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Speciation and Diversification in the Indo-West Pacific

Permanent Link: http://ufdc.ufl.edu/UFE0041941/00001

Material Information

Title: Speciation and Diversification in the Indo-West Pacific Inferences from the Molecular Systematics of Reef-Associated Crustaceans
Physical Description: 1 online resource (186 p.)
Language: english
Creator: Malay, Maria
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: allopatric, archaeobalanidae, armatobalanus, balanomorpha, barnacle, biodiversity, biogeography, calcinus, cirripedia, coral, crab, crustacea, dna, esu, evolution, hermit, host, indo, marine, molecular, neotrevathana, pacific, paguridae, peripatric, phylogenetics, phylogeography, pyrgomatidae, reef, speciation, species, sympatric, systematics, trevathana
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This study integrates molecular phylogenetics, morphological analysis, and biogeographic data to study speciation, phylogeography, and systematics of two reef-associated crustacean taxa: Calcinus hermit crabs and coral dwelling barnacles (Pyrgomatidae). Calcinus is a charismatic genus of colorful hermit crabs that is most diverse on coral reefs. On the other hand, the family Pyrgomatidae is a little-known yet remarkably specialized group of coral-dwelling barnacles. I found that speciation is primarily allopatric in both systems; however, the location of speciation events differed greatly. In Calcinus, most recent speciation events clustered in remote oceanic archipelagos, while in the pyrgomatid barnacle 'genus' Trevathana s.l. recent speciation occurred between the West Indian Ocean and the West Pacific, and between the West Pacific and Central Pacific. From the Calcinus data, I found that allopatric sister-species pairs are younger than sympatric ones, suggesting that > 2 million years are needed for sympatry to develop. From the Trevathana s.l. data, I determined that both geographic isolation and host-switching drive diversification. Younger sister-species are geographically structured while older divergences are structured by host, implying that geographic isolation is a relatively fast driver of speciation while host switching occurs at a much slower rate. Differences in speciation patterns, together with the fact that neither hermit crabs nor coral barnacles exhibited a biodiversity hotspot in the Indo-Malayan 'coral triangle', imply that patterns of marine speciation result from very diverse processes that probably cannot be explained by only a single model of marine speciation. In Calcinus, differences in color patterns have evolved very rapidly between sister-species; while in Trevathana s.l., sister-species exhibit differences in skeletal morphology. In both systems molecular phylogenetic data were able to uncover numerous species that are new to science; further proof of the utility of molecular data in species discovery. In addition, in a separate study on the systematics of the coral barnacles (Pyrgomatidae), I found that several morphological characters traditionally used in pyrgomatid taxonomy are phylogenetically homoplasious, such that the systematics of the entire family needs to be revisited. Major groupings within the Pyrgomatidae sensu stricto did not correspond to the traditional subfamilies, and one supposed 'outgroup' to the pyrgomatids actually fell within the clade. Traditionally, taxonomic limits in the pyrgomatids were defined on the basis of skeletal fusion; however, results from my study indicate that other phenotypic characters related to control of coral overgrowth may be more important in delineating subgroups within the Pyrgomatidae.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maria Malay.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Paulay, Gustav.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041941:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041941/00001

Material Information

Title: Speciation and Diversification in the Indo-West Pacific Inferences from the Molecular Systematics of Reef-Associated Crustaceans
Physical Description: 1 online resource (186 p.)
Language: english
Creator: Malay, Maria
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: allopatric, archaeobalanidae, armatobalanus, balanomorpha, barnacle, biodiversity, biogeography, calcinus, cirripedia, coral, crab, crustacea, dna, esu, evolution, hermit, host, indo, marine, molecular, neotrevathana, pacific, paguridae, peripatric, phylogenetics, phylogeography, pyrgomatidae, reef, speciation, species, sympatric, systematics, trevathana
Biology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This study integrates molecular phylogenetics, morphological analysis, and biogeographic data to study speciation, phylogeography, and systematics of two reef-associated crustacean taxa: Calcinus hermit crabs and coral dwelling barnacles (Pyrgomatidae). Calcinus is a charismatic genus of colorful hermit crabs that is most diverse on coral reefs. On the other hand, the family Pyrgomatidae is a little-known yet remarkably specialized group of coral-dwelling barnacles. I found that speciation is primarily allopatric in both systems; however, the location of speciation events differed greatly. In Calcinus, most recent speciation events clustered in remote oceanic archipelagos, while in the pyrgomatid barnacle 'genus' Trevathana s.l. recent speciation occurred between the West Indian Ocean and the West Pacific, and between the West Pacific and Central Pacific. From the Calcinus data, I found that allopatric sister-species pairs are younger than sympatric ones, suggesting that > 2 million years are needed for sympatry to develop. From the Trevathana s.l. data, I determined that both geographic isolation and host-switching drive diversification. Younger sister-species are geographically structured while older divergences are structured by host, implying that geographic isolation is a relatively fast driver of speciation while host switching occurs at a much slower rate. Differences in speciation patterns, together with the fact that neither hermit crabs nor coral barnacles exhibited a biodiversity hotspot in the Indo-Malayan 'coral triangle', imply that patterns of marine speciation result from very diverse processes that probably cannot be explained by only a single model of marine speciation. In Calcinus, differences in color patterns have evolved very rapidly between sister-species; while in Trevathana s.l., sister-species exhibit differences in skeletal morphology. In both systems molecular phylogenetic data were able to uncover numerous species that are new to science; further proof of the utility of molecular data in species discovery. In addition, in a separate study on the systematics of the coral barnacles (Pyrgomatidae), I found that several morphological characters traditionally used in pyrgomatid taxonomy are phylogenetically homoplasious, such that the systematics of the entire family needs to be revisited. Major groupings within the Pyrgomatidae sensu stricto did not correspond to the traditional subfamilies, and one supposed 'outgroup' to the pyrgomatids actually fell within the clade. Traditionally, taxonomic limits in the pyrgomatids were defined on the basis of skeletal fusion; however, results from my study indicate that other phenotypic characters related to control of coral overgrowth may be more important in delineating subgroups within the Pyrgomatidae.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maria Malay.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Paulay, Gustav.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041941:00001


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1 SPECIATION AND DIVERSIFICATION IN THE INDO WEST PACIFIC: INFERENCES FROM THE MOLECULAR SYSTEMATICS OF REEF -ASSOCIATED CRUSTACEANS By MARIA CELIA DEFRANCE MALAY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIV ERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Maria Celia Defrance Malay

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3 To my extraordinary grandparents, Marie Leprtre Defrance, Lonce Defrance, Paula Carolina Santos Malay, and Armando de Jesus Malay

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4 ACKNOWL EDGMENTS Acknowledgments for Chapter 2: We are deeply grateful to Joseph Poupin for his advice, encouragement, specimens, and for kind permission to use the images and information posted on his excel lent website on Calcinus Yoyo Rahayu kindly lent us a paratype specimen, and Regis Clva facilitated access to the MNHN collections. We thank Patsy McLaughlin and Joseph Poupin for help with some of the identifications. We wish to acknowledge the many people who contributed specimens to this study: D. Eernise, M. Frey, J. Hooper, L. Kirkendale, J. ODonnell, B. Olaivar, C. Pittman, J. Poupin, L. Rocha, W. Sterrer, and P. Wirtz. Photo credits: J. Poupin ( C. californiensis, explorator, imperialis, inconspicu us, mclaughlinae, orchidae, pascuensis, aff. sirius ), L. Albenga ( albengai, aff. albengai ), J. Hoover ( hakahau), W. Sterrer (verrilli), C. dUdekem dAcoz ( tubularis ), P. Wirtz ( talismani), J. Okuno ( anani ), and D.L. Felder (obscurus ). All other photos are by G. Paulay and M. Malay. We are also grateful to Mike Hellberg and 3 anonymous reviewers for their helpful comments on the manuscript. Acknowledgements for Chapter s 3 and 4: I am grateful to Bill Newman, Arnold Ross (1936 -2006), Roger Portell, Yair Ach ituv, Andrew Hosie, Fabio Pitombo, and Benny Chan for patiently answering my questions on barnacle taxonomy, morphology, and evolution. For contributing samples, I thank Patricia Cabezas -Padilla, Camilla Floros, Franois Michonneau, Seabird McKeon, Lisa Ki rkendale, John Starmer, Gustav Paulay, Helmut Zibrowius, Yair Achituv, and Andrew Hosie. For contributing sequences and for logistical support in sequencing, I thank Marcos Prez -Losada, Maegan Finley, Chris Meyer, and Maggie Fagan. I also wish to acknowledge help from Rebecca Kimball, Ed Braun, Mike Miyamoto, and Fan Qiu for advice on topological tests, and

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5 Pam Soltis for advice on how to deal with missing data. For logistical and/or administrative support in field work, I am very grateful to Annete Juinio-Meez, Perry Alio, Edgardo Gomez, Wilfredo Licuanan, the University of the Philippines Marine Science Institute, Keryea Soong, Minghui Chen, Michel Claereboudt, Esther Emmanuelli, Vincent Hollevout, the cole Pratique des Hautes tudes (EPHE), the Centre des Recherches Insulaires et Observatoire de lEnvironnement (CRIOBE) in Moorea, the Gump Station in Moorea, Mireille Guillaume, Henrich Bruggemann, the Universit de la Runion, Philippe Bouchet, Victor Bonito, Miri Tabaiwalu and everyone in the villag e of Votua, Gerald McCormack, and Teina and Jackie Rongo. Jada Simone White generously allowed me to use her larval culturing setup at the Gump Station in Moorea. I thank my two student volunteers, Karina Concha and Austin Perlmutter, for all their hard w orking help in dissecting barnacles and setting up PCRs. I am grateful to the governments of the Republic of the Philippines, the Gouvernement de la Polynsie Franaise, the Republic of the Fiji Islands, the Government of the Cook Islands, Taiwan/the Republic of China, the Sultanate of Oman, Terres Australes et Antartiques Franaises (TAAF), and the Republic of France, for granting permission to sample in their territories. I am exceedingly grateful for financial support I have received through the years fr om the University of Florida Alumni Fellowship, th e Department of Zoology (now Biology), the Brian Riewald Memorial Fund Research Grant, the Leila and William Brayfield Scholarship in Invertebrate Paleontology, the Lerner -Gray Grant at the American Museum of Natural History, the FACE program (Ocean Bridges: A Florida France Training & Research Cooperative in Coral Reef Conservation and Biodiversity)

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6 the Kuroshio Research Fellowship at the National Sun Yat -Sen University of Taiwan, the American Microscopical Society, the Crustacean Society, and the Carcinological Society of Japan. The Florida Museum of Natural History and the Department of Biology at the University of Florida have been excellent academic homes throughout my PhD. I am most grateful to the team at the Invertebrate Zoology lab at FLMNH, especially Mandy Bemis, Sarah McPherson, Jenna Moore, Ch elsey Campbell, Kim Kemppanion, and John Slapcinsky, for all the curatorial support. I also acknowledge Chris Meyer, Lisa Kirkendale, Jessica Light, Franoi s Michonneau, and Nat Evans for advice on phylogenetic programs and analyses; and Matt Gitzendanner and the University of Florida Phyloinformatics Cluster for High Performance Computing in the Life Sciences for computational support My committee members D avid Reed, Pam Soltis, Rebecca Kimball, and Mike Miyamoto were there to help me throughout the long process of the PhD, and I wish to thank them for their support. I most gratefully acknowledge my awesome PhD supervisor, Gustav Paulay, for all the intellec tual, financial, logistical, emotional support through the years, and for getting me started on these projects in the first place. None of these studies would have been possible without him. Lastly, I thank my family; my Mama and Papa, my sister Pat and br other Ali, my cousins near and far, aunts and uncles, and my dear departed grandparents; all of whom I love so much and who love me equally back. My fianc Jimmy Huang gave me all out love and emotional and moral support through these years, and kept me honest and grounded on reality. Jimmy s family, especially Ping have been wonderfully

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7 supportive too. Lastly I thank all my friends, old and new (there are too many to mention!) for their love.

