Astrotoma agassizi collection stations. Significant Tajima?s D values are indicated by (P < 0.05). 84 CHAPTER 4: Phylogeography of the Antarctic planktotrophic britle star Ophionotus victoriae reveals genetic structure inconsistent with early life history 4.1 ABSTRACT In the marine environment, connectivity is influenced by physical oceanography as wel as life history and behavioral traits, which in combination with historical geologic/climatic events, determine population genetic structure. The Antarctic britle star Ophionotus victoriae develops via a feding planktonic larval stage, and therefore has potential for long-distance dispersal throughout its Antarctic/subantarctic range. To evaluate this hypothesis, the phylogeography of this ecologicaly dominant species was elucidated by sequence analyses of two mtDNA genes from individuals collected throughout the Antarctic Peninsula and from two subantarctic islands. Counter to expectations of genetic homogeneity, mtDNA data revealed substantial levels of genetic diferentiation as wel as diversity. While there were some geneticaly homogeneous populations, such as those throughout Bransfield Strait, we found O. victoriae to have significant population genetic structure throughout much of the Antarctic Peninsula, with evidence of potential cryptic speciation betwen the western and eastern Antarctic Peninsula. Furthermore, Antarctic Peninsula populations were geneticaly distinct from subantarctic island populations. The low levels of connectivity implied for O. victoriae 85 contrast with those found for many other Antarctic benthic taxa, suggesting a complex interplay betwen oceanography, recent climate history and larval ecology. 4.2 INTRODUCTION The genetic composition of a population is influenced by biological properties as wel as physical environmental factors and historical proceses. Biological properties include dispersal ability, which is expected to corelate with species range size (Jablonski and Lutz 1983; Jablonski 1986; Jefery and Emlet 2003) and often predicts genetic structure (Burton 1982; Palumbi 1994; Ward et al. 2004). For benthic marine organisms, adults typicaly have low mobility and dispersal occurs predominantly during a planktonic larval stage (Kinlan and Gaines 2003; Gerber and Heppel 2004; Paulay and Meyer 2006). Theoreticaly, species with longer planktonic larval duration should disperse greater distances than brooders or those with a brief larval period (Scheltema 1986; Kinlan and Gaines 2003; Shanks et al. 2003), although some marine organisms are known to raft long distances as larvae and/or adults (Highsmith 1985; Helmuth et al. 1994; O?Foighil et al. 1999). Acordingly, many studies have shown that benthic organisms with long-lived planktonic larvae have les population diferentiation than those with abbreviated larval development (Berger 1973; Crisp 1978; Janson 1987; McMilan et al. 1992; Duffy 1993; Hunt 1993; Helberg 1996; Hoskin 1997; Arndt and Smith 1998). However, mounting evidence suggests the relationship betwen early life history and dispersal is anything but straightforward and predictable. Recent studies have shown that in many cases, marine species with planktonic larvae display genetic paterns 86 consistent with low levels of connectivity and geographic subdivision (Hare and Avise 1996; Palumbi 1996; Taylor and Helberg 2003; Sotka et al. 2004; Galarza et al. 2009). Physical bariers in the marine environment can limit or prevent connectivity in species with otherwise high dispersal potential, resulting in decreased gene flow and increased population diferentiation. Examples of such bariers include oceanographic proceses such as fronts, eddies, prevailing currents (Hedgecock 1986; Scheltema 1986; Cowen et al. 1993; Hare and Cowen 1996; Gaylord and Gaines 2000) and varying temperature/salinity composition of adjacent water mases (Hutchins 1947; Valentine 1966; Gaines et al. 2007), as wel as land bariers (Lesios 1981; Knowlton 1993). Historicaly, Pleistocene glacial cycles subjected benthic fauna to fluctuating sea-levels and cyclic expansions/contractions of large ice sheets in both the Northern and Southern hemispheres (Hewit 2000), with a cumulative efect of increasing divergence and speciation rates while reducing genetic diversity in a number of marine and terestrial species (Hewit 2000). Antarctica and the surrounding Southern Ocean provide an unparaleled system in which to study biological and physical factors afecting marine connectivity. Antarctica has been geographicaly and thermaly isolated for 28-41 milion years (Lawver and Gahagan 2003; Pfuhl and McCave 2005; Scher and Martin 2006), resulting in high endemism (Knox and Lowry 1977; Brandt 1991; Jazdzewski et al. 1991) and stenothermy, with many species unable to survive even slight increases in water temperature (Peck and Conway 2000; Peck et al. 2004; Peck et al. 2009). Dispersal ability of many Antarctic benthic invertebrates is enhanced by the slow development of their larvae in the cold Southern Ocean, alowing longer persistence times in the water 87 column (Bosch et al. 1987; Stanwel-Smith and Peck 1998). For example, Antarctic echinoderm planktotrophic (i.e., feding) larvae are estimated to spend 5-6 months in the water column (Pearse and Bosch 1986; Bosch et al. 1987), and lecithotrophs (i.e., non- feding) may persist for up to 2-3 months (Bosch and Pearse 1990). Circumpolar currents in the Southern Ocean have long been considered to influence the distribution of Antarctic marine organisms (Fel 1962; Del 1972; Arntz et al. 1994; Waters 2008). These include the Antarctic Circumpolar Current (AC), a powerful, easterly-flowing curent, and the Eastwind Drift, a weaker countercurrent that circulates around the Antarctic coast (Philpot 1985; Stein and Heywood 1994). The AC and Eastwind Drift are presumed to have a homogenizing efect on populations by transporting larvae and/or adults around the Antarctic continent (Fel 1962; Del 1972; Arntz et al. 1994; Waters 2008). Large cyclonic gyres in the Weddel and Ross Seas are also thought to play a role in determining levels of connectivity among Antarctic marine organisms (Patarnelo et al. 1996). For example, the Weddel gyre has been implicated in restricting gene flow betwen populations occurring on either side of the gyre (Bargeloni et al. 2000). Additionaly, the Antarctic benthos have been heavily impacted by the expanding/contracting polar ice sheet throughout the Pleistocene, and the continental slope may have acted as a refugium for shelf fauna during glacial maxima when most of the continental shelf was covered by glaciers (Clarke and Crame 1989, 1992; Thatje et al. 2005; Clarke 2008). Studies investigating the evolutionary history of Antarctic marine organisms have, in many cases, recovered greater genetic divergence among populations than would be expected based on dispersal potential (e.g., Bargeloni et al. 2000; Wilson et al. 2007; 88 Thornhil et al. 2008). Britle stars (Ophiuroidea) are abundant throughout Antarctica, yet have received litle atention in terms of their evolutionary history. Ophionotus victoriae is a widely distributed and abundant ophiuroid throughout the Antarctic/subantarctic region, typicaly being the dominant britle star in Antarctic benthic asemblages (Dahm 1996). This species has a circumpolar Antarctic/subantarctic distribution and is found inhabiting a variety of substrates ranging from ud to rocky bottoms at depths of 5-1300 m (Fel 1961; Frat and Dearborn 1984). Ophionotus victoriae produces a typical ophiopluteus planktotrophic larvae, and spawns annualy during November-December, coincident with the austral summer phytoplankton bloom (Grange et al. 2004). Given the ecological and biogeographic importance of this species, combined with its potential for long-distance dispersal, O. victoriae is an ideal candidate for evaluating the efects of life history, oceanography and historical proceses on population genetic structure in Antarctic invertebrates. We sequenced and analyzed two mitochondrial genes (16S rDNA and COI) to determine whether this species conforms to the hypothesized patern of genetic homogeneity across populations, or instead shows evidence of limited connectivity owing to contemporary and/or historical forces. 4.3 MATERIALS AND METHODS 4.3.1 Data collection Ophionotus victoriae individuals from the Antarctic Peninsula were collected during two cruises aboard the R/V Laurence M. Gould during November-December 2004 and May-June 2006. Benthic samples were collected using an epibenthic sled, Blake trawl, or rock dredge. Samples from the subantarctic South Sandwich and Bouvet Islands 89 were collected during the 2004 ICEFISH expedition aboard the R/V Nathaniel B. Palmer using Blake or otter trawls. Samples for DNA analysis were either frozen upon collection at -80?C or preserved in ~85% ethanol. Detailed sampling information is provided in Table 1 and Figure 1. DNA was extracted using the DNeasy ? Tisue Kit (QIAGEN) following manufacturer?s protocol. Two mitochondrial gene fragments, 16S rDNA (16S) and cytochrome c oxidase subunit I (COI), were amplified using standard PCR protocols. Primers 16SarL (5?-CGCTGTTATCAAACAT-3?) and 16SbrH (5?-CGTCTGACTCAGATCACGT-3?) (Palumbi et al. 1991) amplify a ~500 bp fragment from the middle of the 16S gene. For COI, the primer set LCO1490 (5?- GTCACAATCATAAGATATG-3?) and HCO2198 (5?- TAACTCAGGTGACAAATCA-3?) (Folmer et al. 1994) was used to amplify a ~560 bp fragment from the 5? end of the COI gene. Double-stranded PCR products were purified using the QIAquick ? Gel Extraction Kit (QIAGEN) following manufacturer?s protocol. Purified PCR products were bi-directionaly sequenced using a CEQ8000 Genetic Analysis System (Beckman Coulter). Al O. victoriae haplotypes were deposited in GenBank under acesion numbers FJ917290-FJ917354. Sequences were edited in SeqMan (DNA* LASERGENE) and aligned with Clustal W (Thompson et al. 1994) in MegAlign (DNA* LASERGENE). Alignments were examined by eye in MacClade v4.0 (Maddison and Maddison 2000) and COI sequences were translated to ensure stop codons were not present. No gaps were required for the 16S alignment. 16S and COI alignments are available in TreBASE (ww.trebase.org; acesion nos. XX). Given that mitochondrial genes represent a 90 single, non-recombining locus and as such have a single evolutionary history (Avise 2004), 16S and COI gene fragments were concatenated for data analysis. 4.3.2 Population structure analyses Parsimony networks were constructed using mtDNA haplotypes in TCS v1.18 (Clement et al. 2000) with a 95% connection limit betwen haplotypes. To ases levels of genetic diferentiation betwen sampling stations, pairwise ? ST ?s were computed in Arlequin v3.1 (Excoffier et al. 2005). Arlequin was used to perform an analysis of molecular variance (AMOVA) on mtDNA sequences to ases how haplotypic variation was geographicaly partitioned. For the AMOVA, variance was partitioned into thre hierarchical components: within sampling stations (? ST ), among sampling stations within a geographic region (? SC ), and among geographic regions (? CT ). For these analyses, geographic regions were Northern Peninsula, Southern Peninsula and Subantarctic Islands. These regions were determined a priori based on oceanographic discontinuities betwen the Northern and Southern Peninsula and because of the >1800 km separating South Sandwich and Bouvet Islands from the Antarctic Peninsula. For both the pairwise ? ST and AMOVA analyses, the 16S+COI dataset was used with 10,000 permutations, and the Tamura-Nei model (Tamura and Nei 1993) with among site rate variation was chosen because it most closely approximated the model of sequence evolution selected by Modeltest v3.7 (Posada and Crandal 1998). 91 4.3.3 Historical demography and migration analyses Nucleotide (?) and haplotype (h) diversities were calculated in DnaSP v4.1 (Rozas et al. 2003) to quantify levels of genetic diversity within O. victoriae. Tajima?s D (Tajima 1989) test statistic was calculated in DnaSP to evaluate the asumptions of selective neutrality of mtDNA sequences as wel as population equilibrium. Fu?s F S (Fu 1997) neutrality statistic, shown to be particularly sensitive to population demographic expansion as indicated by large, negative F S values (Fu 1997), was calculated in DnaSP and significance asesed by 10,000 permutations. Genetic distances were measured as uncorrected p values in PAUP* v4.0 (Swofford 2002) to determine levels of divergence betwen O. victoriae haplotypes In order to estimate levels of migration throughout the Antarctic Peninsula and betwen the Peninsula and subantarctic islands, an MCMC approach was taken as implemented in the program MDIV (Nielsen and Wakeley 2001; http:/cbsuapps.tc.cornel.edu/mdiv.aspx). Thre independent runs with diferent random number seds, but otherwise identical run conditions, were completed for each comparison and results averaged. For these analyses, the finite-sites model (HKY) was used, with Markov chain length = 5 ? 10 6 , 10% burn-in and M max = 10, 30 or 50. The migration rate per generation was determined by the M value with highest posterior probability. 4.4 RESULTS Mitochondrial 16S (496 bp) and COI (563 bp) data were collected from 134 individuals from 15 sampling stations, resulting in a 1059 bp combined 16S and COI 92 dataset that yielded 60 unique mtDNA haplotypes. The majority of haplotypes (39, 65%) were singletons. Of the remaining 21 haplotypes, 17 were collected from more than one sampling station. 4.4.1 Population structure Parsimony network analysis resulted in two networks (Fig. 2) at the 95% connection limit, which alowed a maximum of 14 mutational steps. One network ( = Clade 1) consisted of the majority of O. victoriae individuals (127 individuals, 58 haplotypes), while the second network ( = Clade 2; average 1.8% sequence divergence betwen Clades 1 and 2) consisted of seven individuals corresponding to two haplotypes. Six of these seven individuals were collected near Eagle Island in the Weddel Sea (St. 40), while one individual was obtained at Station (St.) 21 in the northeast corner of Bransfield Strait (se Fig. 1). Given that individuals collected at St. 40 belonged to two separate clades, this station was divided into 40a (Clade 1 individuals; N = 5) and 40b (Clade 2 individuals; N = 6) for subsequent analyses. Additionaly, the single Clade 2 individual collected from St. 21 was not included in analyses involving this station. Clades 1 and 2 could be joined into a single network when the connection limit was lowered to 93%, where Clade 2 haplotypes were separated from Clade 1 haplotypes by 16 mutational steps. The Clade 1 network revealed substantial genetic diversity and divergence in O. victoriae. A majority of individuals possesed unique mtDNA haplotypes, and a divergent group of haplotypes within Clade 1 was identified. Within Clade 1a, hereafter used to refer to the majority of Clade 1 (se Fig. 2), individuals from the subantarctic 93 islands did not share haplotypes with Antarctic Peninsula individuals, with one exception. Clade 1b, hereafter used to refer to the divergent Clade 1 group, was separated by a minimum of 11 mutational steps from Clade 1a (average 1.5% sequence divergence betwen Clades 1a and 1b). Clade 1b was comprised almost exclusively of individuals from thre sampling stations from the Antarctic Peninsula, Sts. 17, 58 and D8. Clade 1b formed a separate network at the 97% connection limit, which alowed a maximum of 10 mutational steps. Pairwise ? ST values (Table 2) indicated that Stations 21, 51, D5 and D6 from the Northern Peninsula are geneticaly homogeneous. Furthermore, the two southernmost Antarctic Peninsula stations (Sts. 33 and 47) are geneticaly homogeneous with the above-mentioned Northern Peninsula populations. Individuals collected from Deception Island (St. 64) in the Bransfield Strait are geneticaly distinct from al other populations except Bransfield Strait St. 51, the most geographicaly proximate station. Also in the Northern Peninsula, St. 17, situated just outside Bransfield Strait northwest of the South Shetland Islands, had large and significant ? ST values when compared to other Northern Peninsula stations. However, St. 17 was geneticaly homogenous with Sts. 58 and D8 in the Southern Peninsula. These thre populations largely comprised Clade 1b. Pairwise ? ST values betwen the subantarctic islands and Antarctic Peninsula were significant and often large. However, ? ST values were lower (and in some cases not significant) betwen South Sandwich Island stations and Clade 1a stations. Individuals sampled from the South Sandwich Islands showed significant genetic diferentiation when compared to those collected from Bouvet Island. Not surprisingly, ? ST values from comparisons with Clade 2 were high ( ? 0.59) and significant in al cases. 94 The AMOVA (Table 3) confirmed that the greatest proportion of genetic variance (47%, P < 0.0001) was atributable to that among stations within a geographic region. An almost equivalent amount of genetic variation was found within stations (45%, P < 0.0001), while only 8% (P = 0.100) was atributable to betwen geographic regions. 4.4.2 Historical demography Nucleotide diversity indices (Table 1) were similar in magnitude for al populations with the exception of Station 40b from the Weddel Sea, which exhibited lower nucleotide diversity. Similarly, haplotype diversities (Table 1) were typicaly high, with the exception of Station 40b. Tajima?s D, while negative for many populations, was not significant in any case (Table 1). Fu?s F S was also negative for the majority of stations, and significant for four populations (Table 1), indicating that recent expansion is not supported for most O. victoriae populations. When Clade 1 and Clade 1a were analyzed, significant negative Fu?s F S values resulted, possibly because these clades include geneticaly distinct populations (Skibinski 2000). To ases migration betwen Bransfield Strait and other geographic regions, 17 individuals from thre homogenous populations within Bransfield Strait (Sts. 21, 51 and D6) were randomly chosen and pooled to form a composite Bransfield Strait population. Gene flow betwen Bransfield Strait and St. 33 in the southern Peninsula was high, as the posterior probability distribution plateaued around M = 10 migrants per generation (Fig. 3). In contrast, migration betwen Bransfield Strait and St. 17, situated just 100 km northwest of Bransfield Strait, was low (M = 0.34), as was migration betwen Bransfield Strait and the two Anvers Island populations (M = 0.3) (the two homogenous Anvers 95 Island populations, Sts. 58 and D8, were pooled for migration analyses). Gene flow betwen the Anvers Island populations and St. 17 was moderate, around thre migrants per generation. In order to estimate the number of migrants betwen the Antarctic Peninsula and the subantarctic islands, migration analyses were performed using the composite Bransfield Strait population and a pooled South Sandwich Islands population, comprised of al individuals sampled from the South Sandwich Islands (Sts. 51 and 57). The two Bouvet Island populations (Sts. 76 and 81) were similarly pooled. Levels of migration betwen Bransfield Strait and the South Sandwich Islands were very low, les than one migrant per generation (M = 0.2), and were comparable to migration rates betwen Bransfield Strait and Bouvet Island (M = 0.3). 4.5 DISCUSION In this study, a planktotrophic britle star, Ophionotus victoriae, showed evidence of genetic divergence betwen some closely situated populations, while other more distant populations were geneticaly homogeneous. The genetic structure of O. victoriae is complex, with no clear concordance betwen genealogy and geography (Fig. 4). Instead, physical oceanography, bottom topography, recent geologic history and larval ecology appear to have shaped the modern-day population genetic structure of this ecologicaly dominant Antarctic echinoderm. 4.5.1 Interclade genetic relationships Mitochondrial data show that Ophionotus victoriae has been subject to some degre of contemporary or historical isolation throughout the Antarctic Peninsula. 96 Evidence from statistical parsimony indicates at least two distinct genetic lineages occur in this region. One lineage, Clade 1, appears widespread throughout the Antarctic Peninsula and occurs around South Sandwich and Bouvet Islands. In contrast, the second lineage, Clade 2, was sampled from only two stations (Sts. 21 and 40). The restricted geographic region from which Clade 2 haplotypes were sampled encompases the northern and southern margins of the Antarctic Sound (Fig. 1), the body of water flowing betwen the northeast end of the Antarctic Peninsula and Joinvile Island. The Antarctic Sound is approximately 48 km and connects Bransfield Strait on the western side of the Antarctic Peninsula to the Weddel Sea on the eastern side of the Antarctic Peninsula. Clades 1 and 2 have haplotypes distributed on either side of the Antarctic Sound, suggesting recent or ongoing gene flow within clades across this body of water. Gene flow could occur via larval transport in Antarctic Surface Water, which flows from the Weddel Sea into Bransfield Strait through the Antarctic Sound, and is therefore likely unidirectional (Stein and Heywood 1994). Given the majority of Clade 2 individuals were collected from the Weddel Sea, it is possible that Weddel Sea populations of O. victoriae are geneticaly distinct from western Antarctic Peninsula and subantarctic populations. This patern has been recovered for other Antarctic taxa, including several notothenioid fish, the Antarctic kril Euphausia superba, and the giant Antarctic isopod Glyptonotus antarcticus. These taxa similarly show significant genetic diferentiation betwen populations from the western Antarctic Peninsula and/or subantarctic islands compared to the Weddel Sea, despite substantial dispersal potential (Zane et al. 1998; Bargeloni et al. 2000; Patarnelo et al. 2003; Held and W?gele 2005). The Weddel Gyre, which separates much of the Weddel 97 Sea from surrounding waters, and the Weddel-Scotia Confluence, where the AC and Weddel Gyre interact, have been implicated in limiting gene flow in and out of the Weddel Sea (Zane et al. 1998; Bargeloni et al. 2000; D?ez et al. 2004). In the case of O. victoriae, migration across the Antarctic Sound may have been restricted during Pleistocene glacial periods, due to lowered sea levels and ice sheet expansion (Thatje et al. 2005). Isolation of western and eastern Antarctic Peninsula populations during glacial periods could explain present-day divergent lineages. Clades 1 and 2 may have only recently established secondary contact, with contemporary oceanographic bariers reinforcing isolation of Clade 2 in the Weddel Sea. Using an approximate echinoderm tDNA divergence rate of 3.1%-3.5%/my (Lesios et al. 1999; McCartney et al. 2000), separation of Clades 1 and 2 can be dated around 510,000- 580,000 years ago, during the mid-Pleistocene. Interestingly, this timing roughly coincides with the split betwen the two most closely related cryptic lineages of the Antarctic crinoid Promachocrinus kerguelensis (Wilson et al. 2007). Further sampling throughout the Weddel Sea could reveal whether the population at St. 40 represents an anomalous divergence, or is typical of O. victoriae populations in the Weddel Sea. Whether Clades 1 and 2 represent cryptic species not previously recognized on the basis of morphology requires further sampling throughout the Weddel Sea and eastern Antarctic. However, examination of external morphological characters did not reveal any fixed diferences betwen clades. Given the prevalence of cryptic speciation among Antarctic benthic marine invertebrates (e.g., Beaumont and Wei 1991; Held 2003; Held and W?gele 2005; Raupach and W?gele 2006; Linse et al. 2007; Wilson et al. 2007; Hunter and Halanych 2008; Lese and Held 2008; Mahon et al. 2008; Thornhil et al. 98 2008; Wilson et al. 2009), it would not be surprising if additional collections revealed O. victoriae, as currently recognized, to be comprised of two or more cryptic species. 