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES .............................................................................................................. 11 LIST OF FIGURES ............................................................................................................ 12 ABSTRACT ........................................................................................................................ 15 CHAPTER 1 INTRODUCTION ........................................................................................................ 17 Introduction to Indo -West Pacific Marine Biogeography ........................................... 17 Overall Goals .............................................................................................................. 22 2 PERIPATRIC SPECIATION DRIVES DIVERSIFICATION AND DISTRIBUTIONAL PATTERN OF REEF HERMIT CRABS (DECAPODA: DIOGENIDAE: Calcinus ) ............................................................................................ 24 Introduction ................................................................................................................. 24 Materials and Methods ............................................................................................... 26 Specimens ............................................................................................................ 26 Molecular Methods ............................................................................................... 28 Sequence Analysis ............................................................................................... 29 Molecular Clock Analysis ..................................................................................... 31 Analysis of Speciation and Biogeography ........................................................... 32 Results ........................................................................................................................ 34 Sequence Attributes ............................................................................................. 34 Phylogeny Reconstruction and Species Boundaries .......................................... 34 Molecular Clock Results ...................................................................................... 36 Distribution of Calcinus Species .......................................................................... 36 Diversity Patterns in Calcinus .............................................................................. 37 Speciation ............................................................................................................. 37 Discussion ................................................................................................................... 38 Species Boundaries ............................................................................................. 38 Evolution of Color Patterns .................................................................................. 41 Geography of Speciation ..................................................................................... 42 Inter -Regional Comparisons ................................................................................ 46 Ecology ................................................................................................................. 47 Diversity Patterns ................................................................................................. 49 Conclusions ................................................................................................................ 51

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9 3 PHYLOGENETIC SYSTEMATICS OF CORAL DWELLING BARNACLES (BALANOMORPHA: PYRGOMATIDAE) ................................................................... 76 Introduction ................................................................................................................. 76 Taxonomy and Global Biogeogr aphy of the Pyrgomatids .................................. 77 Phylogenetic Hypotheses Regarding the Pyrgomatids ...................................... 80 Materials and Methods ............................................................................................... 82 Taxon Selection and Identification ...................................................................... 82 Molecular Methods ............................................................................................... 83 Phylogenetic Analyses ......................................................................................... 84 Topological Tests ................................................................................................. 86 Character Tracing and Morphological Evolution ................................................. 87 Results ........................................................................................................................ 90 Sequence Characteristics .................................................................................... 90 Phylogenetic Relationships within the Pyrgomatidae and Congruence of Gene Trees ....................................................................................................... 90 Placement of Outgroup Taxa ............................................................................... 91 Topological Tests ................................................................................................. 92 Character Tracin g and Phylogeny -Trait Correlation ........................................... 92 Discussion ................................................................................................................... 94 Systematics .......................................................................................................... 94 Character State Evolution .................................................................................... 96 Evolution of Parasitism ........................................................................................ 99 Conclusions and Recommendations ....................................................................... 100 4 GEOGRAPHY AND HOST -SPECIFICITY BOTH INFLUENCE SPECIATION OF CORAL BARNACLES IN THE CLADE Trevathana SENSU LATO ........................ 132 Introduction ............................................................................................................... 132 Materials and Methods ............................................................................................. 133 Specimens and Morphological Examinations ................................................... 133 Molecular Methods ............................................................................................. 134 Data Analyses .................................................................................................... 135 Results ...................................................................................................................... 136 Host -Specificity Patterns .................................................................................... 137 Biogeographic Patterns ...................................................................................... 138 Discussion ................................................................................................................. 140 Host Specificity ................................................................................................... 140 Biogeography ..................................................................................................... 140 Temporal Difference Between Host Shifts and Geographical Isolation ........... 141 Species Diversity Patterns ................................................................................. 141 What Are Species? ............................................................................................ 142 Status of Neotrevathana .................................................................................... 143 Conclusions .............................................................................................................. 146 5 CONCLUSIONS ........................................................................................................ 169

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10 LIST OF REFERENCES ................................................................................................. 173 BIOGRAPHICAL SKETCH .............................................................................................. 18 6

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11 LIST OF TABLES Table page 2 -1 Known species of Calcinus and new ESUs, including their geographic ranges and accession information for all the sequenced specimens ............................... 54 2 -2 List of ESUs used in biogeographic analyses, and their geographic distributions relative to each oth er ......................................................................... 64 3 -1 List of recent pyrgomatid species ........................................................................ 102 3 -2 Morphological and ecological characters of the genera, as synthesized from the taxonomic lit erature ........................................................................................ 105 3 -3 List of sequenced specimens .............................................................................. 107 3 -4 Sequence attr ibutes for the 5 gene fragments .................................................... 110 3 -5 Topological incongruencies in ML (computed using RAxML) and Bayesian (computed using MrBayes) gene trees ............................................................... 111 3 -6 Pvalues obtained from topological tests ............................................................. 113 3 -7 Results of BaTS analyses of seven phenotypic characters ................................ 114 4 -1 All nominal species within the Trevathana sens u lato subclade. ....................... 148 4 -2 List of sequenced specimens .............................................................................. 150 4 -3 List of ESUs .......................................................................................................... 154 4 -4 List of analyzed ESEs. ......................................................................................... 155

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12 LIST OF FIGURES Figure page 2 -1 Bayesian phylograms constructed using (a)3 concatenated genes and (b)COI only ............................................................................................................. 65 2 -2 Frequency distribution of K2P distances for intraspecific variation and interspecific distances in Calcinus without (a) and with (b) the C. minutus complex .................................................................................................................. 67 2 -3 Distributions, color patterns, and COI phylogeny of Clade I Calcinus species. ... 68 2 -4 Distributions, color patterns, and COI phylogeny of Clade II Calcinus species ... 68 2 -5 Distributions, color patterns, and COI phylogeny of Clade III Calcinus species .. 69 2 -6 Dist ributions, color patterns, and COI phylogeny of Clade IV Calcinus species .................................................................................................................... 69 2 -7 Distributions, color patterns, and COI phylogeny of Clades V and VI Calcinus species .................................................................................................................... 70 2 -8 Distributions, color patterns, and COI phylogeny of Clade VIIa Calcinus species .................................................................................................................... 70 2 -9 Distributions, color patterns, and COI phylogeny of Clade VIIb Calcinus species .................................................................................................................... 71 2 -10 Distributions, color patterns, and COI phylogeny of Clade VIIc Calcinus species .................................................................................................................... 71 2 -11 Distrib utions, color patterns, and COI phylogeny of Clade VIII Calcinus species .................................................................................................................... 72 2 -12 Distributions, color patterns, and COI phylogeny of Clade IXa Calcinus species .................................................................................................................... 72 2 -13 Distributions, color patterns, and COI phylogeny of Clade IXb Calcinus species .................................................................................................................... 73 2 -14 Distributions, color patterns, and COI phylogeny of Clade X Ca lcinus species .. 73 2 -15 Spatial distribution of species richness. ................................................................ 74 2 -16 Approximate distribution of IWP ESEs .................................................................. 74 2 -17 Age distribution (in million years, my) of Calcinus sister species pairs ................ 75

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13 3 -1 Schematic illustrations of pyrgomatid anatomy and grow th process, an d diversity in shell morphology ................................................................................ 115 3 -2 Phenotypic characters traced onto the pyrgomatid phylogeny, with the vario us character states illustrated ...................................................................... 116 3 -3 Maximum likelihood phylogram for COI computed using RAxML ...................... 117 3 -6 Maximum likelihood phylogram for 12S computed using RAxML ...................... 120 3 -8 Maximum likelihood phylogram for all mt genes computed using RAxML ......... 122 3 -9 Maximum likelihood phylogram for all nuc genes computed using RAxML ....... 123 3 -10 Maximum likelihood phylogram for all 5 sequenced genes computed using RAxML .................................................................................................................. 124 3 -11 Reconstruction of ancestral char acter states for opercular valve fusion ............ 125 3 -12 Reconstruction of ancestral character states for number of wall plates ............. 126 3 -1 3 Reconstruction of ancestral character states for wall height .............................. 127 3 -14 Reconstruction of ancestral character states for basis calcareousness ............ 128 3 -15 Reconstruction of ancestral character states for cirrus 3 armature .................... 129 3 -16 Reconstruction of ancestral character states for degree of coral overgrowth .... 130 3 -17 Reconstruction of ancestral character states for mechanism of overgrowth suppression .......................................................................................................... 131 4 -1 RAxML phylogram for Trevathana s.l. ................................................................. 156 4 -2 Geograph ic distribution of Clade I ESUs ............................................................. 157 4 -3 Geographi c distribution of Clade II ESUs ............................................................ 158 4 -4 Geographic distribution of Clade III ESUs ........................................................... 159 4 -5 Geographi c distribution of Clade IV ESUs .......................................................... 160 4 -6 Geographi c distribution of Clade VI ESUs .......................................................... 161 4 -7 Geographic distribution of Clade VII ESUs ......................................................... 162 4 -8 Geographi c distribution of Clade IX ESUs .......................................................... 163 4 -9 Geographic distribution of Clade X ESUs ........................................................... 164

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14 4 -10 Geographi c distribution of Clade XI ESU s .......................................................... 165 4 -11 Geographic distribution of Clade XII ES Us ......................................................... 166 4 -12 ESU richness in sampled localities. ..................................................................... 167 4 -13 Occurrences of unfused (open circle) and fused (filled circle) opercular valves in the phylogeny ........................................................................................ 168

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15 Abstract of Dissertation Presented to the Graduate School of the Univers ity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SPECIATION AND DIVERSIFICATION IN THE INDO WEST PACIFIC: INFERENCES FROM THE MOLECULAR SYSTEMATICS OF REEF -ASSOCIATED CRUSTACEANS By Maria Cel ia Defrance Malay August 2010 Chair: Gustav Paulay Major: Zoology This study integrates molecular phylogenetics, morphological analysis, and biogeographic data to study speciation, phylogeography, and systematics of two reef associated crustacean taxa: Calcinus hermit crabs and coral dwelling barnacles (Pyrgomatidae). Calcinus is a charismatic genus of colorful hermit crabs that is most diverse on coral reefs. On the other hand, the family Pyrgomatidae is a little known yet remarkably specialized group o f coral dwelling barnacles. I found that speciation is primarily allopatric in both systems; however, the location of speciation events differed greatly. In Calcinus most recent speciation events clustered in remote oceanic archipelagos, while in the pyrg omatid barnacle genus Trevathana s.l. recent speciation occurred between the West Indian Ocean and the West Pacific, and between the West Pacific and Central Pacific. From the Calcinus data, I found that allopatric sister -species pairs are younger than s ympatric ones, suggesting that >2 million years are needed for sympatry to develop. From the Trevathana s.l. data, I determined that both geographic isolation and host -switching drive diversification. Younger sister -species are geographically structured wh ile older divergences are structured by host, implying that geographic isolation is a relatively fast driver of speciation while host switching occurs at

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16 a much slower rate. Differences in speciation patterns, together with the fact that neither hermit cra bs nor coral barnacles exhibited a biodiversity hotspot in the IndoMalayan coral triangle, imply that patterns of marine speciation result from very diverse processes that probably cannot be explained by only a single model of marine speciation. In Calc inus differences in color patterns have evolved very rapidly between sister species; while in Trevathana s.l. sister -species exhibit differences in skeletal morphology. In both systems molecular phylogenetic data were able to uncover numerous species tha t are new to science; further proof of the utility of molecular data in species discovery. In addition, in a separate study on the systematics of the coral barnacles (Pyrgomatidae), I found that several morphological characters traditionally used in pyrgom atid taxonomy are phylogenetically homoplasious, such that the systematics of the entire family needs to be revisited. Major groupings within the Pyrgomatidae sensu stricto did not correspond to the traditional subfamilies, and one supposed outgroup to t he pyrgomatids actually fell within the clade. Traditionally, taxonomic limits in the pyrgomatids were defined on the basis of skeletal fusion; however, results from my study indicate that other phenotypic characters related to control of coral overgrowth may be more important in delineating subgroups within the Pyrgomatidae.