4.5.2 Clade 1 relationships: the Antarctic Peninsula Clade 1 is comprised of two geneticaly divergent subclades. Based on our sampling, Clade 1a occurs throughout the Bransfield Strait and more southern regions of the Antarctic Peninsula, as wel as around South Sandwich and Bouvet Islands. Clade 1b was collected primarily from two geographicaly proximate stations near Anvers Island, and from a third station approximately 100 km northwest of the South Shetland Islands. Anvers Island is situated betwen Bransfield Strait and the two southernmost sampling sites along the Antarctic Peninsula. Given that the Antarctic Peninsula continental shelf has been open since the end of the last glacial maxima ~19,000 years ago (Gersonde et al. 2005), it is likely that present-day mechanisms, rather than historical efects, are responsible for this distributional patern. Although a wel-established oceanographic barier does not exist that could explain this distribution of mtDNA haplotypes, water mases of diferent origins flowing at diferent depths over complex topography throughout the Antarctic Peninsula may be involved. For example, water from the AC, Eastwind Drift, and Weddel Sea flows throughout the Antarctic Peninsula as Antarctic Surface Water. In contrast, Antarctic Bottom Water, formed primarily in the Weddel Sea, flows north at deep depths along the continental shelf and slope, while Circumpolar Dep Water flows south and upward towards the continent at intermediate depths (El- Sayed 1985; Stein and Heywood 1994). 99 In general, it is thought that limited water exchange occurs along the Antarctic Peninsula continental shelf, due to narow shelf topography and water mas structure (Smith et al. 1999). Additionaly, the shalow topography of the southwest entrance to Bransfield Strait likely prevents southern Antarctic Peninsula water from entering Bransfield Strait (Smith et al. 1999). However, some AC water flowing from the southwest does enter Bransfield Strait through a deep gap betwen two islands at the southwest margin (Zhou et al. 2002). Given this, planktonic larvae of O. victoriae from the southern Peninsula could potentialy become entrained in the AC, and occasionaly transported into Bransfield Strait in Antarctic Surface Water. This transport mechanism could acount for gene flow occurring betwen Sts. 33 and 47 in the southern Antarctic Peninsula and Bransfield Strait. The distinct genetic signature of the populations near Anvers Island implies that oceanographic paterns in this region difer from those farther south along the Antarctic Peninsula, or that larvae are being transported in diferent water mases. Larvae in this region may be transported ofshore in Antarctic Surface Water as it flows northward (El-Sayed 1985), and enter the AC beyond the point where some AC water enters Bransfield Strait. Genetic homogeneity and moderate gene flow betwen the Anvers Island populations and the station north of the South Shetland Islands (St. 17) support this hypothesis, given that St. 17 is offshore and directly in the path of the AC. In contrast to O. victoriae, other studies of Antarctic planktotrophic marine invertebrates have recovered identical mtDNA haplotypes across large distances throughout the Antarctic Peninsula (Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.), and even Antarctic brooders show evidence of extensive 100 connectivity throughout the Antarctic Peninsula (Hunter and Halanych 2008; Mahon et al. 2008). One notable exception is the lecithotrophic crinoid Promachocrinus kerguelensis, characterized by multiple cryptic lineages, several of which are not shared betwen Bransfield Strait and the southern Antarctic Peninsula (Wilson et al. 2007). While litle is known about O. victoriae ophioplutei, it is possible that larval ecology is limiting dispersal betwen certain geographic regions. For example, vertical migration behavior may expose O. victoriae ophioplutei to diferent water mases compared to other planktotrophic larvae. Additionaly, temporal diferences in spawning may influence dispersal. Two planktotroph species that display extensive genetic connectivity throughout the Antarctic Peninsula (i.e., Odontaster validus and Parborlasia corrugatus) spawn at diferent times of the year compared to O. victoriae (Pearse et al. 1991; Shreve and Peck 1995; Stanwel-Smith and Peck 1998), which may expose their larvae to diferent oceanographic and environmental conditions. Indeed, Antarctic zooplankton tend to be more abundant in coastal waters in the summer, and in deeper, open-ocean waters in the winter (El-Sayed 1985). 4.5.3 Clade 1 relationships: Subantarctic Islands Gene flow betwen the subantarctic South Sandwich and Bouvet Islands and the Antarctic Peninsula appears to be infrequent. Only a single haplotype was shared betwen these regions and significant genetic structure was recovered. The South Sandwich Islands are situated approximately 1800 km from Bransfield Strait, whereas Bouvet Island is approximately 3300 km from Bransfield Strait. Significant genetic structure was also recovered betwen the South Sandwich Islands and Bouvet Island, 101 separated by about 1500 km. These results contrast with phylogeographic paterns found for several other Antarctic taxa, where multiple mtDNA haplotypes were shared betwen the Antarctic Peninsula and these subantarctic islands (Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.; but se Wilson et al. 2007). Due to the eastward circumpolar flow of the AC, any gene flow occurring betwen the Antarctic Peninsula and subantarctic islands would most likely be unidirectional. Larvae could be transported in Antarctic Surface Water as it exits the northeast margin of the Bransfield Strait, pases by Elephant Island, and merges with the AC, which then flows past the subantarctic islands (Stein and Heywood 1994). While planktonic larval duration has not been measured in O. victoriae, the Antarctic planktotroph echinoderms Sterechinus neumayeri and Odontaster validus have larval durations of around 115 days and 165 days, respectively (Pearse and Bosch 1986; Bosch et al. 1987). Thus, the duration of O. victoriae ophioplutei development is likely within the same range. The AC has surface speeds ranging from 0.25-0.4 m s -1 (Klinck and Nowland 2001). Flow of that magnitude, coupled with a development time potentialy greater than 100 days, would alow ample time for larvae to disperse from the Antarctic Peninsula to the subantarctic islands. Again, larval behavior may explain the apparent low levels of genetic connectivity if ophioplutei regularly migrate to deeper depths where current speeds decline to only a few centimeters per second near the botom (Klinck and Nowland 2001). 102 4.5.4 Clade 1 relationships: Deception Island Individuals from St. 64 in the Bransfield Strait were collected from Deception Island, an active volcano with a sunken, water-filed caldera inhabited by marine organisms. Thre large eruption events occurred from 1967-1970 (Lovel and Trego 2003), resulting in a mas mortality event thought to have largely eradicated the benthic flora and fauna inhabiting the drowned caldera (Galardo and Castilo 1968, 1970). In a recent survey of community structure at Deception Island, O. victoriae was determined to be the dominant epibenthic organism (Galardo and Castilo 1968, 1970; Arnaud et al. 1998; Lovel and Trego 2003) and the only ophiuroid species occurring in this highly disturbed locality (Manj?n-Cabeza and Ramos 2003). The Deception Island population showed significant genetic diferentiation to al other populations except for the most geographic proximate population, St. 51, separated by only 57 km. Due to the recent volcanic disturbance, individuals inhabiting Deception Island might have recently imigrated from other existing O. victoriae populations, despite limited water exchange betwen Deception Island and the rest of Bransfield Strait (Lovel and Trego 2003). Along with this, a recent expansion is suggested for this population, as evident by significantly negative values of Fu?s F S . Negative values for Fu?s F S can be explained by purifying selection, population expansion imediately following a bottleneck, or by a low level of migration from a geneticaly distinct population(s) (Skibinski 2000). Given the recent geologic history of Deception Island, combined with genetic homogeneity with St. 51, the O. victoriae population at Deception Island is likely experiencing spatial expansion following re-colonization by closely-related individuals from St. 51 in Bransfield Strait. 103 In summary, Ophionotus victoriae is characterized by previously unrecognized levels of genetic diversity and divergence, and is not geneticaly homogeneous throughout the sampled range. It should be emphasized that this study sampled only a portion of the supposedly circumpolar distribution of this species, yet revealed levels of geographic subdivision that appear to be atypical among Antarctic benthic marine invertebrates. Thus, additional sampling around the Antarctic continent would surely reveal even greater genetic diversity and likely cryptic speciation. 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Mol Ecol 18:965?984 116 Zane L, Ostelari L, Macatrozzo L, Bargeloni L, Bataglia B, Patarnelo T (1998) Molecular evidence for genetic subdivision of Antarctic kril (Euphausia superba Dana) populations. Proc R Soc Lond B 265:2387?2391 Zhou M, Niler P, Hu J-H (2002) Surface currents in the Bransfield and Gerlache Straits, Antarctica. Dep-Sea Res Part I 49:267?280 117 Table 1 Population summary statistics for Ophionotus victoriae (derived from the 16S+COI concatenated dataset): N refers to number of individuals, H is the number of haplotypes, ? refers to nucleotide diversity, and h is haplotype diversity. Tajima?s D and Fu?s F S refer to results of neutrality tests. Station numbers correspond with Figure 1. Region Station Lat/Long Depth N H ? ? SD h ? SD Tajima?s D Fu?s F S Northern Peninsula 17 ? South Shetland Is. S 62?19', W 61?45' 34 m 8 5 0.048 ? 0.023 0.86 ? 0.1 ? 1.421 ? 0.807 21 ? Bransfield Strait S 63?09', 57?07' 192 m 5 4 0.019 ? 0.008 0.83 ? 0.2 ? 0.780 0.134 40a ? Eagle Is. S 63?40', W 57?20' 35 m 5 5 0.04 ? 0.009 1.0 ? 0.13 0.461 ? 1.481 40b ? Eagle Is. S 63?40', 57?20' 35 m 6 2 0.003 ? 0.002 0.3 ? 0.2 ? 0.93 ? 0.03 51 ? Bransfield Strait S 63?23', W 60?03' 27 m 15 11 0.040 ? 0.005 0.95 ? 0.04 ? 0.36 ? 4.086 * 64 ? Deception Is. S 62?57', 60?39' 161 m 10 7 0.026 ? 0.006 0.91 ? 0.08 ? 0.97 ? 2.291 * D5 ? Elephant Is. S 61?12', W 54?4' 239 m 6 6 0.078 ? 0.02 1.0 ? 0.10 ? 0.63 ? 1.258 D6 ? South Shetland Is. S 62?17', 58?27' 192 m 10 10 0.041 ? 0.005 1.0 ? 0.05 ? 0.861 ? 6.56 ** Southern Peninsula 33 ? Adelaide Is. S 67?4', W 69?17' 12 m 11 7 0.037 ? 0.007 0.87 ? 0.09 0.13 ? 1.01 47 ? Adelaide Is. S 67?40', 68?15' 170 m 10 6 0.062 ? 0.020 0.89 ? 0.08 0.141 1.039 58 ? Peterman Is. S 65?1', W 64?15' 285 m 10 5 0.016 ? 0.003 0.76 ? 0.13 ? 0.29 ? 1.021 D8 ? near Anvers Is. S 64?53', 62?54' 187 m 5 3 0.065 ? 0.031 0.80 ? 0.16 ? 0.840 2.942 Subantarctic Islands 51 ? South Sandwich Is. S 58?29', W 26?12' 270 m 9 8 0.036 ? 0.004 0.97 ? 0.06 ? 0.249 ? 3.517 * 57 ? South Sandwich Is. S 57?03', 26?45' 130 m 6 5 0.036 ? 0.007 0.93 ? 0.12 0.49 ? 0.839 76 ? Bouvet Is. S 54?38', W 03?18' 648 m 8 5 0.020 ? 0.007 0.79 ? 0.15 ? 1.045 ? 1.037 81 ? Bouvet Is. S 54?29', 03?18' 169 m 10 5 0.027 ? 0.003 0.84 ? 0.08 0.710 0.235 Al Clade 1a 103 49 0.047 ? 0.002 0.97 ? 0.01 ? 1.094 ? 39.158 ** Clade 1b 24 8 0.015 ? 0.002 0.74 ? 0.06 ? 0.826 ? 2.582 Clade 1 Total 127 57 0.076 ? 0.004 0.97 ? 0.01 ? 0.458 ? 31.73 ** Clade 2 Total 7 3 0.049 ? 0.030 0.67 ? 0.16 0.59 0.589 *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 118 Table 2 Pairwise ? ST values betwen Ophionotus victoriae Antarctic and subantarctic stations. Station numbers correspond with Figure 1. Northern Peninsula Southern Peninsula Subantarctic Islands 17 21 40a 40b 51 64 D5 D6 33 47 58 D8 51 57 76 81 Northern Peninsula 17 ? 21 0.67 ** ? 40a 0.63 * ? 0.10 ? 40b 0.68 *** 0.70 * 0.6 * ? 51 0.68 *** 0.02 ? 0.0 0.72 *** ? 64 0.73 *** 0.29 * 0.2 * 0.75 *** 0.06 ? D5 0.48 * ? 0.05 ? 0.10 0.59 ** 0.0 0.12 * ? D6 0.67 *** ? 0.05 ? 0.05 0.70 *** 0.0 0.19 ** ? 0.04 ? Southern Peninsula 33 0.70 *** 0.17 0.07 0.73 *** 0.09 0.34 ** 0.01 0.04 ? 47 0.50 ** 0.06 0.01 0.62 *** 0.09 * 0.1 * ? 0.04 0.10 * 0.24 ** ? 58 0.06 0.8 ** 0.84 *** 0.80 *** 0.81 *** 0.86 *** 0.70 *** 0.81 *** 0.83 *** 0.69 *** ? D8 ? 0.12 0.59 * 0.54 * 0.64 ** 0.64 *** 0.69 *** 0.37 * 0.61 *** 0.65 *** 0.42 ** 0.06 ? Subantarctic Islands 51 0.67 *** 0.17 * 0.08 0.70 *** 0.27 *** 0.40 *** 0.18 ** 0.27 *** 0.37 *** 0.24 *** 0.82 *** 0.61 *** ? 57 0.6 ** 0.20 0.1 0.68 ** 0.28 *** 0.41 *** 0.17 ** 0.28 *** 0.41 *** 0.21 * 0.84 *** 0.60 ** ? 0.10 ? 76 0.73 *** 0.59 ** 0.51 ** 0.76 *** 0.53 *** 0.65 *** 0.42 *** 0.54 *** 0.60 *** 0.45 *** 0.87 *** 0.69 ** 0.43 *** 0.47 ** ? 81 0.73 *** 0.48 ** 0.4 *** 0.75 *** 0.47 *** 0.60 *** 0.39 *** 0.47 *** 0.54 *** 0.43 *** 0.86 *** 0.68 *** 0.42 *** 0.4 *** 0.13 ? *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 119 Table 3 Hierarchical analysis of molecular variance (AMOVA) for Antarctic and subantarctic populations of Ophionotus victoriae. Source of variation % variation ? statistic P value Among regions 8.21 ? CT =0.08 0.10 Among stations 47.24 ? SC =0.51 * within a region Within stations 4.54 ? ST =0.5 * *P < 0.001 120 Figure 1 Map showing collection localities for Ophionotus victoriae from the Antarctic Peninsula and subantarctic South Sandwich Islands and Bouvet Island (inset). 121 Figure 2 16S + COI parsimony networks of 134 O. victoriae individuals collected throughout the Antarctic Peninsula and subantarctic islands. Haplotype circles are coded by geographic region and sized acording to relative abundance. Mising (unsampled) haplotypes are denoted by smal black circles. 122 Figure 3 Posterior probability distributions for the number of migrants per generation (M) betwen certain O. victoriae stations/regions. 123 Figure 4 Map showing the distribution of O. victoriae clades throughout the Antarctic Peninsula. Given that al subantarctic populations belonged to Clade 1a, these geographic localities were not included. 124 CHAPTER 5: Geographical subdivision and demographic history in two Antarctic ophiuroids: the role of Pleistocene glacial cycles and contemporary oceanography 5.1 ABSTRACT Two common ophiuroid species were collected from a portion of their range in order to ases their population connectivity and genetic diversity. Ophiurolepis gelida was sampled from the Antarctic Peninsula, Ross Sea, Weddel Sea and several subantarctic islands, and O. brevirima was sampled from the northern Antarctic Peninsula (Bransfield Strait) and Weddel Sea. Both species are thought to produce lecithotrophic larvae and therefore have potential for larval dispersal. Mitochondrial (16S rDNA) data were analyzed using coalescent and frequency-based methods. These data showed evidence of significant population genetic structure in both species betwen major geographic regions. Within regions however, population connectivity was evident, and parsimony network analyses and neutrality tests indicated a recent population expansion in Bransfield Strait for both O. gelida and O. brevirima. These results suggest that populations within distinct geographic regions have a unique genetic signature, highlighting the potential for cryptic speciation and underestimation of genetic diversity and divergence in Antarctic benthic invertebrates. 125 5.2 INTRODUCTION Connectivity and distributional paterns of benthic organisms inhabiting the Antarctic continental shelf are influenced by contemporaneous factors including oceanography and life history, as wel as historical forces such as palaeoclimatology (e.g., Pleistocene glacial cycles). For example, the Antarctic Circumpolar Current (AC), a fast-flowing easterly curent, is thought to play an important role in dispersing marine organisms around Antarctica (Fel 1962; Del 1972; Arntz et al. 1994; Thornhil et al. 2008; Waters 2008), while the Antarctic Polar Front (APF), a frontal region asociated with the AC, is considered to prevent exchange betwen Antarctic and temperate populations (Patarnelo et al. 1996; Shaw et al. 2004; Clarke et al. 2005; Hunter and Halanych 2008; Thornhil et al. 2008). Coastal currents (e.g., Eastwind Drift) and large oceanic gyres (e.g., Weddel and Ross Sea gyres) in the Southern Ocean also act to structure populations (Patarnelo et al. 1996; Bargeloni et al. 2000; Linse et al. 2007). The Southern Hemisphere has experienced four major episodes of ice sheet expansion during the past 1.2 my (Eliot 1985), with the most recent glacial maximum occurring roughly 17 kya to 21 kya (Gersonde et al. 2005). During glacial periods, grounded ice sheets covered the majority of the Antarctic continental shelf (Anderson et al. 2002; Huybrechts 2002; Hodgson et al. 2003), and any open shelf regions were frequently disturbed by iceberg scour (Beaman and Haris 2003). Expanding ice sheets displaced or eradicated continental shelf fauna, forcing species onto the continental slope and/or deep sea, or into isolated shelf refugia (Thatje et al. 2005). During interglacial periods, benthic organisms with dispersal ability could recolonize continental shelf habitat as it opened up around Antarctica (Poulin et al. 2002; Thatje et al. 2005). The 126 extent to which glacial/interglacial cycles impacted the genetic signature of Antarctic benthos is not fully understood, but increased genetic diversity and radiations in several taxonomic groups have been atributed to Pleistocene climatic proceses (Eastman and Clarke 1998; Held 2000; Alcock 2005; Raupach et al. 2007; Wilson et al. 2007; Wilson et al. 2009). Life history traits such as presence/absence of a dispersive larval stage interact with physical forces in Antarctica to determine population connectivity. Historicaly, cold-water invertebrates were considered to be almost exclusively brooders (Thomson 1878, 1885; Murray 1885; Thorson 1936), however it is currently recognized that many Antarctic taxa produce pelagic larvae (Pearse et al. 1991; Clarke 1992; Pearse 1994). For example, over 70% of Antarctic echinoderms have a planktonic larval stage (Pearse 1994). Additionaly, Antarctic organisms have delayed development in the cold Southern Ocean, which increases persistence times of their larvae in the water column, presumably enhancing dispersal ability (Bosch et al. 1987; Stanwel-Smith and Peck 1998). Many taxonomic groups in Antarctica remain relatively unstudied in terms of their biogeography, levels of population connectivity and genetic diversity. Ophiuroids, commonly known as britle stars, the most diverse group of echinoderms with over 90 species recognized in Antarctic/subantarctic waters (Smirnov 1994), are one conspicuous example. Ophiurolepis gelida and Ophiurolepis brevirima are closely-related ophiuroids that are abundant and dominant throughout the Antarctic benthos (Dahm 1996; Piepenburg et al. 1997; Manj?n-Cabeza and Ramos 2003). Ophiurolepis gelida occurs throughout the Antarctic/subantarctic (Mortensen 1936; Fel 1961; Madsen 1967), whereas O. brevirima is restricted to Antarctica. Both O. gelida and O. brevirima are 127 reported to have circumpolar distributions, a common hypothesized distributional patern among Antarctic benthic organisms (Fel et al. 1969; Dayton et al. 1990; Del 1990). The reproductive biology of these two species is not definitively known, but Mortensen (1936) postulated that neither species were brooders, based on an absence of brooded embryos and the presence of numerous eggs of moderate size (~0.3 m) (RL Hunter, pers. obs.). He suggested that they were lecithotrophic, producing a non-feding planktonic larval form that could persist in the plankton for up to 2-3 months (Bosch and Pearse 1990). Ophiurolepis gelida and O. brevirima occur sympatricaly in some regions of Antarctica, including the Antarctic Peninsula (Mortensen 1936). Given the similar life history of these species, we compared sympatric populations to evaluate the efect of Pleistocene glacial cycles on population structure and genetic diversity within each species, and to ases the role of oceanography in structuring present-day populations. Additionaly, O. gelida was collected from a wide range around Antarctica to determine if O. gelida larvae are realizing their dispersal potential, or whether ongoing and/or historical factors have limited circumpolar gene flow in this widespread species. For O. brevirima, in addition to evaluating connectivity within the Antarctic Peninsula, a population from the Weddel Sea was sampled to ases whether the Weddel Sea gyre is restricting gene flow betwen Antarctic Peninsula and Weddel Sea populations. 128 5.3 MATERIALS AND METHODS 5.3.1 Data collection Ophiurolepis gelida and O. brevirima were collected during cruises to the Antarctic Peninsula in 2004 and 2006 aboard the R/V Laurence M. Gould. Benthic samples were collected with an epibenthic sled, Blake trawl, or rock dredge. O. gelida was collected from the subantarctic South Sandwich and Bouvet Islands during the 2004 ICEFISH expedition, and from the Ross Sea by SCUBA. Both species were collected from the Weddel Sea during the 2005 ANDEP-3 cruise. Samples for DNA analysis were either frozen upon collection at -80?C or preserved in ~85% ethanol. Sampling information is provided in Table 1 and Figure 1. DNA was extracted using the DNeasy ? Tisue Kit (QIAGEN) following manufacturer?s protocol. The mitochondrial gene fragment 16S rDNA (16S) was amplified using standard PCR protocols. Primers 16SarL (5?- CGCTGTTATCAAACAT-3?) and 16SbrH (5?- CGTCTGACTCAGATCACGT-3?) (Palumbi et al. 1991) amplify a ~500 bp fragment from the middle of 16S. Double-stranded PCR products were purified using the QIAquick ? Gel Extraction Kit (QIAGEN) following manufacturer?s protocol. Purified PCR products were bi-directionaly sequenced using a CEQ8000 Genetic Analysis System (Beckman Coulter). All O. gelida and O. brevirima haplotypes wil be deposited in GenBank. Sequences were edited in SeqMan (DNA* Lasergene) and aligned with Clustal W (Thompson et al. 1994) in MegAlign (DNA* Lasergene). For O. gelida, a smal number of gaps were required for alignment, and no gaps were necesary to align O. brevirima 129 sequences. Alignments were examined visualy in MacClade v4.0 (Maddison and Maddison 2000) and wil be available in TreBASE (ww.trebase.org). 5.3.2 Population structure analyses Parsimony networks were constructed using mtDNA haplotypes in TCS v1.18 (Clement et al. 2000) with a 95% connection limit betwen haplotypes. Gaps were treated as mising data. To ases levels of genetic diferentiation betwen sampling stations (Table 1), pairwise ? ST ?s were computed (where N ? 4) in Arlequin v3.1 (Excoffier et al. 2005). Given the close proximity of the two South Sandwich Islands stations, and the thre Bouvet Island stations, sites within these regions were pooled for pairwise analysis. Arlequin was used to perform an analysis of molecular variance (AMOVA) to evaluate the relationship betwen haplotype variation and geography in O. gelida. An AMOVA was not caried out for O. brevirima due to sampling limitations (only a single population was sampled from the Weddel Sea region). For the AMOVA, variance was partitioned into thre hierarchical components: within sampling stations (? ST ), among sampling stations within a geographic region (? SC ), and among geographic regions (? CT ). For O. gelida, geographic regions were Bransfield Strait, the Southern Peninsula (stations south of Low Is.) and Subantarctic Islands, and were determined a priori based on oceanographic discontinuities in the Antarctic Peninsula (Zhou et al. 2002) and due to the large distance (>1800 km) separating the subantarctic islands from the Antarctic Peninsula. For both pairwise ? ST ?s and the AMOVA, the Tamura-Nei model (Tamura and Nei 1993) was selected in Arlequin as it most closely approximated the model of sequence evolution chosen by Modeltest v3.7 (TrN + I for O. gelida; TIM for O. 130 brevirima) (Posada and Crandal 1998), and 10,000 permutations were caried out to ases significance. For subsequent analyses, geneticaly homogenous populations (determined by non-significant pairwise ? ST ?s) within a geographic region were pooled. With the exception of the haplotype network analysis, analyses were done only using collection stations or pooled regions where four or more individuals were sampled. 