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17 CHAPTER 1 INTRODUCTION Introduction to Indo-West Pacific Marine Biogeography The tropical Indo -West Pacific (IWP) is the single largest marine biogeographic region in the world, stre tching half the globe from the eastern coast of Africa to the central Pacific. IWP biodiversity peaks in the comparatively small triangle formed by Indonesia, the Philippines, and New Guinea. This well known IndoMalayan biodiversity triangle or coral t riangle is the center of species richness for a wide variety of marine taxa, including reef fishes, corals, gastropods, and crustaceans. Species and genus level diversity progressively diminishes as one moves away from this central region (reviewed in Paulay 1997). The location of the Indo Malayan biodiversity hotspot is correlated with the amount of habitat area: the Indo Malayan region has very extensive tracts of shallow water reef habitat, which may in turn sustain elevated levels of biodiversity (Bellwood and Hughes 2001, Karlson et al. 2004, Bellwood et al. 2005). Yet despite its fame, there is still no consensus on the question of how the hotspot originated. Why is species diversity so high in the IndoMalayan triangle, and what mechanism(s) of species origination gave rise to current -day patterns of species distribution in the region? Aside from species area relationships and middomain effects (e.g., Bellwood and Hughes 2001, Bellwood et al. 2005), many hypotheses incorporating ideas on species or igination have been proposed (see review in Rosen 1988). While they all propose allopatric or peripatric models of speciation, these theories fall into 3 main categories that disagree about whether new species form (a) in remote islands at the peripheries of the IWP (e.g., Center of Accumulation theory); (b) in the complex sub-basins of the IndoMalayan

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18 triangle (Center of Origin theory); or (c) whether high diversity is simply due to the overlap of two separate biogeographic provinces, the Western Pacific and Indian Ocean (Center of Overlap theory; reviewed in Palumbi 1997, Paulay 1997). The issue is made more confusing by the fact that these 3 main hypotheses can each be further subdivided into dispersal and vicariance-based explanations, which differ in their predictions on the timing of speciation across different taxa. For instance, one variant of the Center of Overlap theory may predict that faunas on the W Pacific and Indian Ocean diverged simultaneously because of a single major vicariant event, whi le another variant of the theory would argue that the pattern resulted from an accumulation of chance migrations across a current driven dispersal barrier during periods of low sea levels (Paulay 1997). A Center of Overlap scenario could also have arisen a s a result of divergence across an oceanic vs. continental reef ecological gradient. Similarly, the Center of Accumulation scenario could have resulted from rare founder speciation events in remote islands of the central Pacific, or from the fragmentation of previously more extensive species ranges followed by reinvasion of the central IWP by the newly -formed species (Paulay and Meyer 2002). Finally, some proponents of the Center of Origin theory posit synchronous patterns of speciation in different faunal groups during periods of reduced connectivity in the Indo-Malayan triangle (e.g., during Plio Pleistocene low sea level stands; Barber and Bellwood 2005). However, it is also possible that some in situ speciation events occurred as a result of the escalat ing complexity of ecological interactions in the Indo-Malayan diversity hotspot (i.e., existing diversity begetting even more diversity; Emerson and Kolm 2005).

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19 Evaluating the different hypotheses is very difficult because different evolutionary scenarios may result in identical biogeographic patterns (Palumbi 1997, Paulay 1997, Kirkendale and Meyer 2004). Patterns of speciation are overlaid by subsequent migrations and local extinctions, such that present day species distributions alone may not be sufficie nt to establish where speciation took place. While some studies make the assumption that present -day centers of species ranges are equivalent to the region of species origination (Mora et al. 2003), such assumptions may very likely be erroneous (e.g., Barb er and Bellwood 2005). For instance, while the remote central Pacific islands have surprisingly high numbers of endemic fore -reef species, fossils of these species from the Indo Malayan region prove that this pattern is a result of reliction rather than sp eciation, presumably because the relict species are unable to cope with escalating ecological pressures in the more diverse regions of the IWP. Conversely, the depauperate inner -reef fauna in the central Pacific has resulted from localized extinctions of l agoonar specialists from remote Pacific atolls as a result of fluctuating sea levels (Paulay 1990, 1996). These conclusions were made on the basis of detailed examinations of bivalve fossil records; however, good records are lacking for many IWP marine tax a, thus the use of fossils is taxonomically limited. Another important consideration is that similar distributional patterns are not necessarily the result of identical speciation mechanisms different processes may give rise to concordant patterns in dif ferent taxa. Neither can one rule out the possibility that more than one speciation mechanism is operating within a single taxonomic group (Meyer et al. 2005, Paulay 1997, Palumbi 1997, Randall 1998) Thus it does not seem likely that marine biogeographers will soon agree on a single unambigous explanation for the existence of

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20 the Indo -Malayan biodiversity hotspot; and I believe there should be no reason to expect a single, simple explanation for this complex pattern. Molecular phylogenetics and phylogeogra phy are powerful tools for inferring the evolutionary histories of species and clades. The application of phylogenetic techniques to marine systems has already caused major shifts in our understanding of marine biogeography, despite the fact that the entir e field of phylogeography is a little over two decades old (Avise et al. 1987). The overall picture emerging from studies of IWP phylogeography is that both species diversity and population level diversity are much greater than previously thought (e.g., Kn owlton 1993, Knowlton 2000, Meyer et al. 2005). Moreover, populations and species exhibit more geographic structuring than previously thought (e.g., Williams and Reid 2004, Meyer et al. 2005, Barber et al. 2006). These new realizations challenge the old paradigm that marine biota comprise open systems (compared to terrestrial environments), with very few barriers to dispersal and thus less opportunities for allopatric diversification. In reality, we are only now starting to appreciate how diverse, and how geographically structured, the IWP truly is. Aside from revising our understanding of diversification, what other insights have molecular phylogenetics provided? Can we now use phylogenetic information to infer the mechanisms by which IWP diversity was ge nerated? Different studies do not agree on a single mechanism of diversification. There have been studies claiming support for the Center of Overlap theory (e.g., in organisms such as starfish, snapping shrimp, coconut crabs, patelloid limpets; Williams 2000, Williams et al. 2002, Lavery et al. 1996, Kirkendale and Meyer 2004); yet there are also studies that favor a Center of Origin (in reef fish, Briggs 1999 and Mora et al. 2003) or a Center of Accumulation scenario (e.g.,

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21 in Echinometra oblonga sea urchi ns, Landry et al. 2003; and reef fish, Bellwood and Wainwright 2002, Hughes et al. 2002). In several cases, a combination of different processes have been proposed to operate in a single taxonomic system (in turbinid gastropods, Meyer et al. 2005; wrasses, Barber and Bellwood 2005; and cowries, Meyer 2003). Different geological, evolutionary, and ecological processes have interacted in complex manners to produce the patterns we observe today. Thus, the best way to understand what has transpired (and is transpiring) in the IWP is by applying the timehonored comparative method: studying the phylogenetics and phylogeography of a wide variety of taxonomic groups, and hopefully gaining an understanding of the overall picture of diversification in the IWP through an inductive process. Phylogenetic studies in the IWP have so far focused mainly on geographic isolation (i.e., allopatry through dispersal and vicariance) as a mechanism of species origination; few have given more than a passing mention to the possible i nfluence of ecological adaptations on the process of speciation (see Paulay 1996 for discussion of adaptations to different reef habitats; Landry et al. 2003 on presence or absence of congeneric species), and very few have directly investigated such a hypothesis (but see Duffy 1996; Faucci et al. 2007). Divergent ecological pressures can give rise to speciation in populations connected by gene flow, or through reinforcement of reproductive barriers following secondary overlap of allopatric populations (Rice and Hostert 1993, Coyne and Orr 2004). There is also theoretical (Diekmann and Doebeli 1999, Gavrilets 2000) and limited empirical evidence supporting speciation in wholly sympatric situations (e.g., Schliewen et al. 1994, Eastman and McCune 2000, Turelli et

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22 al. 2001, Via 2001). In particular, in situ speciation through host -race formation has been receiving much attention recently (e.g., Feder et al. 1994, Abrahamson et al. 2002, Emelianov et al. 2004). Such a speciation mechanism would be expected in com mensals, parasites, and mutualists with limited mobility and highly evolved interactions with specific hosts ecological guilds that are particularly species -rich in coral reefs, as in all hyperdiverse communities. However, as of present there have been e xtremely few phylogenetic studies of obligately symbiotic reef associated organisms. Overall Goals For my dissertation, I studied the phylogenetics and phylogeography of two representative reef associated crustacean taxa: hermit crabs in the genus Calcinu s and coral dwelling barnacles in the family Pyrgomatidae. One objective was to evaluate what new data can tell us about existing biogeographic hypotheses about diversification in the IWP. In the case of the Pyrgomatidae, another major objective was to re evaluate the systematics of the family and analyze the evolution of character traits, including traits related to adaptation to a symbiotic coral dwelling lifestyle. Towards these goals, in Chapter 2 I discuss global speciation patterns in my first study system, the hermit crab Calcinus In Chapter 3 I tackle the systematics of the pyrgomatid coral dwelling barnacles. Then in Chapter 4 I focus on one of the phylogenetically well supported clades, the Trevathana sensu lato group, and analyze the patterns of distribution and speciation of all members of the clade. These patterns are summarized and synthesized in the last chapter, Chapter 5. The overarching goal of this work is to contribute to ongoing efforts to understand mechanisms of species diversificatio n in the sea, with a special emphasis on IndoWest

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23 Pacific coral reef crustacean taxa. Just as research on phytophagous insects has helped terrestrial biologists to understand the diversity of tropical rainforests (reviewed in Coyne and Orr 2004), I employ ed my work on crustacean speciation to make inferences on the origins of species in coral reefs, the rainforests of the sea.

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24 CHAPTER 2 PERIPATRIC SPECIATION DRIVES DIVERSIFICATION AND DISTRIBUTIONAL PATTERN OF REEF HERMIT CRABS (DECAPODA: DIOGENIDAE: C a lcinus ) Introduction The marine tropics can be divided into four broad regions defined by largely endemic biotas: the East Atlantic (EA; West African tropical coastline and offshore islands, Mediterranean), West Atlantic (WA; East American tropical coastli ne, Caribbean, and offshore islands including Bermuda), East Pacific (EP; West American tropical coastline to offshore islands including Galapagos & Clipperton), and IndoWest Pacific (IWP; from East Africa to Easter Island) regions (Ekman 1953, Briggs 1974). Diversity is lowest in the EA and highest, by about an order of magnitude, in the IWP (Paulay 1997). Further patterns are evident within the vast IWP, where marine biodiversity peaks in the IndoMalayan triangle bounded by the Philippines, Indonesia, a nd New Guinea and decreases in a striking manner toward the central Pacific (Stehli & Wells 1971). Much early work focused on these striking spatial patterns and attempted to find single or at least dominant processes to explain them. While numerous hypotheses have been proposed to explain observed spatial patterns in reef diversity (often focused on the high diversity of Indo Malaya) (Rosen 1988), three have been most emphasized: the center of origin, center of overlap, and center of accumulation hypothese s. These have attributed the Indo Malayan diversity peak to in situ diversification, overlap in the ranges of Indian and Pacific basin species, and accumulation of species originating elsewhere, respectively. Increasing documentation of variation in spatial diversity patterns as well as modes of speciation have, however, led to the realization that multiple processes must be involved in generating the observed patterns of diversity (Palumbi 1997, Paulay 1997, Williams 2007).