5.3.3 Historical demography and divergence dating In order to quantify genetic diversity within O. gelida and O. brevirima, nucleotide (?) and haplotype (h) diversities were calculated in DnaSP v4.1 (Rozas et al. 2003). Tajima?s D (Tajima 1989) test statistic was calculated in DnaSP to evaluate the asumption of selective neutrality of 16S sequences as wel as population equilibrium. Fu?s F S (Fu 1997) neutrality statistic, shown to be particularly sensitive to population demographic expansion as indicated by large, negative F S values (Fu 1997), was computed in DnaSP and significance asesed by 10,000 data permutations. Mismatch distributions for each species were examined in Arlequin, and 10,000 coalescent simulations, based on parameters estimated from a sudden demographic expansion, were done to ases significance of test statistics. The mismatch distribution compares the observed versus expected distribution of pairwise nucleotide diferences and was used to determine if population expansion had occurred in the history of O. gelida or O. brevirima. Divergence dates were computed in BEAST v1.4.8 (Drummond and Rambaut 2007), which uses a coalescent-based MCMC approach to estimate time to the most recent common ancestor (tMRCA). BEAST was used to calculate coalescense times 131 within lineages and to date spliting events betwen lineages recovered from statistical parsimony. In BEAST, the HKY + I model of sequence evolution was used and a strict clock was employed with runs conducted under two diferent starting conditions. In the first set of runs, the mean mutation rate per site per year was set to 2.5 ? 10 ?9 , and in the second set of runs, 1.5 ? 10 ?8 was used. These values span the typical range of average pairwise sequence divergences reported for the 16S gene for invertebrates (0.5-3.0%/my; Held 2001; Govindarajan et al. 2005; Johnson 2005). Fiften milion MCMC generations were run to ensure efective sampling sizes exceded 200 (Drumond et al. 2007). MCMC chains were sampled every 500 iterations and 10% were discarded as burn-in. Dating times and confidence intervals were filtered using Tracer v1.4 (Rambaut and Drummond 2007). 5.4 RESULTS DNA sequences from a portion (503 bp) of the mitochondrial 16S rDNA gene were obtained from 118 Ophiurolepis gelida individuals and 87 O. brevirima individuals. Within both species, the majority of 16S haplotypes were distributed across multiple sampling localities. In O. gelida, 118 individuals yielded 29 unique haplotypes, and in O. brevirima, 87 individuals yielded 17 haplotypes, one of which was found at every collection site except the Weddel Sea. 5.4.1 Population structure Statistical parsimony analysis of O. gelida sequences indicated at least four geographic ?subclades? are present within this species, corresponding to Bransfield Strait 132 (plus Southern Peninsula St. 29), Southern Peninsula, Subantarctic Islands and Ross Sea/Weddel Sea (Fig. 2). O. gelida and O. brevirima were comparable in the portion of their haplotype networks representing individuals collected from the Bransfield Strait, the geographic region where their distributions overlapped. In this region, both species were characterized by a numericaly dominant, geographicaly widespread 16S haplotype, with a few additional haplotypes difering by only one or two substitutions. Also in both species, individuals collected from St. 29 in the Southern Peninsula, situated imediately southwest of the west entrance to Bransfield Strait, clustered with Bransfield Strait individuals, as did O. brevirima St. 72 individuals (in close proximity to St. 29). None of the five O. brevirima individuals from the eastern Weddel Sea shared haplotypes with Antarctic Peninsula individuals, nor was the single O. gelida individual from the Weddel Sea identical to any other O. gelida individuals. For O. gelida, genetic subdivision was recovered betwen al major geographic regions where sample sizes were sufficient to conduct analyses (Table 2a). ? ST values betwen populations in Bransfield Strait (including St. 29), the Southern Peninsula, and subantarctic islands were large (? 0.54) and significant in al cases. Conversely, within geographic regions, ? ST values were typicaly low and non-significant. For O. brevirima, populations within and imediately southwest of Bransfield Strait were geneticaly homogenous (Table 2b), with one exception (St. 51). Genetic diferentiation betwen the Antarctic Peninsula and Weddel Sea was supported by large, significant ? ST values (? 0.72) in al pairwise comparisons, owing to a lack of shared mtDNA haplotypes. AMOVA based on a priori geographic regions resulted in similar levels of genetic variation among geographic regions (41%, P < 0.05) and among stations within a 133 geographic region (34%, P < 0.0001) in O. gelida. Les genetic variation (25%, P < 0.0001) was atributed to within collection stations. Because pairwise ? ST values were low and non-significant betwen Bransfield Strait populations and one Southern Peninsula population, an AMOVA was performed that included St. 29 in the Bransfield Strait region. Under these conditions, the majority of genetic variation existed among geographic regions (79%, P < 0.01), whereas almost no variation was present among stations within a geographic region (0.1%, P > 0.05). The remainder of the variation was at the within collection station level (20%, P < 0.0001). 5.4.2 Historical demography and divergence Nucleotide and haplotype diversity estimates were low (? ? 0.0011, h ? 0.45; Table 3a) for most geographic regions for both species, with the exception of O. gelida in the Southern Peninsula (? = 0.0076, h = 0.76), which was moderately diverse. Both Tajima?s D and Fu?s F S were significantly negative for the Bransfield Strait region in both species, as wel as for the subantarctic island region for O. gelida. Significant negative values for neutrality statistics can be explained by purifying selection, a selective swep, or by a population expansion imediately following a bottleneck (Skibinski 2000). The distribution of pairwise diferences betwen haplotypes within a geographic region closely modeled the expected distribution under a sudden demographic expansion in al cases except O. gelida in the Southern Peninsula (Fig. 3). The sum of square deviations (SSD) betwen the observed and expected distribution of a sudden demographic expansion, and raggednes statistic (r) (Harpending 1994) for the mismatch distribution were not statisticaly significant (Table 3b), indicating that the sudden 134 expansion model could not be rejected for either species in any geographic region (Harpending 1994; Schneider and Excoffier 1999). Estimation of divergence dates for O. gelida and O. brevirima lineages resulted in means with large confidence intervals (Table 4), possibly due to using a single locus with too few informative sites. For O. gelida, the Bransfield Strait-Southern Peninsula split was dated to the early Pliocene, prior to the onset of Pliocene-Pleistocene glacial cycles, using the slower mutation rate (0.5%/MY), while the faster mutation rate (3%/MY) recovered a much ealier split during the Miocene. The Bransfield Strait-Subantarctic separation was estimated to have occurred during the late-Pliocene to mid-Pleistocene. Estimation of coalescence times within lineages suggested that O. gelida lineages were typicaly of Pleistocene origin, or originated just prior to the Pleistocene. Similar date estimations were recovered for O. brevirima. The split betwen Bransfield Strait and Weddel Sea lineages was dated as late-Pliocene to mid-Pleistocene, and the age of the Bransfield Strait clade fel within the same range. 5.5 DISCUSION Phylogeographic analyses of mtDNA data from Ophiurolepis gelida and O. brevirima resulted in a clear patern of genetic heterogeneity betwen geographic regions in contrast to genetic homogeneity within geographic regions. In the case of O. gelida, genetic diferentiation was found betwen populations in Bransfield Strait (+ St. 29), the Southern Peninsula and the subantarctic South Sandwich and Bouvet Islands. Parsimony analysis suggested a distinct Ross Sea/Weddel Sea population as wel, although limited sampling in these regions prevents robust conclusions. For O. brevirima, populations 135 from the Antarctic Peninsula and eastern Weddel Sea appear to be limited in their connectivity. Overal, these results cal into question the previously reported circumpolarity of these species. 5.5.1 O. gelida A north-south phylogeographic break exists within O. gelida along the Antarctic Peninsula, separating Bransfield Strait individuals from those southwest of Brabant Island (se Fig. 1). There were only four cases (3.4% of individuals) where ?Bransfield Strait? haplotypes were collected south of Brabant Island. This north-south break could be atributed to the mostly shalow bottom topography of the entrance to Bransfield Strait, which restricts intermediate and deep water exchange betwen Bransfield Strait and the Southern Peninsula (Zhou et al. 2002). Limited water flow betwen these regions would reduce or efectively eliminate larval transport as a mechanism for maintaining connectivity. Diferentiation betwen Bransfield Strait populations and at least some Southern Peninsula populations has been found in the ophiuroid Ophionotus victoriae (Hunter and Halanych submited) and crinoid Promachocrinus kerguelensis (Wilson et al. 2007). However, this patern contrasts with results from other phylogeographic studies in which populations from Bransfield Strait and the Southern Peninsula were geneticaly homogeneous (Hunter and Halanych 2008; Mahon et al. 2008; Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.). Divergence estimation suggested that the Bransfield Strait and Southern Peninsula lineages may have split prior to the onset of Pleistocene glacial cycles, which began around 1.2 mya in the Southern Hemisphere (Eliot 1985). However, 95% confidence 136 intervals from both slow and fast mutation rate estimates include the early-mid Pleistocene. Therefore, Pleistocene glacial cycles cannot be excluded as a potential mechanism driving the divergence of these lineages. Populations in the northern and southern peninsula may have been isolated in separate refugia during early glacial periods, and subsequently acumulated sufficient genetic diferences to prevent mixing during interglacial periods. Fragmentation into separate glacial refugia has been invoked to explain present-day population structure in a notothenioid fish (Janko et al. 2007) and Antarctic sea slug (Wilson et al. 2009), as wel as in a number of Northern Hemisphere taxa (Hewit 1996). Given limited water exchange throughout the Antarctic Peninsula, genetic structure of O. gelida in this region may be the result of historical isolation combined with low potential for contemporary dispersal. However, there was some evidence of ongoing or historical gene flow over large distances throughout the Antarctic Peninsula. The fact that thre ?Bransfield Strait? haplotypes were collected from one of the southernmost sampled populations (Sts. 33/45) suggests low levels of ongoing or historical transport from Bransfield southwest along the peninsula. Weddel and Ross Sea mtDNA haplotypes were les divergent from Southern Peninsula haplotypes than from those collected from Bransfield Strait. However, there were no shared haplotypes betwen Weddel Sea, Ross Sea or Southern Peninsula individuals. The single Weddel Sea individual was separated by only a single mutation from a Ross Sea individual, and other studies have similarly uncovered a closer genetic relationship betwen Ross Sea and Weddel Sea samples compared to Antarctic Peninsula samples (Held and W?gele 2005; Linse et al. 2007). A hypothesized historical seaway linking the Ross and