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25 Molecular phylogenetics provide s a powerful tool for understanding the origins of observed patterns of species richness. By analyzing diverse taxa we can address questions of diversification from a quantitative, mechanistic perspective: what is the relative importance of different modes of speciation in generating species richness and spatial patterns of diversity? In the past, analyses of spatial patterns of diversity were largely inferential (i.e., top-down): by examining biota -level patterns, researchers inferred likely processes of d iversification. In contrast a quantitative phylogenetic approach provides a mechanistic (i.e., bottom -up) perspective: by documenting numerous speciation events, we can investigate how regional -level diversity patterns arise. Such an approach necessitates thorough spatial and taxonomic sampling, so that most or all speciation events in a clade are identified and characterized. Thorough taxonomic coverage is also one of the most important factors in recovering an accurate tree topology, as shown by both empi rical and simulation studies (e.g., Graybeal 1998, Zwickl & Hillis 2002, Soltis et al. 2004). The objectives of this study are to pursue a comprehensive phylogenetic and biogeographic analysis of the reef associated hermit crab genus Calcinus to: (1) dete rmine spatial and temporal patterns of diversification, (2) evaluate the relative importance of different modes of speciation and how they gave rise to observed patterns of diversity and distribution, and (3) assess the roles of color and ecology in divers ification. Calcinus are diverse, medium -sized, diurnal, conspicuous, colorful, and abundant diogenids. All known species are tropical or subtropical, most live on coral reefs, and several are facultative coral associates, frequently encountered within branching corals. The genus is circumtropical, with 41 recognized species (Table 21):

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26 33 in the IWP and 2-4 in each of the remaining regions (EA, WA, EP). There is substantial variation in ranges among IWP species, with some extending from East Africa to Hawaii, while others are known from single islands or archipelagos (Table 2-1). Calcinus species are most readily identified from, and a few can only be reliably differentiated based on, their color pattern (e.g., Poupin and McLaughlin 1998, Poupin and Lemaitr e 2003). Partly because colors fade in preserved specimens, coloration has been underutilized in crustacean taxonomy in the past. However, more effective field methods, including SCUBA, field photography, increased sampling, and appreciation of color differences, have substantially improved our knowledge of Calcinus in recent decades. Alpha taxonomy and geographic distributions are now comparatively well documented (Poupin 2003), making Calcinus an excellent focus for evolutionary and biogeographic study. W e constructed a phylogeny of Calcinus based on most described species in the genus, including samples from multiple locations spanning the known ranges of most widespread species. Sequence data provide evidence for substantial cryptic diversity in the genu s. In some species color pattern appears to have evolved so rapidly that sister species with strikingly different color patterns are only slightly or not genetically differentiated. Most young sister species pairs have allopatric distributions, indicating that allopatric speciation is the main or only mechanism for diversification. Isolation on remote island groups appears to be the most common cause of speciation. Materials and Methods Specimens We sampled 37 of the 43 nominal species of Calcinus and 9 add itional, undescribed phylogenetic species recognizable on the basis of sequence data (Table 2-

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27 1). The species not sequenced are Calcinus urabaensis known from a single specimen in Colombia, Calcinus kurozumii known only from a single collection on Pagan Island (Marianas), C. tropidomanus known from a single collection in Somalia, and C. sirius from Australia. We also did not sample Calcinus paradoxus a species based on a single specimen collected in much deeper (500 m) water than any other Calcinus w hose generic assignment even its author questioned (Bouvier, 1922); nor the dwarf species C. revi suspected to be the juvenile of more common Calcinus species (Poupin, pers. comm.). Much of the material was collected by reef walking, snorkeling, or scuba-diving, fixed in 75 -95% ethanol, and deposited in the Invertebrate Collections of the Florida Museum of Natural History, University of Florida (UF; Table 2-1). Additional specimens were borrowed from other institutions (Table 21). Whenever possible living animals were photographed to record color pattern. We identified specimens using Poupins (2003) interactive taxonomic key, the primary taxonomic literature, and in consultation with J. Poupin and P. McLaughlin. Data on geographic ranges and ecology of species were compiled from the taxonomic literature, Poupins (2003) website on the genus, the UF specimen database, and the authors field observations. The diogenid hermit crab genera Ciliopagurus and Dardanus were chosen as the closest outgroup taxa based on a phylogenetic analysis (not shown) of a larger set of hermit crab genera. Samples for sequencing were selected to span as much of the geographic range of each species as available material permitted (Table 21, Figs. 2-4 to 215). We collected DNA seq uence data from 150 operational taxonomic units (OTUs). All but 1 (C. talismani ) of the 150 OTUs were sequenced for the cytochrome oxidase I (COI)

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28 mitochondrial gene fragment. We generated phylogenetic trees for the COI only dataset, and on the basis of th ese trees we selected a subset of 96 OTUs for further sequencing of 16S rDNA and Histone 3 (H3) genes. The 96-OTU subset was comprised of only the 2 genetically most divergent individuals in each species or genetically distinct putative new species. Thus t he full 150-OTU taxon set was utilized for constructing the COI only tree while a pruned subset of 96 OTUs was used for constructing individual gene trees and a concatenated 3gene tree. The ILD test for data combinability (see below) was also performed on the 96-OTU subset. Lastly, molecular clock analyses (see below) were performed on a further reduced 50-OTU taxon subset, in order to keep computations manageable. Molecular Methods DNA was extracted from muscle tissue using DNAzol and proteinase K foll owing the protocol given in Meyer 2003. Sequence data were collected for two mitochondrial DNA markers (COI and 16S) and one nuclear marker (H3). Average length of the amplified fragments and PCR primers used are as follows: COI: ~645 base pairs (bp), prim ers dgLCO (5 -GGT CAA CAA ATC ATA AAG AYA TYG G -3) and dgHCO (5 -TAA ACT TCA GGG TGA CCA AAR AAY CA3; Meyer 2003). 16S: ~550 bp, primers 16SAR (5 -CGC CTG TTT ATC AAA AAC AT -3) and 16SBR (5 -GCC GGT CTG AAC TCA GAT CAC GT -3; Palumbi 1996). H3: ~350 bp, primers H3af (5 ATG GCT CGT ACC AAG CAG ACV GC -3) and H3ar (5 ATA TCC TTR GGC ATR ATR GTG AC -3; Colgan et al. 1998). PCR thermocycler profiles for COI and 16S were as in Meyer (2003), while the PCR profile for H3 followed Prez Losada et al. (2004). PCR products were either (a) cleaned using Wizard PCR Preps (Promega) and sequenced using the ABI Big Dye protocol and a Perkin-Elmer ABI Automated Sequencer; or (b) cleaned

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29 using the exo-sap cleanup protocol and sequenced at the high-throughput sequencing facility of the University of Floridas Interdisciplinary Center for Biotechnology research (ICBR) in a 96well format using BigDyeTerminator cycle sequencing reactions, employing an ABI 3730 XL for electrophoresis. Initially, mitochondrial DNA sequencing was done along both directions of a DNA fragment, and as our confidence in base calls increased in later stages, only 1 strand was sequenced (unless base ambiguities were noted, in which case the 2nd direction was sequenced). Histone 3 sequencing was always done on both directions. Sequence Analysis Chromatograms of the sequences were manually checked and edited using the software Sequencher ver. 4.2 (Gene Codes). Sequence alignment was done by eye using Se-Al v2.0a11 (Rambaut, http://tree.bio.ed.ac.uk/software/seal/ ). Sequences are available in GenBank (accession nos. FJ620149FJ620493, EF683559 -EF683561). We also included COI data from GenBank for Calcinus obscurus (AF436039 ). In all analyses, all sites were weighted equally, characters were unordered, and gaps were treated as missing data. We used two approaches to decide whether or not to pool the 3 gene fragments into a single analysis. Firstly, we used the incongruence length difference (ILD) test (Farris et al 1995), a parsimony based statistical test of data combinability commonly employed in phylogenetic studies. We used PAUP* ver. 4.0b10 (Swofford 2002) to perform the ILD test simultaneously for the 3 data partitions. No significant incongruences were noted among the 3 gene trees. However, the usefulness of the ILD test for evaluating data combinability has been called into question (e.g., Yoder et al 2001, Barker & Lutzoni 2002). To address these concerns, and to explore our data

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30 further, we also visually compared Bayesian tree topologies resulting from independent searches for each of the 3 gene regions. A visual comparison of the gene trees showed no conflict with each other nor with the 3-gene concatenated analysis (data not shown). Bas ed on this evidence, we decided that a combined analysis was appropriate. We determined the simplest model of evolution that best fit our COI only dataset as well as our 3-gene dataset using the Akaike Information Criterion (AIC) as implemented by the program Modeltest 3.6 (Posada & Crandall 1998). Phylogenetic relationships were estimated using maximum likelihood (ML), maximum parsimony (MP), and Bayesian statistics (BS). Parsimony analyses were done using PAUP, ML analyses were implemented using both PAU P and GARLI v0.951-1 (Zwickl 2006, http://www.bio.utexas.edu/faculty/antisense/garli/Garli.html ), while Bayesian analyses were implemented using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). In the MP and ML analyses using PAUP, heuristic searches started with random addition of taxa replicated 10 times using the treebisection-reconnection (TBR) branch-swapping algorithm. Branch support in the MP anal yses was estimated by bootstrap support values, calculated as above with 1,000 (for the 3-gene tree) or 200 (for the COI tree) replicates. ML branch support values were not calculated using PAUP due to computational constraints. In the ML analyses using GA RLI, we used random starting trees and performed 5 -7 independent runs to obtain the best tree. Branch support values were estimated in GARLI using 2,200 and 1,300 bootstrap replicates for the COI only and 3-gene datasets, respectively. In the Bayesian anal yses, we ran 2 independent chains for 1 million generations each; each chain was sampled every 100 generations. The MCMC runs reached stationarity in 60k generations or less. We

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31 discarded the initial 25% of the trees as the burn in phase. Bayesian posterior probabilities were calculated based on the remaining 75% of the trees. We calculated pairwise COI genetic distances for each sister species pair identified in our phylogenetic trees using PAUP. We used Kimuras (1980) K2P distance metric to facilitate comparison with earlier studies. Molecular Clock Analysis We used BEAST 1.4.8 (Drummond & Rambaut 2007) to estimate divergence times of Calcinus sister taxa. BEAST employs a Bayesian statistical framework to simultaneously estimate phylogenetic trees and div ergence times, thus it is capable of integrating uncertainty in topology in the divergence date estimation. BEAST also allows for incorporation of uncertainty in calibration points. We did a partitioned analysis for all 3 genes (3 -nucleotide codon partitions for the coding regions COI and H3, and 1 partition for the non -coding 16S, for a total of 7 partitions) using an uncorrelated log normal relaxed clock. For each data partition we specified a GTR+I+G model of sequence evolution. We asked BEAST to estimat e the time to most recent common ancestor (TMRCA) of each pair of sister species in the phylogeny. We used a Yule tree prior, specified a UPGMA starting tree, and did 2 independent runs of 1x107 generations each. We sampled the posterior distributions of t he dates being estimated by sampling the runs every 1,000 generations, after removing the first 10% of the MCMC chain as the burnin period. Convergence of the results was checked by loading the posterior distributions into the program Tracer. The analysis was calibrated by specifying a prior on the date of divergence of the transisthmian species pair Calcinus tibicen and C. explorator The timing of vicariance of transisthmian sister species varies substantially among taxa, with many falling around the tim e of final severing of the land bridge

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32 around 3.1 my (Coates & Obando 1996), but others are older (cf. Knowlton & Weigt 1998, Lessios 2008). As a preliminary approximation we set a prior with a lognormal distribution with a mean of 3.5 my and a standard deviation of 1.0 (this was approximated by specifying a lognormal mean of 1.21352716 and a lognormal standard deviation of 0.28012786). In our analysis a normal distribution was not appropriate because a transisthmian divergence time of zero would then have a positive probability, which is an unrealistic prior and would cause calculation problems (AJ Drummond, personal communication). Using a lognormal prior ensures that a divergence time of zero is excluded from the analysis, while allowing for a TMRCA subst antially older than 3.5 my. Analysis of Speciation and Biogeography Our analysis of speciation patterns focuses on recognized species as well as previously unrecognized, but genetically distinct Evolutionary Significant Units (ESUs; sensu Moritz 1994). ESU s are defined as reciprocally monophyletic populations for the locus investigated (here 16S and COI mtDNA; H3 was not considered due to low levels of interspecific divergence), that have at least one other independent, defining attribute such as distinct c olor pattern, structural morphology, distribution, or reciprocal monophyly in another, independent marker. ESUs satisfy the phylogenetic species concept, and are clades with an evolutionary history separate from other ESUs. Some ESUs are as morphologically and genetically distinctive as recognized species; conversely a few recognized species are not reciprocally monophyletic in mtDNA (see below). ESUs are thus species -level units which, unlike biological species, can be defined in allopatric as well as in s ympatric settings without experimental tests of interbreeding.