Weddel Seas (Lawver and Gahagan 2003) is one 137 explanation for this phenomenon (Linse et al. 2007). Additional sampling from the Ross and Weddel Seas wil aid in determining levels of connectivity betwen these geographic regions, and with the Antarctic Peninsula. Genetic discontinuity was also recovered betwen Antarctic Peninsula and subantarctic island populations of O. gelida. Even though the Antarctic Peninsula and subantarctic islands are linked by the AC, approximately 1800 km separates the South Sandwich Islands from the Antarctic Peninsula, while a 3300 km distance separates the peninsula from Bouvet Island. Despite O. gelida?s dispersal potential, long-distance connectivity may be limited by a combination of high larval mortality and low probability of larvae reaching suitable shelf habitat on the subantarctic islands. Nonetheles, other studies have recovered multiple shared mtDNA haplotypes betwen the subantarctic islands and Antarctic Peninsula (Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.). The South Sandwich and Bouvet Island populations of O. gelida may be the result of a single colonization event from the Antarctic continental shelf during the Pleistocene. Some Antarctic invertebrates with pelagic larvae may have found refuge on the subantarctic islands during glacial periods (Thatje et al. 2005). O. gelida could have dispersed to one or more of these islands as glacial expansion impacted the Antarctic continental shelf, and following colonization, underwent a range expansion. The presumed ancestral haplotype of the subantarctic island subclade was found equaly distributed among both island localities, therefore it was not possible to determine the direction in which colonization occurred. However, given that the AC flows from the 138 South Sandwich Islands to Bouvet Island, dispersal likely occurred in an eastward direction. 5.5.2 O. brevirima For O. brevirima, 16S data reveal that litle to no connectivity has occurred betwen Antarctic Peninsula populations and the eastern Weddel Sea population most likely since the Pleistocene. Antarctic Peninsula and Weddel Sea populations may have been isolated in separate glacial refugia in their respective geographic regions. Additionaly, oceanographic features such as the Weddel Gyre, separating the Weddel Sea from surrounding waters, and/or the Weddel-Scotia Confluence, where the AC and Weddel Gyre interact, may have played a role in historical and/or contemporary isolation of Weddel Sea populations. Temperature and salinity diferences betwen Weddel Sea and Antarctic Peninsula water mases (Stein and Heywood 1994) may have further reduced mixing betwen these regions. Genetic divergence betwen western Antarctic Peninsula and Weddel Sea populations has been found in several other Antarctic taxa (Zane et al. 1998; Bargeloni et al. 2000; Patarnelo et al. 2003; Held and W?gele 2005; Hunter and Halanych submited). Another potential isolating factor is the depth from which Weddel Sea individuals were sampled. O. brevirima was collected from ? 500 m depths from the Antarctic Peninsula, while the depth of the single Weddel Sea station was 1017 m. Eurybathy is a common phenomenon among Antarctic marine invertebrates (Hempel 1985), but the degre to which populations are homogeneous over large depth ranges has not been wel studied. There are, however, preliminary indications that depth is not 139 typicaly a limiting factor for dispersal in the Antarctic, and species distributed over moderately wide depth ranges (e.g., 100-1200 m) have been shown to be geneticaly homogenous (Wilson et al. 2007; Hunter and Halanych 2008). Given that we have only characterized a single Weddel Sea population with few individuals, additional sampling is necesary to fully document the extent of genetic divergence, and to determine if depth plays any role in structuring O. brevirima populations in the Weddel Sea and throughout the Antarctic. Additionaly, although O. brevirima has been recorded as being circumpolar (Madsen 1967), we did not find this species south of Brabrant Island. 5.5.3 Phylogeography within Bransfield Strait Where sampling of O. gelida and O. brevirima overlapped, similar phylogeographic paterns were recovered. Both species were geneticaly homogenous throughout Bransfield Strait, with the exception of one O. brevirima population. Furthermore, stations situated just outside the western margin of Bransfield Strait (St. 29 for O. gelida; Sts. 29 and 72 for O. brevirima) were geneticaly homogenous with Bransfield Strait populations. Even though Bransfield Strait is semi-enclosed, some water does enter through a deep gap betwen Brabant and Smith Islands (Zhou et al. 2002), right in the vicinity of stations 29 and 72. Clearly, these populations are ?Bransfield Strait? in composition, and not alied with other Southern Peninsula populations. The low levels of genetic variation and significantly negative neutrality statistics recovered for O. gelida and O. brevirima in Bransfield Strait can be explained by either a recent range expansion or selection acting on the mitochondrial genome (Skibinski 2000). Selection, while not ruled out, sems les likely given that it would have had to similarly 140 afect both species. A more likely explanation for the correspondence in geography and genetics betwen these species is that both species have recently experienced range expansions throughout Bransfield Strait, as suggested by mismatch distributions. The Antarctic continental shelf and slope are considered to have been the most heavily impacted environments on earth during Pleistocene glacial cycles, and were largely uninhabitable during glacial periods (Thatje et al. 2005). Presently, Antarctic benthic habitats recently disturbed by iceberg scour are recolonized first by benthic invertebrates possesing a pelagic larval stage (Thatje 2005; Thatje et al. 2005). Acordingly, Antarctic invertebrates with planktonic larvae may have been among the few to survive on the continental shelf during glacial periods, persisting by migrating from refugia to refugia. Antarctic species with such dispersal potential would have been the first to re-colonize from deep sea/slope habitats during interglacial periods as wel (Thatje 2005; Thatje et al. 2005). Given that O. gelida and O. brevirima likely produce a lecithotrophic larvae, both species could have persisted in isolated refugia in the Bransfield Strait, or on the continental slope/deep sea, during a glacial period and subsequently expanded during an interglacial period. The age of these lineages appears to pre-date the last glacial period in Antarctica (70 kya-10 kya; Anderson et al. 2002), suggesting that both species underwent population expansions prior to the last glacial maximum (17 kya to 21 kya; Gersonde et al. 2005). Kril and notothenioid fish have similarly been shown to have undergone population expansions prior to the last glacial maximum (Zane et al. 1998; Zane et al. 2006). While our circumpolar sampling is not complete, especialy for O. brevirima, data from this study suggest that neither O. gelida nor O. brevirima are geneticaly 141 homogeneous circumpolar species. 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Region/Station Lat/Long Depth N H Ophiurolepis gelida Bransfield Strait Low Is. ? St. 13 S 63?25', W 61?51' 132 m 14 3 near Livingston Is. ? St. 14 S 62?56', 61?29' 18 m 3 2 Trinity Peninsula ? St. 21 S 63?09', W 57?07' 192 m 14 2 near D?Urvile Is. ? St. 38 S 62?45', 56?45' 207 m 10 2 mid-Bransfield ? St. 51 S 63?23', W 60?03' 27 m 4 3 mid-Bransfield ? St. D2 S 63?21', 60?0' 246 m 3 2 Elephant Is. ? St. D5 S 61?12', W 54?4' 239 m 3 1 Southern Peninsula Brabant Is. ? St. 29 S 64?08', W 62?46' 156 m 13 5 Adelaide Is. ? St. 31 S 6?37', 68?19' 261 m 3 1 Adelaide Is. ? Sts. 3/45 S 67?4', W 69?17' 12/195 m 15 8 Marguerite Bay ? St. 37 S 68?1', 67?36' 232 m 2 1 Renaud Is. ? St. 53 S 65?56', W 6?54' 183 m 3 3 near Adelaide Is. ? St. 80 S 6?32', 69?59' 50 m 1 1 near Renaud Is. ? St. 82 S 65?40', W 68?02' 278 m 1 1 near Anvers Is. ? St. 85 S 64?41', 65?56' 368 m 6 2 near Anvers Is. ? St. 89 S 64?46', W 62?43' 45 m 1 1 near Anvers Is. ? St. D8 S 64?53', 62?54' 187 m 2 2 Subantarctic Islands South Sandwich Is. ? St. 51 S 58?29', W 26?12' 270 m 4 2 South Sandwich Is. ? St. 52 S 58?56', 26?30' 120 m 4 2 Bouvet Is. ? St. 71 S 54?2', W 03?23' 20 m 3 2 Bouvet Is. ? St. 80 S 54?30', 03?28' 159 m 2 2 Bouvet Is. ? St. 81 S 54?29', W 03?18' 169 m 3 1 Ros Sea 3 3 Wedel Sea ? St. 74_2 S 71?10', W 13?34' 1017 m 1 1 Total 118 29 Ophiurolepis brevirima Bransfield Strait Low Is. ? St. 13 S 63?25', W 61?51' 132 m 17 3 near Livingston Is. ? St. 14 S 62?56', 61?29' 18 m 11 5 Trinity Peninsula ? St. 21 S 63?09', W 57?07' 192 m 16 5 near D?Urvile Is. ? St. 38 S 62?45', 56?45' 207 m 8 1 mid-Bransfield ? St. 49 S 63?14', W 58?45' 87 m 3 2 mid-Bransfield ? St. 51 S 63?23', 60?03' 27 m 7 3 Southern Peninsula Brabant Is. ? St. 29 S 64?08', W 62?46' 156 m 15 3 Brabant Is. ? St. 72 S 63?51', 62?38' 256 m 5 3 Wedel Sea ? St. 74_2 S 71?10', W 13?34' 1017 m 5 2 Total 87 17 N number of individuals, H number of haplotypes 153 Table 2a Pairwise ? ST values betwen O. gelida stations (where N ? 4) from the Antarctic Peninsula and Subantarctic Islands. Station numbers correspond with Figure 1 and Table 1. Bransfield Strait Southern Peninsula Subantarctic Islands Bransfield Strait St. 13 St. 21 St. 38 St. 51 St. 29 Sts. 3/45 St. 85 SSI BI St. 13 ? St. 21 0.0 ? St. 38 ?0.01 0.01 ? St. 51 0.14 0.21 0.14 ? Southern Peninsula St. 29 0.01 0.08* 0.05 0.02 ? Sts. 3/45 0.6** 0.67** 0.63** 0.54* 0.63** ? St. 85 0.96** 0.97** 0.96** 0.92* 0.92** 0.02 ? Subantarctic Islands SSI 0.85** 0.8** 0.86** 0.76* 0.73** 0.65** 0.95** ? BI 0.85** 0.8** 0.86** 0.76* 0.73** 0.64** 0.95** 0.0 ? *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 Significant values (P < 0.05) are in boldface type SI poled South Sandwich Islands population, BI poled Bouvet Island population 154 Table 2b Pairwise ? ST values betwen O. brevirima stations (where N ? 4) from the Antarctic Peninsula and Weddel Sea. Bransfield Strait Southern Peninsula Wedel Sea Bransfield Strait St. 13 St. 14 St. 21 St. 38 St. 51 St. 29 St. 72 WS St. 13 ? St. 14 0.01 ? St. 21 0.02 0.02 ? St. 38 ?0.05 ?0.03 ?0.01 ? St. 51 0.30* 0.14* 0.18* 0.28* ? Southern Peninsula St. 29 0.03 0.03 0.04 ?0.01 0.25* ? St. 72 0.05 ?0.05 0.0 0.10 0.15 0.08 ? Wedel Sea WS 0.8** 0.72** 0.75** 0.93** 0.76* 0.84** 0.7* ? *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 Significant values (P < 0.05) are in boldface type 155 Table 3a Results of diversity estimates and neutrality statistics for Ophiurolepis gelida and O. brevirima. Diversity Estimates Neutrality Tests N H ? ? SD h ? SD Tajima?s D Fu?s F S Ophiurolepis gelida Bransfield Strait (including St. 29) 55 9 0.009 ? 0.002 0.36 ? 0.08 ?2.026* ?8.819** Southern Peninsula (excluding St. 29) 21 8 0.076 ? 0.021 0.76 ? 0.07 ?0.818 ?0.281 Subantarctic Islands 16 5 0.010 ? 0.004 0.45 ? 0.15 ?1.831* ?3.314** Al Individuals 118 24 0.086 ? 0.007 0.75 ? 0.04 ?1.127 ?7.421* Ophiurolepis brevirima Bransfield Strait (excluding St. 51) + Southern Peninsula 72 12 0.010 ? 0.002 0.40 ? 0.07 ?2.230* ?13.06*** St. 51 4 3 0.020 ? 0.007 0.83 ? 0.2 ?0.710 ?0.87 Wedel Sea 5 2 0.008 ? 0.005 0.40 ? 0.24 ?0.817 0.090 Al Individuals 87 17 0.015 ? 0.003 0.51 ? 0.07 ?2.281* ?18.05** *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 Significant values (P < 0.05) are in boldface type N number of individuals, H number of haplotypes, ? nucleotide diversity, h haplotype diversity 156 Table 3b Results of mismatch distribution for Ophiurolepis gelida and O. brevirima. Mismatch Distribution ? 0 (95% CI) ? 1 (95% CI) SSD r Ophiurolepis gelida Bransfield Strait (including St. 29) 0.00 (0.00?0.04) 0.543 (0.00?99999) 0.014 0.251 Southern Peninsula (excluding St. 29) 0.00 (0.00?0.90) 2.72 (1.250?99999) 0.052 0.089 Subantarctic Islands 0.00 (0.00?0.054) 999 (41.89?999) 0.07 0.148 Al Individuals 0.00 (0.00?0.319) 334 (14.38?999) 0.024 0.162 Ophiurolepis brevirima Bransfield Strait (excluding St. 51) + Southern Peninsula 0.00 (0.00?0.03) 999 (15.29?999) 0.00 0.146 St. 51 0.02 (0.00?0.012) 999 (24.08?999) 0.048 0.286 Wedel Sea 0.05 (0.00?0.09) 999 (2.540?999) 0.07 0.20 Al Individuals 0.02 (0.00?0.018) 999 (13.97?999) 0.018 0.21 *0.05 ? P ? 