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33 We call the divergence of ESUs from each other Evolutionary Significant Events (ESEs). ESEs are to speciation what ESUs are to species: they are objectively defined diversification events that give rise to ESUs. To quantify the relative importance of different modes of diversification, we enumerated all identifiable ESEs that have given rise to at least one individual ESU (or recognized species). That is, we considered ESEs that have given rise to either two separate ESUs, or led to the separation of one ESU from a clade that subsequently further diversified. Species occurrence records were mapped in ArcGIS, and species ranges inferred by drawing a polygon around bordering record points. Species were considered allopatric when they had separate ranges; such ranges may end on adjacent islands, but are then separated by open ocean. Species ranges that truly abut, or overlap for <10% of the range of the narrower sister taxons range, were termed par apatric. Diversity contour maps were generated from these data by superimposing inferred distributional range of each species. Such diversity contour maps can be biased in that 1) diversity in interior areas can be overestimated when species are actually absent from there but inferred to occur because of peripheral records, and 2) lack of sampling of marginal occurrence will lead to an underestimation of marginal range, but lack of sampling of central occurrence will not lead to an underestimation of centr al range. As a second method for estimating local diversity, we also assembled species lists for relatively well studied areas and have indicated the number of species known from these on the contour maps. The latter method is prone to the biases of geographically varied sampling methods and efforts.

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34 Results Sequence Attributes The COI region sequenced was 609 base pairs (bp) long, with 368 invariable and 238 parsimony -informative sites. Mean base frequencies were: 0.25A, 0.17C, 0.23G, 0.35T, showing an A -T bias of 60%. The model that best fit these data was a GTR+I+G model. The 16S gene fragment contained some regions that could not be confidently aligned across all taxa. We tested the importance of these hypervariable regions by running separate analyses with and without them. Inclusion or exclusion of hypervariable regions did not result in substantial topological differences, thus they were included in the final analyses. The 16S gene fragment was 459 bp long, with 276 bp invariable and 125 bp parsimony -informative sites, and mean base frequencies of 0.32A, 0.18C, 0.13G, 0.36T (A -T bias 68%). The H3 gene fragment was 336 bp long, with 279 bp invariable and 53 bp parsimony informative sites, and mean base frequencies of 0.19A, 0.34C, 0.28G, 0.19T (A -T bias 38%). The best fit models were GTR+I+G for COI, 16S, and the combined 3gene set, and GTR -I for H3. Phylogeny Reconstruction and Species Boundaries The 3 methods of phylogenetic analyses used (MP, ML, BS) gave congruent results, and the topologies genera ted from the 3-gene and COI only datasets were likewise congruent (fig. 2-1 a & b). Bootstrap values were higher in the 3gene trees (particularly at the deeper nodes), as expected. We thus used the 3gene trees to identify supra -specific clades within Cal cinus Ten strongly supported clades were identifiable within the genus. We defined strong phylogenetic support as >60% bootstrap values in the MP and ML trees and >90% posterior probabilities in the BS trees (see clades I X in fig. 2 1a; the sole exception to our criterion for defining clades

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35 was clade IV, which was supported by both ML and BS analysis, but had no bootstrap support under MP; nonetheless, this grouping was recovered in all methods of analysis used). Relationships of ESUs within these clades were generally well resolved, but the relationships of the clades to each other was generally poorly resolved. Thus these clades served as the basic units for our analyses of speciation patterns. Because the COI analyses (fig. 21b) covered more individuals from more geographic locations, these were used to delineate species and ESUs. Analyses revealed 9 ESUs (22% of the sampled IWP fauna) that do not correspond to previously described species. Eight of these 9 are allopatrically divergent populations of r ecognized species, while one is co -distributed with its sister -species but has a non overlapping depth range. Three traditionally recognized species were not reciprocally monophyletic: Calcinus minutus, C. nitidus and C. rosaceus are interdigitated in a m ostly unresolved species complex. All other nominal species for which multiple individuals were sequenced were recovered as monophyletic units with high bootstrap/posterior probability support values. Thus most named species fulfilled the ESU criterion and phylogenetic species concept (Wheeler and Meier 2000). Excluding the C. minutus complex (see Discussion), intra-specific K2P distances ranged from 0 -6% (1.3+/ 1.0%), with only one outlier with K2P>4%. Pairwise, interspecific K2P distances within clades r anged from 425% (K2P) (Fig. 22a). Thus there was no barcoding gap (Hebert et al. 2003, Meyer & Paulay 2005), but also little overlap between intra-specific and inter -specific distances. Including the C. minutus complex creates a much larger overlap between intraand interspecific differences (Fig. 2 -2b).

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36 Out of 267 pairwise intraspecific K2P distance comparisons, 10% had values >2.7%. These were within C. argus, C. pulcher s.s C. haigae, and C. anani These species appear to exhibit substantial geogra phic structuring across their range (see fig. 2 -1b): C. argus appears to have divergent populations in Reunion Island and Hawaii; C. pulcher has a distinct population in the Philippines; C. haigae shows divergence in the Tuamotus; and C. anani from the Mar quesas and Papua New Guinea appear genetically differentiated. Interestingly, we observed distinct color morphs for a C. anani specimen from the Philippines (not sequenced) and for juvenile C. haigae from the Tuamotus (illustrated in Poupin 2003). However, the distinct groupings were not consistently supported across all methods of analysis and small sample sizes also limit our ability to further investigate differentiation within these species. Molecular Clock Results The molecular clock analyses showed t hat allopatrically -distributed sister species pairs were significantly younger than sympatric sister species (allopatric sister species: mean=2.0 my, range=0.4 -6.3 my; sympatric sister species: mean=5.8 my, range=2.210.2 my; fig. 23; p>0.05, t -test) and all young (<2.5 my) divergences were among allopatric sister taxa. There is considerable spread in TMRCAs, particularly for sympatric sister species pairs. There is no temporal gap dividing the ages of strictly allopatric species from sister species that h ave broadly overlapping geographic ranges. Distribution of Calcinus Species Our surveys led to numerous new geographic records and substantial improvement in the documentation of the distribution of Calcinus species (see http://www.flmnh.ufl.edu/scripts/dbs/malacol_pub.asp for source of records). Figures 24 to 2 -15 show the presently known geographic range of each species.

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37 Diversity Patterns in Calcinus The species richness of Calcinus is highest in the oceanic Pacific, and does not peak in the Indo Malayan triangle (Fig. 2-16). Both projected and known diversity peak in the Mariana and Tuamotu Islands, at the NW and SE Oceania. Sixteen species have been recorded from the Marianas and 15 from the Tuamotus. Diversity in the IndoMalayan triangle is substantially lower, with 8 species recorded from the Philippines, 9 from Indonesia, and 10 from all of New Guinea. Only 12 species have been recorded from the entire IndoMalayan archipelago, c ompared with 21 species from SE Polynesia. Speciation Twenty -four ESEs were identified in the IWP and 4 in other regions. Six of the IWP ESEs separate sympatric sister taxa, others are geographically structured. Of the 22 geographically structured ESEs 20 separate allopatric sister taxa and two split parapatric sisters with narrow areas of distributional overlap. Sympatric sister taxa are generally separated by deeper genetic distances than allopatric or parapatric taxa (see above). All 21 pairs of allopat ric or parapatric sister taxa appear to have adjacent ranges as far as current sampling can document. Geographically -structured ESEs span the globe, but cluster in some areas (Fig. 217); most fall in areas previously recognized as potentially important in speciation, as evidenced by a high proportion of endemics. Within the IWP, four ESEs separate ESUs in Hawaii. Three ESEs each separate ESUs between the tropical and subtropical S Pacific, and across the Indian Ocean (although the exact locations of the latter separations are poorly constrained because of sparse sampling in the Indian Ocean). Two ESEs each separate ESUs in the Marquesas, SE Polynesia, and at subtropical

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38 latitudes across Australia. Single ESEs separate ESUs in Arabia and Easter Island (Fig. 2 -17). Outside the IWP, 2 ESEs separate ESUs between adjacent regions (EP WA, and Bermuda EA), and two separate sister taxa within the EP: along the central American coast, and between Clipperton Island and the central American coast. Discussion Specie s Boundaries Although there is general correspondence between described species and genetically -defined ESUs, 22% of the taxa examined were not concordant. Three of the named species were not reciprocally monophyletic, while 9 ESUs represent previously und escribed (and mostly unrecognized) forms. Lack of correspondence between named morphological species and ESUs delineated with genetic methods is commonly encountered in genetic surveys of well studied taxa, and can have multiple causes (Funk & Omland 2003, Meyer & Paulay 2005). Calcinus minutus, C. nitidus and C. rosaceus failed to sort into monophyletic units. Most specimens in this complex form a tight cluster (K2P<2%, with all species combinations represented at K2P C. rosaceus fr om Reunion in the Mascarene Islands (K2P=10%). These three nominal species have allopatric, abutting ranges, are very similar in structural morphology, with C. minutus and C. nitidus in particular, nearly impossible to distinguish except by color (Fig. 2 6 ; Morgan 1991, Poupin and McLaughlin 1998, Poupin 2003) Several factors can cause species level non -monophyly (Funk & Omland 2003). First, there may be insufficient differences in the marker used to differentiate species. We consider this unlikely because mitochondrial gene regions used cleanly resolve other Calcinus species. Second, ancestral polymorphisms may have been retained, because of a slow rate of evolution

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39 or recent speciation. While there is no evidence for a slow down in the rate of evolution in this lineage, species divergence may have been so recent that ancestral haplotypes have not had sufficient time to sort into monophyletic clades. The virtual lack of morphological differentiation other than color between C. nitidus and C. minutus is su ggestive of recent divergence. Third, mitochondrial haplotypes could have introgressed across species boundaries. The occurrence of a divergent sequence in one C. rosaceus specimen, sister to all others in the complex, suggests that introgression is a plausible explanation. Structurally as well as in color pattern C. rosaceus is closest to C. haigae the sister taxon to this complex (Poupin 2003; Asakura and Tachikawa 2003). This suggests that the divergent sequence may represent the original C. rosaceus ge notype, which has largely been replaced by a sweep of C. minutus haplotypes. Independent markers could provide a test of this hypothesis. The H3 nuclear sequences show slow rates of evolution and are not variable across this complex, and appear to lack the power to resolve this problem. The distinct color patterns are likely under genetic, nuclear control, thus they represent an independent marker; however, color may be under selection and could thus have evolved more rapidly than potentially neutral mitochondrial markers (see below). Future work with other markers is needed to resolve the status of these species. In contrast, six previously -recognized species show marked differentiation into 2 or 3 ESUs each. In two ( C. albengai, C. elegans ), the different iated ESUs show conspicuous and previously noted color forms that have not been taxonomically recognized (Poupin & Lemaitre 2003, Haig & McLaughlin 1984). In the six others ( C. hazletti, C. vachoni X2 C. latens X2, and C. pulcher ), no color differences we re noted

PAGE 40

40 during collection, but are evident in four of the five for which live images were taken. Color differences could not be discerned only in photographs of C. hazletti ESUs in Micronesia and the Hawaiian Islands (although color polymorphism has been reported in this species in Japan; Asakura 2004). No images were available for the SE Polynesian C. vachoni ESU. Much of the incongruence between morphology based species and genetic ESUs result from changing taxonomic traditions, and reflect a lack of sys tematic revision. Historically, carcinologists hesitated to describe species distinguished solely by color pattern; thus, the strikingly distinctive Hawaiian color form of C. elegans has not been named (Haig & McLaughlin 1984). More recently, workers have tended to recognize such structurally -similar color forms, such as the Marquesan endemic C. hakahau as distinct species (Poupin & McLaughlin 1998). A well executed revision should rectify alternate species concepts currently in use. Species boundaries ca n be defined based on a variety of criteria and characters (e.g., Wheeler & Meier 2000). When taxa are sympatric and co occurring, species limits are usually straightforward; however, species limits are more subjective for allopatric taxa not subject to potential interbreeding. Genetics, color pattern, structural morphology, and/or geography can all inform taxonomic delineations. We defined ESUs as reciprocally monophyletic taxa in a genetic marker, that are also distinguishable by at least one additional i ndependent character. Three recognized species do not meet this definition, as they are not demonstrably reciprocally monophyletic with the genetic markers used. However, these three forms do have other, independent characters that