0.01; *0.01 > P ? 0.01; **P < 0.01 ? 0 = 2?N 0 and ? 1 = 2?N 1 where N 0 and N 1 are the population sizes before and after expansion, SSD sum of squared deviations, r ragednes statistic 157 Table 4 Time of the most recent common ancestor for O. gelida and O. brevirima lineages. Top date was obtained using a 3%/MY mutation rate, and the bottom number resulted from a slower mutation rate of 0.5%/Y. 95% CI given in parentheses. O. gelida Bransfield Strait Southern Peninsula Subantarctic Bransfield Strait 0.26 MY (0.06?0.57) 2.93 MY (0.07?7.30) ? Southern Peninsula 2.17 MY (0.35?5.36) 0.48 MY (0.1?1.01) 13.1 MY (0.42?32.4) 5.63 MY (0.14?13.7) ? Subantarctic 0.31 MY (0.07?0.67) 0.09 MY (0.01?0.2) 0.6 MY (0.07?1.74) 1.04 MY (0.02?2.51) O. brevirima Bransfield Strait Bransfield Strait 0.28 MY (0.07?0.59) 2.42 MY (0.08?6.42) Wedel Sea 0.31 MY (0.07?0.64) 2.69 MY (0.09?7.24) 158 Figure 1 Map showing collection localities for Ophiurolepis gelida and O. brevirima from Antarctica and subantarctic South Sandwich Islands and Bouvet Island. 159 Figure 2 16S parsimony networks of 118 O. gelida individuals collected throughout Antarctica and from two subantarctic islands; and 87 O. brevirima individuals collected from the Antarctic Peninsula and Weddel Sea. Haplotype circles are coded by geographic region and sized acording to relative abundance. Mising (unsampled) haplotypes are denoted by smal black circles. 160 Figure 3 Observed and expected mismatch distributions for pooled O. gelida and O. brevirima geographic regions. 161 6.1 CONCLUSIONS The primary goal of phylogeography studies is to understand the relationship betwen genealogy and geography within and among closely related species (Avise 2009). Specificaly, these studies aim to determine the relative roles of contemporary and historical proceses afecting the distribution and genetic composition of populations (Avise 2004). In the marine environment, phylogeography is influenced by oceanography (Wares 2002; Palumbi 2004), organismal biology and ecology (Vermeij 1989; Lindberg 1991; Duffy 1996; Helberg 1996; Wares and Cunningham 2001; Uthicke and Benzie 2003), land bariers (Knowlton 1993), and paleoclimatology (Hewit 2000). Major conclusions of marine phylogeographic studies to date include: 1) many species with high dispersal potential are characterized by substantial levels of population genetic structure (Avise 1994; Palumbi 1997; Benzie 1999a), 2) concordant phylogeographic breaks in multiple taxa correspond to major biogeographic boundaries in the ocean (Reb and Avise 1990; Burton 1998; Benzie 1999a, b; Dawson 2001), and 3) cryptic speciation appears to be common (Knowlton 1993). While comparatively few phylogeography studies have focused on Antarctic/ Southern Ocean marine organisms, recent work has revealed numerous cases of intraspecific geographic subdivision and cryptic speciation (e.g., Held 2003; Held and W?gele 2005; Linse et al. 2007; Wilson et al. 2007; Wilson et al. 2009). However, examples of long-distance connectivity and genetic homogeneity across major 162 biogeographic bariers in the Southern Ocean exist as wel (e.g., Hunter and Halanych 2008; Mahon et al. 2008; Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.). The research presented herein examined phylogeographic paterns in four Antarctic britle star (ophiuroid) species, which span the range of invertebrate developmental modes and have been subject to similar oceanographic and historical climatic conditions, at least where their ranges overlap. Although sampling in the Southern Ocean varied across species, a concordant patern of restricted connectivity betwen major geographic regions emerged. South American and Antarctic populations of the brooding britle star Astrotoma agassizi are geneticaly distinct, and gene flow is not occurring across the Drake Pasage. Levels of mtDNA genetic divergence betwen geographicaly restricted clades were sufficiently high to suggest cryptic speciation in A. agassizi. Despite possesing planktonic larvae, both Ophiurolepis gelida and Ophionotus victoriae showed evidence of limited connectivity betwen Antarctic Peninsula populations and those from the subantarctic South Sandwich and Bouvet Islands. Furthermore, divergent Weddel Sea lineages were recovered in Ophiurolepis brevirima, O. gelida and Ophionotus victoriae. Additionaly, O. gelida individuals in the Ross Sea are geneticaly distinct from Antarctic Peninsula and subantarctic individuals. These results suggest that britle stars in the Southern Ocean often do not disperse in acordance with predictions based on life history, and mtDNA data have provided strong evidence of cryptic divergence and/or speciation for al ophiuroids studied to date. In some instances, britle stars have lower levels of connectivity compared with other Antarctic marine invertebrates with similar life histories (se Thornhil et al. 2008; LN Cox pers. comm.; AM Janosik pers. comm.). 163 Until more is known about the life history, ecology and behavior of these ecologicaly important members of the Antarctic benthos, explanations remain tentative. A more varied picture resulted from analysis of intraspecific mtDNA data within major geographic regions. My data indicated that the brooding A. agassizi was the only ophiuroid examined with geneticaly homogeneous populations throughout the Antarctic Peninsula (over at least 500 km). In contrast, the planktonic developing O. gelida and O. victoriae exhibited genetic subdivision betwen northern and southern Antarctic Peninsula populations, although some O. victoriae northern and southern populations were geneticaly similar. This semingly paradoxical result, where a brooder displays evidence of greater connectivity than species with pelagic larvae, may be explained by the lifetime dispersal potential of these species, not just the capacity for dispersal during development. A. agassizi?s habit of wrapping its curled arms around epifauna subject to being dislodged (Bartsch 1982; Dearborn et al. 1986; Ferari and Dearborn 1989), may confer greater dispersal ability to adult and juvenile A. agassizi individuals compared to O. gelida and O. victoriae. Acordingly, evidence is emerging that suggests the relationship betwen dispersal ability, based on presence/absence of a pelagic larval stage, and genetic structure is complex and not easily predicted (Benzie 1999a, 2000; Helberg et al. 2002). Realized dispersal distances likely depend on multiple interacting biotic factors including spawning characteristics, larval behavior, interaction with conspecifics, and specific habitat requirements (Shulman and Bermingham 1995; Barber et al. 2002; Kirkendale and Meyer 2004; Imron et al. 2007), often unknown parameters for marine organisms living in extreme environments. 164 O. gelida and O. victoriae posses planktonic larvae and theoreticaly should be capable of long-distance dispersal (but se above). Despite their life history, both species showed restricted connectivity betwen northern and southern regions of the Antarctic Peninsula. However, the patern of geographic subdivision was discordant betwen these species, and diferent phylogeographic breaks emerged. O. gelida is characterized by a sharp north-south phylogeographic break, separating Bransfield Strait and southern peninsula populations. In O. victoriae, genetic structure exists betwen some north-south populations, while others are geneticaly homogeneous. The longer-lived planktotrophic larvae of O. victoriae may facilitate connectivity betwen distant northern and southern populations (separated by ~800 km), while local oceanographic conditions may be contributing to the genetic divergence recovered betwen others. For O. gelida, multiple lines of evidence supported divergence betwen northern-southern populations sometime during the early Pleistocene, and populations within both regions showed evidence of fluctuations corresponding to Pleistocene glacial oscilations. While oceanography presumably plays a role in influencing contemporary levels of gene flow in O. gelida, the genetic signature of this species has likely been shaped primarily by recent climate conditions in Antarctica. Overal, this work suggests that life history alone cannot be used to predict connectivity and genetic structure in Antarctic marine invertebrates. Rather, a complex interplay of life history, regional oceanography and recent climate history has likely shaped the genetic composition of most Antarctic invertebrates, and cryptic divergence/speciation is probably commonplace. This research should caution against the use of sweping generalizations when characterizing paterns of connectivity and 165 diversity in the Southern Ocean, as these do not sem to be easily predictable. However, one generalization is likely to hold, future studies in the Antarctic wil continue to reveal biodiversity far exceding previous expectations. 166 6.2 REFERENCES Avise JC (2004) Molecular markers, natural history and evolution, 2 nd edition. Sinaeur Asociates, Sunderland, Masachusets Avise JC (2009) Phylogeography: retrospect and prospect. 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Homalophiura is intermediate to these two groups in having the arm spine character of Ophiurolepis and Theodoria but the low disc character of Ophiuroglypha (Fel 1960). 2. Disc plates: 0 = tumid to varying degres; 1 = iregularly thickened; 2 = flatened The dorsal and ventral disc plates in Ophiuroglypha are evenly swollen to varying degres. Among Ophiurolepis, some species exhibit an uneven thickening of the disc plates while other species have flatened disc plates, as is characteristic of Homalophiura and Theodoria. 3. Disc plate texture: 0 = smooth; 1 = concentric growth lines; 2 = granular Homalophiura, Theodoria and Ophiuroglypha typicaly have smooth disc plating. Species within Ophiurolepis have smooth disc plates or plates with conspicuous circular growth lines, with the exception of O. granulifera which has fine granules covering the disc (Bernasconi & D?Agostino 1973). 4. Aboral disc patern: 0 = six primary plates conspicuous but smal and widely separated by even smaler plates; 1 = six primary plates conspicuous and separated by smaler plates usualy in single or double rows; 2 = rounded plates of varying size separated by a thickened skin; 3 = six primary plates form a compact pentagon in the center of the disc The characteristic disc patern of Ophiurolepis and Theodoria is one primary plate located centraly with five surrounding primary plates in a rosete patern. These six primaries are separated by smaler acesory plates in belts of one or more rows. A similar patern is found in Ophiuroglypha, but the six primaries are much smaler relative to the overal disc size and more widely separated by acesory plates. 5. Radial shield texture: 0 = smooth; 1 = concentric growth lines; 2 = granular Homalophiura, Theodoria and Ophiuroglypha typicaly have a smooth appearance to the radial shields. Within Ophiurolepis, species have either a smooth 171 appearance or conspicuous growth lines, with the exception of O. granulifera which has fine granules covering the radial shields (Bernasconi & D?Agostino 1973). 6. Radial shields: 0 = tumid to varying degres; 1 = flat; 2 = depresed The radials shields in Ophiuroglypha are swollen to varying degres in a similar manner to the other disc plates. In Theodoria and Homalophiura, the radial shields are flatened, while in Ophiurolepis they are either flatened or slightly concave. 7. Radial shield size: 0 = 3x or larger than the primary plates; 1 = same size to 1.5x size of primary plates The radial shields are approximately the same size or slightly larger than the six primary plates in Ophiurolepis, Theodoria and Homalophiura. The radial shields are thre times or greater the size of the primary plates in Ophiuroglypha. 