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41 correlate: color pattern and geography, implying that they are on independent evolutionary trajectories. Evolution of Color Patterns The general correspondence between color forms and ESUs indicates that color patterns are almost always reliable and sufficient for differentiating Calcinus species. C olor pattern-level differentiation between morphologically -similar sister species is common among Crustacea (e.g., Knowlton 1993; Macpherson and Machordom 2000; Ravago and Juinio Meez 2003) as well as in other taxa, such as reef fish (e.g. McMillan et al. 1999, Bowen et al. 2006; reviewed in Knowlton 1993). Among reef fishes, there have also been documented cases of closely -related species that differ strikingly in color and yet show few (if any) structural differences and are not reci procally monophyletic at the mitochondrial level (e.g., McMillan et al. 1999, Bowen et al. 2006). If the lack of monophyly is not due to introgression, these findings imply that the r ate of color pattern evolution can outpace mitochondrial sequence divergence, which suggests that differentiation in coloration may be driven by selection. In Calcinus coloration is so conspicuous and varied that it can be reasonably assumed to serve a purpose and thus be acted upon by natural selection. For example, it has b een demonstrated that the size of the white chelar patch in Calcinus laevimanus influences success in interspecific agonistic encounters (Dunham 1978). It is likely that other Calcinus species utilize color patterns in adaptive ways. If coloration is invol ved in conspecific interactions, then strong selection on these visual cues could result in the rapid color evolution, and genetically isolated populations may diverge in these cues over relatively short periods of time. Moreover, if color patterns are used for species recognition, then divergence in color may lead to the development of reproductive

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42 isolation barriers and thus speciation. Color patterns have been shown to serve in species recognition and mate choice in other marine groups, including fiddler crabs (Detto et al. 2006) and fish (McMillan et al. 1999, Puebla et al. 2007, Seehausen et al. 2008). Geography of Speciation Speciation appears to be largely or exclusively allopatric in Calcinus as in most animals (Coyne & Orr 2004), and allopatric sep aration of sister taxa is retained for more than 2 million years (fig. 2-3). The narrowly allopatric to parapatric ranges of all young sister taxa imply either that the geography of the original speciation event has been maintained in these taxa and there has been little post -speciational changes in distribution, or that such changes were reciprocal; i.e., expansion in the range of one ESU was associated with contraction in the range of the other. Although the latter hypothesis is difficult to falsify, the former is much more parsimonious and also more likely because boundaries between sister ESUs tend to fall at recognized zones of transition associated with major dispersal or ecological barriers. Narrowly allopatric ranges also imply that localized endemi cs are the result of speciation rather than reliction. Endemism can be high on peripheral island groups, but endemics can result from either local (typically peripatric) speciation (e.g., neoendemics) or reliction (paleoendemics; see Ladd 1960, Stehli & We lls 1971, Newman & Foster 1987). Reliction refers to the survival of formerly widespread taxa often in remote, biologically less intense, safe places (Vermeij 1987). As relicts are generally older taxa that have undergone substantial reduction in their r ange, they are not expected to be narrowly allopatric with their sister taxa, but to show disjunct or

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43 sympatric ranges. None (except C. albengai ) of the insular endemics are sympatric or have disjunct distribution with their sister taxa. The location of geographic speciation events span the globe, but are not randomly distributed. Peripatric speciation on remote islands is most prevalent, while speciation events within the Indo Malayan triangle or between the Indian and Pacific ocean basins are absent / rar e. Speciation across ecological gradients, such as latitude and depth, and between the four tropical regions is also evident. Isolation on remote islands and archipelagos appears to be the most prevalent cause of speciation in Calcinus : 60% of the ESEs ha ve resulted in at least one of the sister taxa becoming restricted to a remote island group. A similar pattern of predominantly peripatric speciation has been found in Thalassoma wrasses (Bernardi et al. 2004). Insular endemics have evolved in Hawaii (4), Marquesas (2), SE Polynesia (2), Mascarenes (2), Easter Island (1), Clipperton (1), and Bermuda (1); an additional endemic putatively assigned to the genus ( C. paradoxus ; see above) in the Azores has not been sampled. These are some of the most remote islands in the world, renowned for high endemism (e.g., Briggs 1974, Randall 1998), thus it is not surprising that they also host endemic Calcinus Among fish, the highest levels of endemism in the IWP are encountered in peripheral areas: Easter, Hawaiian, Mar quesas, Mascarene islands and the Red Sea (4.423% endemics; Randall 1998, Allen 2007). Among these remote island groups, we have sampled the Hawaiian Islands most thoroughly, and have sequenced 9 of 10 species known from there. Of the 9, four (44%) are endemic: C. laurentae, and endemic ESUs of the widespread C. hazletti, C. latens, and C. elegans Calcinus isabellae (known from two Hawaiian records) remains untested. In the

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44 Marquesas, two of four recorded species are endemic, while one of three from Easte r Island are. However, the status of widespread species in the Marquesas and Easter remain to be genetically evaluated. Four clades appear to have given rise to multiple peripheral endemics. In two, peripatric speciation from a widespread form appears to have been the source of these endemics, while in two others, insular endemics appear to have undergone local diversification within a basin. Calcinus elegans and C. latens both ranging from East Africa to Polynesia, gave rise to four peripatric endemics: three on remote islands and one on the Arabian peninsula. The wide ranging ESU is a terminal branch in both clades, implying it was the source of successive peripheral endemics. In contrast the insular sister endemics C. laurentae-C. hakahau-C. gouti and H awaiianMicronesian ESUs of C. hazletti represent lineages diversifying within the central Pacific, and are only more distantly related to widespread taxa ( C. lineapropodus -pulcher and C. minutus -complex, respectively). Calcinus also includes several spec ies restricted to relatively cool, subtropical or moderately deep (100300m) waters. The following species are known only from subtropical latitudes in the southern IWP: C. sirius, C. aff. sirius, C. albengai, C. aff. albengai -shallow, C. dapsiles (clade X ), C. spicatus, C. pascuensis (clade V) and C. imperialis, C. vanninii (clade VII). The origin of these taxa is predominantly by in situ diversification within the subtropics. Similar latitude based niche conservatism has been demonstrated in gastropods (F rey & Vermeij 2008, Williams et al. 2003, Williams 2007). Only the last clade has a relatively recent and thus readily identifiable origin in the tropics, sister to the parapatric C. isabellae

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45 All deep water species investigated ( C. anani, C. albengai deep, C. aff. sirius ) are members of clade X, suggesting that invasion of deep reef habitats may have occurred only once. Interestingly this clade also includes a large portion of subtropically -restricted Calcinus implying that temperature may be an important factor limiting their distribution. Our field observations show that even the two clade X members known from relatively shallow, tropical waters ( C. argus and C. anani ) are rare in those habitats, but also occur in the subtropics or deep water. Sequence and/or morphological data suggest incipient differentiation in four of five recognized species in this clade ( C. anani, C. argus C. sirius, C. albengai ), the only exception being the geographically -restricted W Australian endemic C. dapsiles Moreover, subtropical and deep reef habitats remain substantially undersampled for Calcinus and future explorations will likely result in discovery of numerous new forms and document additional radiation. The small number of samples on hand prevent detailed analysis of speciation in this clade. In contrast to the abundance of peripheral speciation, Calcinus show no diversification within the Indo Malayan area: no ESEs are identified within the area, and only one species, C. gaimardii, is (largely) confined to it. Thi s contrasts with many marine taxa that have numerous endemics in Indo Malaya, some with substantial in situ diversification (e.g., Paulay 1997, Meyer et al. 2005, Barber et al. 2006, Williams & Reid 2004). Overall, speciation along continental shorelines appears to be uncommon in Calcinus with the EP species C. californiensis (Gulf of California to El Salvador) and C. obscurus (El Salvador to Peru) the only known example. Calcinus species show little differentiation between the Indian and Pacific Ocean ba sins. In contrast the restricted seaway between the Indian and Pacific basins is one of

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46 the most important sites of speciation for other marine taxa, with numerous well known as well as cryptic speciespairs differentiating across the boundary between thes e great basins (e.g., Randall 1998, Read et al. 2006, Barber et al. 2000). In Calcinus only 2 ESEs are known that may fall in this area, i.e., in the C. pulcher and C. vachoni complexes. However, the location of the boundary between western and eastern ESU s of both species are poorly constrained, as no samples have been genetically tested between the Philippines/Ryukyus and Mascarenes (Figs. 27 and 2 -14). In contrast none of the other 5 widespread species tested ( C. laevimanus, C. argus, C. elegans, C. guamensis, C. latens ) show much genetic differentiation between populations in the Indian and Pacific Ocean basins. The genetic homogeneity of such wide-ranging species, prevalence of peripatric speciation on remote archipelagos, and diverse Calcinus assemblages on the worlds most isolated islands imply that these crabs have great powers of dispersal, that has influenced their modes of speciation. Inter -Regional Comparisons While the diversity of Calcinus in the IWP and EP are largely the result of in situ r adiation, inter -regional speciation was the source of Atlantic diversity. All non -IWP species studied are in two clades (I and VI). Clade I is comprised of C. tubularis (EA) and C. verrilli (Bermuda). The eastward relationship of the Bermudan endemic is un usual, as the majority of marine organisms in Bermuda originated from the WA, a result of the Gulf Stream facilitating dispersal (Sterrer 1986, Floeter et al. 2007). Among fishes, only a single Bermudan species appears to be clearly of EA in origin (SmithVaniz et al 1999). Clade VI (Fig. 2 -8) is comprised of 4 EP, 1 WA and 1 EA species. Species in this clade are nearly identical in structural morphology, but readily distinguished by color

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47 pattern. The EA Calcinus talismani is not represented in the COI only phylogeny because only the 16S region could be amplified from available specimens. In 16S only, 2 and 3gene trees, C. talismani is sister to C. tibicen Among the remaining clade VI species, C. tibicen (WA) and C. explorator (EP) appear to be geminate species isolated by the emergence of the Isthmus of Panama. The other subclade is comprised of EP species only ( C. californiensis, C. obscurus, and C. mclaughlinae ). Calcinus mclaughlinae is endemic to Clipperton Island, ~1100 km offshore of central Ameri ca, while C. californiensis and C. obscurus have parapatric ranges along the central American coast and are absent from EP oceanic islands. Offshore EP islands (Clarion, Socorro, Clipperton, Cocos, Galapagos) mostly harbor C. explorator a species also pre sent in and near the Gulf of California, but not along the continental coast further south (Fig. 28). Ecology Species distributional boundaries can be set by ecological limitations as well as dispersal barriers (with dispersal barriers themselves a type of ecological limitation). A prevalent form of distributional restriction in the IWP is to continental or oceanic habitats (Abbott 1960, George 1974, Paulay 1994, Reid et al. 2006). While both can be caused by dispersal as well as ecological limitations, ecological restriction is implied for species that range widely among remote islands, but are absent from nearby continents. Pacific -plate endemism (Springer 1982, Kay 1984) is a well documented example of such ecological oceanic restriction (Paulay 199 7). Continental and oceanic habitats differ in many ways, including levels of primary productivity, terrigenous influence, habitat diversity, and presence/absence of predators and competitors that are restricted to continental shores by dispersal limitations.

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48 Oceanic restriction is prevalent in Calcinus Thus only 7 of 17 species of Calcinus recorded from Australian territories are known from the continent, the remainder are recorded only from offshore islands (Morgan 1991). While 12 species are recorded f rom Cocos Keeling and Christmas Islands, small oceanic islands just SW of Indonesia, only 9 species have been recorded from all of Indonesia, the most diverse marine archipelago in the world. Similar continental avoidance occurs in some Australian brachyur ans and the terrestrial hermit crab Coenobita (Paulay & Starmer in prep). Calcinus isabellae is a classic widespread Pacific -plate endemic (Fig. 2-11), and five other species appear to be regionally widespread, yet largely confined to islands: C. sirius in the South Pacific, C. argus and C. seurati across the IWP, C. explorator in the EP, and C. talismani in the EA. An additional 16 species are restricted to one or a few neighboring oceanic island groups, but could be so restricted by dispersal limitation a s well as ecology. Conversely, only three species show largely continental restriction: C. gaimardii in the IWP and C. californiensis and C. obscurus in the EP. There is substantial niche conservatism in Calcinus but also interesting ecological shifts bet ween sister species. As discussed above, several clades are restricted to cool waters. Sister species C. tubularis and C. verrilli are the only Calcinus known with sexually dimorphic behavior, with females commonly adopting a sessile habit, living in tubes of sessile turritellid and vermetid gastropods (Markham 1977; Gherardi 2004). Sister species C. s eurati and C. laevimanus are the only high intertidal/supratidal Calcinus a habitat otherwise occupied by the related (but competitively inferior Hazlett 1 981) diogenid genus Clibanarius However, while C. laevimanus lives in the upper intertidal, C. seurati is restricted to supratidal splash pools. Two ESUs of C.

PAGE 49

49 albengai separate by depth: one ranging from shore to <50 m depths, while the other is exclusiv ely deep water (50 -280 m; Poupin and Lemaitre 2003). The role of ecology vs. geography in the divergence of these species deserves further attention; the latter, with both forms only known from one small island, has potential as sympatric speciation through niche differentiation. Diversity Patterns Calcinus species diversity is about an order of magnitude higher in the IWP than other regions, a pattern typical for reef organisms (Paulay 1997). The IWP is home to 40 ESUs, the EP, WA, and EA to 4, 3, and 2 respectively. Local diversity shows similar inter -regional differences, with up to 16 species coexisting in one archipelago (Marianas) in the IWP, but at most 2 in other regions. Calcinus species richness does not peak within the Indo Malayan triangle, but is highest in Oceania, peaking at two locations: the Mariana and Tuamotu Islands, with 16 and 15 species (Fig. 216). This contrasts with the majority of marine taxa that reach their diversity peak in Indo Malaya, from where richness decreases in all direc tions, but most conspicuously across the Pacific basin (Stehli et al. 1967, Briggs 1974, Hoeksema 2007). Nevertheless, diversity patterns, especially the steepness of the diversity increase towards Indo Malaya varies greatly among taxa, and in large groups it is the composite of different, clade-specific, underlying patterns (see Fig. 4 in Paulay & Meyer 2006). Thus it is not surprising that particular smaller taxa, like Calcinus deviate from the predominant pattern. We propose that the diversity pattern of Calcinus is a reflection of the genus affinity to oceanic conditions. Calcinus species tend to have great dispersal ability, broad ranges, and reach remote islands. The 10 species diversity contour ranges from

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50 the Mascarene to the Hawaiian Islands, and although 13 species are recorded from across all of SE Asia, only 10 are known from any one country in the IndoMalayan archipelago. As noted above, many species avoid continental habitats, such that diversity is higher on oceanic islands immediately outs ide this diversity center, than on the more terrigenous and continental settings of Indo Malaya and Australia. Finally the predominance of peripatric speciation has lead to local diversity hotspots in peripheral locations, like in SE Polynesia, where 15 species are known from the Tuamotus, and 21 when the faunas of immediately adjacent (and biogeographically contiguous) are included. The high diversity in the Indo Malayan triangle has been attributed to three different hypotheses: center of origin, center of accumulation, or center of overlap. The center of origin hypothesis posits that species arise preferentially in Indo -Malaya. Such a pattern is clearly seen in some taxa with limited dispersal capacity, with species commonly restricted to, and arising wi thin, the Indo Malayan triangle (Paulay 1997, Meyer et al. 2005). With at most one Indo Malayanrestricted species and no in situ diversification, this hypothesis fails for Calcinus The center of overlap hypothesis posits that the high diversity in the I ndo-Malayan area is the result of overlapping ranges of Indian and Pacific basin sister species (Woodland 1983). Differentiation between the Indian and Pacific basins is prevalent in marine organisms (e.g., Randall 1998, Read et al. 2006, Reid et al. 2006) and since such sister taxa often show distributional overlap in Indo Malaya, this hypothesis is supported as an important contributor to the diversity peak in the area (Woodland, 1983). In contrast Calcinus shows little speciation between the Indian and Pacific basins

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51 (0 -2 examples). Furthermore no sister species are known to have overlapping ranges in the Indo -Malaya, albeit we have very limited sampling in the area. The center of accumulation hypothesis posits that species predominantly originate in pe ripheral areas but subsequently accumulate in Indo Malaya by distributional expansion followed by reliction (Ladd 1960, Jokiel & Martinelli 1992). Calcinus includes an abundance of peripheral endemics. Such endemics may follow one of two trajectories: (1) range expansion after establishment of reproductive barriers with their sister species, leading to buildup of regional species diversity, or (2) maintenance of restriction until eventual extinction in their isolated ranges. It is difficult to test the impo rtance of these two alternatives, although the predominance of peripatric speciation in Calcinus combined with high diversity of widespread taxa and substantially greater age of sympatric sister species, suggest that the first trajectory may occur reasonably frequently. In contrast, reliction to IndoMalaya has not been an important process, as demonstrated by the paucity of IndoMalayan Calcinus endemics. As noted above, many Calcinus species appear to prefer oceanic habitats. With an abundance and local radiations of oceanic taxa, combined with substantial dispersal ability of many Calcinus (most IWP -wide species are genetically relatively homogeneous across the two ocean basins), it is not surprising that species richness in Calcinus peaks in oceanic parts of the western Pacific. Conclusions Our study has uncovered a wealth of unrecognized diversity in a relatively well known reef dweller, Calcinus The number of ESUs in the IWP was augmented by 22%. This large increase in ESUs was made possible by our approach of exhaustively sampling populations of every accessible species across their entire range. Through

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52 photo-documentation of live specimens, we conclude that differences in coloration correspond to boundaries between ESUs, thus documenting color patt erns is very important in species delineations. We show that color pattern differences evolve extremely rapidly, and we hypothesize that coloration may serve an adaptive purpose. Perhaps coloration is important in intraspecific recognition and mate selecti on. This hypothesis deserves further investigation, for instance by studying the evolution of genes responsible for differences in decapod coloration. The geographic distributions of Calcinus species are now well documented and illustrate several patterns atypical for reef fauna. Non-IWP species all fall in 2 clades. One clade connects a Bermudan endemic to an EA species, a rarely observed pattern. The second non-IWP clade groups together species from EP, WA, and EA (including one geminate species pair). Th is clade contains the only known instances in Calcinus of speciation along a continental margin. Among IWP species, we show that the center of species diversity is not in the IndoMalayan triangle, but further east in the Marianas islands. This may be the result of a tendency in Calcinus to prefer oceanic habitats. We found no support for either center of origin or center of overlap theories. Instead, our results show a history of generally high dispersal abilities coupled with bouts of peripatric speciation in remote areas. The youngest sister species pairs all have narrowly allopatric distributions, and a substantial amount of time (>2 million years, usually much longer) is needed for sister species to develop sympatric distributions. Contrary to the predi ctions of the center of accumulation hypothesis, the Indo Malayan triangle is not the center of Calcinus diversity and does not act as a refuge for relictual taxa.

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53 Ecological factors have also played a role in Calcinus distribution and speciation. Distrib utions are shaped in part by restriction of species to oceanic environments (common) or continental environments (rare). Phylogenetic conservatism of ecological niches is common; however, there are also a few cases of large ecological shifts between sister species. In one instance, a shift between a shallow water and a deep water morph may have occurred in sympatry. None of the 3 dominant IWP biogeographic models succeed in fully explaining species diversity patterns in Calcinus This is to be expected, as all these models simplistically propose that the Indo -Malayan diversity hotspot can be explained by a single vicariance or dispersal driven mechanism. The reality is much more complex. A diverse range of factors, including both historical and ecological mechanisms, influence species distributions. Given this complexity, biogeographers should expect different taxa to show non-identical biogeographic patterns, reflecting the unique histories and ecological adaptations of the different groups. The overall top down picture of marine biodiversity is a summation of all these individual histories.

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54 Table 2 1. Known species of Calcinus and new ESUs, including their geographic ranges and accession information for all the sequenced specimens (including outgroup specimens). Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 1 Calcinus albengai Poupin & Lemaitre 2003 deep morph IWP Austral Ids. Austral Ids Austral Ids. H92 MNHN Pg.6378 + + + 2 Calcinus albengai aff. Poupin & Lemaitre 2003 shallow morph IWP Austral Ids. Austral Ids. Austral Ids. H94 MNHN Pg.6385 + + + 3 Calcinus anani Poupin & McLaughlin, 1998 IWP Japan Tuamot us; Marque sas Bismar ck Arch (PNG) H77 UF 4808 + + + Marqu esas H95 MNHN Pg.6357 + + + Bismar ck Arch. (PNG) H324 UF 11740 + 4 Calcinus argus Wooster, 1984 IWP Mascar ene Ids. Hawaii Mascar ene Ids. H62 UF 5437 + Mascar ene Ids. H114 UF 5446 + N Marian as H 96b UF 5714 + Hawaii H146 UF 7364 + + + Hawaii H192 UF 8038 + New Caledo nia H203 MNHN + Mascar ene Ids. H312 UF 12814 + Mascar ene Ids. H313 UF 13022 + + + 5 Calcinus dapsiles Morgan, 1989 IWP W Australi a W Australi a W Australi a H101 UF 6297 + + + W Australi a H133 UF 6297 + + +

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55 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 6 Ca lcinus elegans H. Milne Edwards, 1836 IWP S Africa Tuamot us; Marque sas Marian as H5 UF 325 + + + Mascar ene Ids. H67 UF 5504 + Tuamo tus IP31 UF 1351 + + + Line Ids. H306 UF 11487 + Line Ids. H317 UF 11204 + 7 Calcinus elegans a ff. Hawaii IWP Hawaii Hawaii Hawaii H15 UF 3216 + + + Hawaii H113 UF 8350 + + + NW Hawaii an Ids. H292 UF 12060 + NW Hawaii an Ids. H293 UF 12064 + NW Hawaii an Ids. H294 UF 12064 + NW Hawaii an Ids. H304 UF 12068 + Hawaii H307 UF 14838 + 8 Calcinus gaimardii H. Milne Edwards, 1848 IWP Maldiv es Fiji Palau H42 UF 3924 + + + Philippi nes H136 UF 6744 + + + 9 Calcinus gouti Poupin, 1997 IWP Line Ids. Tuamot us Tuamo tus H25 UF 1349 + Tuamo tus IP5 UF 18 63 + + + Line Ids. H190 UF 8604 + + +

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56 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 10 Calcinus guamensis Woos ter, 1984 IWP Somali a Hawaii; Marque sas Am. Samoa H23 UF 3224 + Hawaii H60b UF 3219 + Marqu esas H49 UF 5171 + + + Society Ids. IP44 UF 1888 + Mascar ene Ids. H58 UF 5418 + + + Hawaii H142 UF 3219 + 11 Calcinus haigae Wooster, 1984 IWP Red Sea Hawaii; Tuamot us Am. Samoa H41 UF 3225 + Marian as H83 UF 5713 + + + Tuamo tus H82 UF 1744 + Tuamo tus H120 UF 1332 + + + Tuamo tus H232 UF 9270 + + + Line Ids. H175 UF 8372 + Hawaii H139 U F 8035 + Hawaii H230 UF 8035 + + + Line Ids. H176 UF 8379 + Tuamo tus H231 UF 9269 + 12 Calcinus hakahau Poupin & Mclaughlin, 1998 IWP Marque sas Marque sas Marqu esas H51 UF 5175 + + + Marqu esas H117 UF 5166 + + + 13 Calcinus hazletti Haig & McLaughlin, 1984 IWP Hawaii Hawaii Hawaii H119 UF 8349 + + + NW Hawaii an Ids. H295 UF 12157 + + + NW Hawaii an Ids. H302 UF 12158 +

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57 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 14 Calcinus hazletti aff. Northern Marianas Japan? / N Marian as N Marian as N Marian as H79 UF 5732 + + + N Marian as H90 UF 5728 + + + Wake Atoll H191 UF 8438 + 15 Calcinus imperialis Whitelegge, 1901 IWP E Australi a Easter Is. Easter Is. H38 UF 3646 + + + 16 Calcinus inconspicuus Morgan, 1991 IWP E Australi a New Caledo nia New Caledo nia H107 MNHN + + + 17 Calcinus isabellae Poupin, 1997 IWP Marian a s Hawaii; Pitcairn Marian as IP42 UF 732 + + + Tuamo tus IP20 UF 1758 + Line Ids. H174 UF 8371 + Wake Atoll H199 UF 8449 + + + Cook Ids. H228 UF 10354 + 18 Calcinus kurozumii Asakura & Tachikawa, 2000 IWP N Marian as (Pagan onl y) N Marian as (Pagan only) 19 Calcinus laevimanus Randall, 1840 IWP S Africa Hawaii; Tuamot us Hawaii H75 UF 3221 + Mascar ene Ids. H66 UF 5426 + + + Marian as Ids. IP28 UF 601 + Tuamo tus H76 UF 1720 + Hawaii H13 UF 3221 + Wake Atoll H198 UF 8445 + + +