8. Arm comb: 0 = wel-developed; 1 = rudimentary The genera Ophiurolepis, Theodoria and Homalophiura were originaly described as having reduced or rudimentary arm combs. However, some Homalophiura species, such as H. euryplax and H. intorta, have wel-developed arm combs. This character conflict as wel as several others has been used in an atempt to revise the genus Homalophiura (Paterson 1985). 9. Ventral interadius: 0 = occupied by numerous imbricating smal plates; 1 = occupied by the oral shield and several other plates of similar or smaler size; 2 = occupied by large thin scales The ventral interadius is similar among Ophiurolepis, Homalophiura and Theodoria species. It is characterized by a conspicuous oral shield and several other plates of varying size, one which is similar in size to the oral shield. In contrast, in Ophiuroglypha the ventral interadius is characterized by many overlapping scales of similar size and a smal oral shield. 10. Ventral disc plates separated by deep grooves: 0 = absent; 1 = present One of the characters typicaly used to separate Theodoria relegata from the other two Antarctic Theodoria species, T. wallini and T. partita, is the presence of ventral disc plates separated by deep grooves (Fel 1961). Homalophiura intorta also displays this character (Lyman 1878). 11. Length of genital slit: 0 = extending to ambitus; 1 = not longer than basal arm joint The length of the genital slit is a common diagnostic character used to separate several closely related Ophiurolepis species. In the species included in this study, the genital slit extends either to the edge of the disc or just to the end of the first arm joint. 12. Genital slit ornamentation: 0 = papilae along interadial margin; 1 = naked Whether the genital slit extends to the edge of the disc or the first arm joint, its interadial edge is usualy adorned with smal papilae (Matsumoto 1917). These papilae often continue to the aboral surface to form a rudimentary arm comb. 172 13. Number of oral papilae: 0 = 7-10; 1 = 3-6 The number of oral papilae on one side of the jaw ranges from thre to a maximum of six in the ingroup taxa, more typicaly being four or five. In Ophiuroglypha, the oral papilae are more numerous. 14. Shape of oral papilae: 0 = conical within and more blunt towards outside; 1 = block-like The oral papilae are block-like and appear soldered together in Ophiurolepis, Homalophiura and Theodoria, whereas in Ophiuroglypha they are spiniform. 15. Shape of the infradental papila: 0 = spiniform; 1 = diamond-shaped The species used in this study typicaly have one infradental papila at the apex of the jaw. This papila is diamond to triangular-shaped in the ingroup species and spiniform in Ophiuroglypha. 16. Oral shield fragmentation: 0 = absent; 1 = present Fragmentation of the oral shields is present in several species of Theodoria, Ophiurolepis and Homalophiura. Theodoria partita and T. wallini are distinguished in part from T. relegata by the presence of fragmented oral shields (Fel 1961). Homalophiura inornata is also known to have fragmented oral shields (Mortensen 1936). Among Ophiurolepis, O. tuberosa, O. scisa and O. mordax commonly exhibit fragmented oral shields (Koehler 1908, 1922; Mortensen 1936). 17. Jaw excavate on midline: 0 = absent; 1 = present The genus Theodoria, most closely related to Ophiurolepis, was erected to acommodate species that had jaws that were excavate along the midline and that had conspicuous tentacle pores on the thre basal arm joints (Fel 1961). The thre species placed in Theodoria were originaly described as Amphiophiura relegata (Koehler 1922), Ophiurolepis wallini (Mortensen 1925) and Ophioglypha partita (Koehler 1908). There are currently four species in the genus, T. relegata, T. wallini, T. partita and T. madseni. 18. Shape of arm segments in cross section: 0 = sharply triangular; 1 = circular Ophiuroglypha species have a sharp dorsal crest in cross section (Bartsch 1982) whereas the ingroup taxa have circular arm segments in cross section. 19. Size of arm spines: 0 = one-third length of arm joint; 1 = les than one-third length of arm joint In addition to the elevation of the disc, the other morphological character that distinguishes Ophiurolepis and Theodoria from Ophiuroglypha is arm spine size. Ophiurolepis, Homalophiura and Theodoria have minute arm spines while Ophiuroglypha has conspicuous arm spines that are approximately one-third the length of one arm joint (Fel 1960, 1961). 173 20. Number of arm spines: 0 = 3; 1 = 2 The genus Ophiurolepis was originaly described as having two minute, peg-like arm spines and two similar tentacle scales (Matsumoto 1915). The same character state occurs in Homalophiura (Mortensen 1936; Paterson 1985) but difers in Theodoria which has thre arm spines (Fel 1961). 21. Shape of arm spines: 0 = blunt; 1 = conical Ophiurolepis was originaly described as having peg-like arm spines (Matsumoto 1915). The same character state occurs in both Homalophiura (Mortensen 1936; Paterson 1985) and Theodoria (Fel 1961). 22. Arm spine arangement: 0 = contiguous; 1 = widely but evenly spaced; 2 = dorsal- most spine separated from other arm spine and tentacle scales by a gap Most Ophiurolepis species have a dorsal arm spine that is separated from the other arm spine and tentacle scales by a gap. Several Homalophiura species share this character. Theodoria and Ophiuroglypha species have arm spines that are evenly spaced across the distal margin of the lateral plate. 23. Middle arm spine formed into a hyaline hook: 0 = present; 1 = absent The genus Ophiuroglypha characteristicaly has the middle arm spine transformed into a hyaline hooklet in the distal part of the arms (Hertz 1926; Fel 1961). 24. Distal arm spine modified into a hook: 0 = absent; 1 = present Homalophiura euryplax and H. intorta belong to the group of Homalophiura species that are referable to Ophiura, acording to Paterson (1985). This group includes Homalophiura species that share several morphological characters, one of which is the distalmost arm spine being transformed into a hook. 25. Basal tentacle pores: 0 = conspicuous; 1 = not conspicuous The diagnostic morphological characters for Theodoria are conspicuous tentacle pores on the thre basal arm joints and jaws that are excavate along the midline of each jaw. Ophiurolepis has inconspicuous tentacle pores throughout the length of the arm (Fel 1961). 26. Number of tentacle scales beyond proximal joints: 0 = 1; 1 = 2 Ophiurolepis was originaly described as having two minute, peg-like arm spines and two similar tentacle scales (Matsumoto 1915). The same character state occurs in Homalophiura (Mortensen 1936; Paterson 1985) and Theodoria (Fel 1961). 27. Dorsal arm plates: 0 = contiguous throughout most of arm length; 1 = contiguous only proximaly; 2 = not contiguous The dorsal arm plates in Ophiurolepis vary betwen being contiguous throughout the entire length of the arm to being contiguous only in the proximal arm joints. In Theodoria, the dorsal arm plates are proximaly contiguous, while amongst Homalophiura they can be contiguous throughout, only proximaly contiguous or discontinuous throughout. 174 28. Shape of dorsal arm plates beyond proximal joints: 0 = rectangular; 1 = lozenge shaped The dorsal arm plates become lozenge shaped after the first several arm joints in Ophiurolepis, Theodoria and Homalophiura. In Ophiuroglypha, they become markedly rectangular. 29. Dorsal arm plates modified into hook-like knobs: 0 = absent; 1 = present Ophiurolepis olstadi and O. anceps are similar morphologicaly to O. gelida and O. brevirima, but can be distinguished in part by their conspicuous dorsal arm plates, which form hook-like knobs. 30. Dorsal arm plates with distal tubercle: 0 = absent; 1 = present Among Ophiurolepis, O. brevirima, O. gelida, O. tuberosa, O. turgida, O. olstadi and O. anceps are distinct in having the distal edge of the dorsal arm plates raised into a tubercle (Mortensen 1936). 31. Dorsal arm plate fragmentation: 0 = absent; 1 = present This feature of the dorsal arm plates is found in several Theodoria, Ophiurolepis and Homalophiura species. Theodoria partita is distinguished from T. wallini by its fragmented dorsal arm plates (Fel 1961). Homalophiura inornata is known to have fragmented dorsal arm plates (Mortensen 1936) as is H. clasta (Clark 1911). Among Ophiurolepis, O. tuberosa and O. scisa commonly show this feature (Koehler 1908; Mortensen 1936). 32. Ventral arm plates: 0 = broadly contiguous throughout; 1= contiguous only proximaly; 2 = not contiguous The ventral arm plates are contiguous on the proximal arm joints only or discontinuous throughout in Ophiurolepis and Homalophiura. In Theodoria, the ventral arm plates are most commonly discontinuous on al arm joints. 33. Iophon known to parasitize: 0 = absent; 1 = present The parasitic sponge, Iophon radiatus, is known to infest only Ophiurolepis gelida and O. brevirima. Fel (1961) suggested that the degre of infestation could even be used to distinguish betwen O. gelida and O. brevirima, since O. gelida was more heavily infested than O. brevirima in specimens examined. It is unknown why this sponge species infests only two Ophiurolepis species when several other species are common (Dearborn et al. 1973). 34. Gonads: 0 = numerous gonads along adradial and interadial side of genital slits; 1 = two to four gonads along adradial and interadial side of genital slits; 2 = two gonads at interadial side of genital slit with one to two gonads at the adradial side of genital slit Mortensen (1936) commented on the gonad character of the Ophiurolepis, Theodoria, Homalophiura and Ophiuroglypha species he examined. Ophiurolepis species are variable for this character, with some species having numerous gonads and others only a few. 175 35. Genital plate shape: 0 = narow and bar-like; 1 = half-moon shape The genital plate has a wide crescent shape in Ophiurolepis species and in T. partita and T. wallini. This character is variable in Homalophiura, with some species possesing a narow, straight genital plate and others the crescent shape typical of Ophiurolepis. 36. Second oral tentacle pore opens into oral opening: 0 = present; 1 = absent In Ophiuroglypha, the second oral tentacle pore opening is positioned towards and opens into the oral opening. In Ophiurolepis, Theodoria and Homalophiura, the second oral tentacle pore is separate from the oral opening. 176 Select morphological characters used in analysis. Character numbers correspond with Appendix. A. O. gelida lateral arm view. B. O. scisa dorsal arm view. C. T. wallini oral view. D. O. tuberosa oral view. E. O. gelida oral view. F. T. relegata oral view. G. T. wallini oral view. H. O. olstadi lateral arm view. I. O. gelida aboral view. J. O. banzarei aboral view. 177 178 Matrix of morphology characters 000000000111111111122222222223333333 123456789012345678901234567890123456 O. accomodata 110101011000111101111210111100010?11 O. anceps 11111211101011100111121011?111010?11 O. banzarei 110102111010111001111210111101010?11 O. brevirima 111112111010111001111210110101011111 O. carinata 111112111000111001111210110100010011 O. gelida 111112111000111001111210110101011111 O. granulifera 112122111010111001111210111101010?11 O. martensi 120101111000111001111210111100010211 O. mordax 120101111010111101111210111100020?11 O. olstadi 111112111010111001111210110111010?11 O. scissa 020101111010111101111110111100110?11 O. tuberosa 120101111010111101111210112101120?11 O. tumescens 110102111000111001111210111101010?11 O. turgida 11010111101011100111210110101010?11 H. brucei 020101111011111001111?10111100020?11 H. confragosa 020101111010111001111210111100110?11 H. inornata 020101111010111101111210111100110011 H. clasta 02020101100011101111210111100120?11 H. euryplax 020301102001100001011111102100020?01 H. intorta 01010110110011100111111111??00010?01 T. partita 120101111010111111111110011100120211 T. relegata 120101111100111011111110011100020?01 T. wallini 120101111010110111111110011100020211 T. madseni ?111?11111??1110111?1?100???0?020??? Ophiuroglypha carinifera 000000000000000000000000000000000000 Ophiuroglphya lymani 000000000000000000000000000000000000