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58 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq ? 20 Calcinus latens Randall, 1840 IWP Mozam bique; Yemen Tuamot us Marian as Ids. IP39 UF 460 + Mascar ene Ids. H316 UF 12564 Mascar ene Ids. H110 UF 5450 + + + Tuamo tus IP7 UF 1712 + Tuamo tus IP9 UF 1712 + Line Ids. H322 UF 10805 + Cook Ids. H297 UF 10339 + Wake Atoll H320 UF 8440 + Line Ids. H308 UF 10686 + Cook Ids. H298 UF 10339 + + + Wake Atoll H321 UF 8440 + 21 Calcinus latens aff. Hawaii IWP Hawaii Hawaii Hawaii H16 UF 3217 + + + Hawaii H109 UF 3217 + + + NW Hawaii an Ids. H299 UF 12066 + NW Hawaii an Ids. H300 UF 12066 + 22 Calcinus latens aff. Oman IWP Oman Oman Oman H81 UF 5428 + + + Oman H314 UF 5416 + + + 23 Calcinus laurentae Haig & McLaughlin, 1984 IWP Hawaii Hawaii Hawai i H39 UF 3625 + + + NW Hawaii an Ids. H291 UF 12059 + + + NW Hawaii an Ids. H303 UF 12278 +

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59 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 24 Calcinus lineapropodus Morgan & Forest, 1991 IWP Cocos Keeling Tuamot us Am. Samoa H84 UF3255 + Marian as Ids. IP19 UF 1322 + + + Ryukyu s H137 UF 6990 + + + Line Ids. H177 UF 8600 + 25 Calcinus minu tus Buitendijk, 1937 IWP Cocos Keeling Samoa Am. Samoa H86 UF3263 + + + Marian as IP32 UF 1321 + Philippi nes H140 UF 6511 + Ryukyu s H149 UF 6982 + + + 26 Calcinus morgani Rahayu & Forest, 1999 IWP S Africa Tuamot us Am. Samoa H27 UF 3236 + + + Society Ids. IP33 UF 1350 + Marian as Ids. IP43 UF 652 + + + Palau H130 UF 3992 + + + Ryukyu s H145 UF 6995 + + + Ryukyu s H147 UF 7237 + 27 Calcinus nitidus Heller, 1865 IWP Society Tuamot us Society Ids. H26 UF 1 334 + + + Society Ids. H121 UF 1334 + Society Ids. H129 UF 6886 + Tuamo tus IP2 UF 1347 + + + 28 Calcinus orchidae Poupin, 1997 IWP Marque sas Marque sas Marqu esas H50 UF 5177 + + + 29 Calcinus pascuensis Haig, 1974 IWP Easter Is. East er Is. Easter Is. H37 UF 3648 + + +

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60 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 30 Calcinus pulcher Forest, 1958 IW P Seyche lles New Caledo nia Palau H40 UF 3890 + + + Mascar ene Ids. H59 UF 5430 + + + Philippi nes H135 UF 8357 + + + Micron esia H193 UF 5396 + Philippi nes H194 UF 6531 + + + Papua New Guinea (Milne Bay) H148 UF 5553 + + + 31 Calcinus pulcher aff. Mascarenes IWP Mascar ene Ids. Mascar ene Ids. Mascar ene Ids. H309 UF 12741 + Mascar ene Ids. H144 UF 5430 + + + 32 Calcinus revi Poupin & McLaughlin, 1998 IWP Japan Tuamot us Tuamo tus 33 Calcinus rosaceus Heller, 1861 I WP Red Sea Gulf of Oman; Mauriti us Oman H63 UF 5427 + + + Oman H118b UF 5435 + + + Mascar ene Ids. H310 UF 12781 + Mascar ene Ids. H305 UF 12635 + + + 34 Calcinus seurati Forest, 1951 IWP Somali a Hawaii; Tuamot us Marian as IP36 UF 562 + + + Hawaii H14 UF3223 + + + 35 Calcinus sirius Morgan, 1991 IWP W Australi a E Australi a 36 Calcinus sirius aff. Poupin 1997 IWP W Australi a Austral Ids. Austral Ids. H93b MNHN Pg.6395 + + +

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61 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 37 Calcinus spicatus Forest, 1951 IWP E Australi a Pitcairn Is. New Caledo nia H106 MNHN + + + Cook Is. H229 UF 10337 + + + 38 Calcinus tropidomanus Lewinsohn, 1981 IWP Somali a Somali a 39 Calcinus vachoni Forest, 1958 IWP Mascar ene Ids. Easter N Marian as H88 UF 5742 + + + Ryukyu s H131 UF 6992 + Philippi nes H132 UF 6748 + + + 40 Calcinus vachoni aff. Cook Islands IWP Cook Ids. Cook Ids. Cook Ids. H47 UF 1377 + + + Cook Ids. H301 UF 11702 + 41 Calcinus vachoni aff. Runion IWP Mascar ene Ids. Mascar ene Ids. Mascar ene Ids. H311 UF 12634 + + + Mascar ene Ids. H318 UF 13011 + Mascar ene Ids. H319 UF 13011 + + + 42 Calcinus vanninii Gherardi & McLaughlin, 1994 IWP Mascar ene Ids. Mauriti us Mascar ene Ids. H80 UF 5425 + + + Mascar ene Ids. H87 UF 5412 + + + 43 Calcinus californiensis Bouvier, 1898 EP Baja Califor nia El Salvad or Baj a CA Sur H98 UF 8367 + + + Baja CA Sur H99 UF 15221 + + + 44 Calcinus explorator Boone, 1930 EP Gulf of CA Galapa gos Clipper ton Atoll H179 MNHN Pg.7617 + Clipper ton H204 MNHN + + +

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62 Table 2 1. Continued Reported geographic range Specim en info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? 45 Calcinus obscurus Stimpson, 1859 EP El Salvad or Ecuado r; Colom bia Panam a H105 UF 8359 + + + Panam a H111 UF 8359 + + + 46 Calcinus mclaughlinae Poupin, 2006 Clipper ton Atoll H178 MNHN Pg.7622 + + + 47 Calcinus tibicen Herbst, 1791 WA Belize Ubatub a Brazil Florida H102 UF 8363 + + + Florida H103 UF 8364 + Tobag o H124 UF 8358 + + + 48 Calcinus urabaensis Camp os & Lemaitre, 1994 WA Colom bia Colom bia 49 Calcinus verrilli Rathbun, 1901 WA Bermu da Bermu da Bermu da H138 UF 8365 + + + 50 Calcinus paradoxus Bouvier, 1922 EA Azores Azores 51 Calcinus talismani A. Milne Edwards & Bouvier, 1892 EA Cape Ver de Guinea Cape Verde H206 MNHN + + 52 Calcinus tubularis Linnaeus, 1767 EA Ascens ion Is.; Madeir a Lebano n Madeir a H91 UF 8361 + + + Madeir a H97 UF 8361 + + + Outgroups Ciliopagurus strigatus (Herbst, 1804) Marian as Ids. IP21 UF 1871 + + + Ciliopagurus tricolor Forest, 1995 Mascar ene Ids. H68 UF 5433 + + + Ciliopagurus galzini Poupin & Malay, 2009 Tuamo tus H32 UF 1742 + + +

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63 Table 2 1. Continued Reported geographic range Specimen info ES U cou nt Species Regi on W E Specim en proven ance Speci men no. Museum Catalog no. COI seq? 16S seq? H3 seq? Dardanus lagopodes (Forskal, 1775) Marian as Ids. IP15 UF 326 + + + Tuamo tus IP18 UF 1760 + + + Dardanus sanguinocarpus Degener, 1925 Hawaii H45 UF 3507 + + + Dardanus longior Asakura, 2006 Marqu esas H56 UF 3639 + + +

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64 Table 2 2. List of ESUs used in biogeographic analyses, and their geographic distributions relative to each other. Clade Distribution ESU pair I allopatric C. verrili C. tubularis II allopatric C. latens C. aff. latens Hawaii II allopatric C. latens C. aff. latens Oman III allopatric C. hazletti C. aff. hazletti N Marianas III allopatric C. minutus C. rosaceus III allopatric C. minutus C. nitidus III sympatric C. haigae C. minutus/C. rosaceus/C. nitidus III parapatric / slightly sympatric C. inconspicuus rest of clade III IV allopatric C. vachoni C. aff. vachoni Cooks IV allopatric C. vachoni C. aff. vachoni Mascarenes V allopatric C. spicatus C. pascuensis VI allopatric C. mclaughlinae C. obscurus VI parapatric / slightly sympatric C. californiensis C. mclaughlinae/C. obscurus VI allopatric C. tibicen C. talismani VI allopatric C. explorator C. tibicen/C. talismani VII sympatric C. gaimardii C. morgani VII allopatric C. elegans C. aff. elegans Hawaii VII allopatric C. imperialis C. vaninii VII parapatric / slightly sympatric C. isabellae C. imperialis/C. vaninii VIII sympatric (depth separated) C. laevimanus C. seurati IX allopatric C. pulcher C. aff. pulcher Mascarenes IX allopatric C. hakahau C. gouti IX allopatric C. laurentae C. hakahau/C. gouti IX sympatric C. lineapropodus rest of clade IX X sympatric (depth separated) C. albengai C. aff albengai deep X allopatric C. dapsiles C. albengai complex X allopatric C. argus C. aff. sirius X sympatric C. anani C. argus/C. sirius

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65 Figure 21. Bayesian phylograms constructed using (a)3 concatenated genes and (b)COI only. The valu es above the branches represent parsimony bootstraps / maximum likelihood bootstraps / Bayesian posterior probabilities, respectively. A

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66 Figure 21. Continued B

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6 7 Figure 22. Frequency distribution of K2P distances for intraspecific variation and interspec ific distances in Calcinus without (a) and with (b) the C. minutus complex.

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68 Figure 23. Distributions, color patterns, and COI phylogeny of Clade I Calcinus species. Colored symbols represent specimens available in the FLMNH collection; unfilled/black symbols represent records derived from the literature. Figure 24. Distributions, color patterns, and COI phylogeny of Clade II Calcinus species. Symbols follow Fig. 3.

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69 Figure 25. Distributions, color patterns, and COI phylogeny of Clade III Calcinu s species. Symbols follow Fig. 3. Figure 26. Distributions, color patterns, and COI phylogeny of Clade IV Calcinus species. Symbols follow Fig. 3.

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70 Figure 27. Distributions, color patterns, and COI phylogeny of Clades V and VI Calcinus species. Symbo ls follow Fig. 3. Figure 28. Distributions, color patterns, and COI phylogeny of Clade VIIa Calcinus species. Symbols follow Fig. 3.

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71 Figure 29. Distributions, color patterns, and COI phylogeny of Clade VIIb Calcinus species. Symbols follow Fig. 3. Figure 210. Distributions, color patterns, and COI phylogeny of Clade VIIc Calcinus species. Symbols follow Fig. 3.

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72 Figure 211. Distributions, color patterns, and COI phylogeny of Clade VIII Calcinus species. Symbols follow Fig. 3. Figure 212. Distributions, color patterns, and COI phylogeny of Clade IXa Calcinus species. Symbols follow Fig. 3.

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73 Figure 213. Distributions, color patterns, and COI phylogeny of Clade IXb Calcinus species. Symbols follow Fig. 3. Figure 214. Distributions, color patterns, and COI phylogeny of Clade X Calcinus species. Symbols follow Fig. 3.

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74 Figure 215. Spatial distribution of species richness. Contour lines represent number of species expected in area based on overlay of species ranges (Figs. 3 -14, see methods). Contours are drawn for 4, 10, 13, and 17 species in increasingly darker shades. Numbers represent number of documented species within the following regions and archipelagoes: Australia, Austral -Rapa, Caroline, Cocos -Christmas, Cook, Fiji, Gilbert, Haw aii, Indonesia, Japan, Line, Madagascar, Mariana, Marquesa, Marshall, Mascarene, Nauru, New Caledonia, New Guinea, Ogasawara, Palau, Philippine, Pitcairn, Ryukyu, Samoa, Society, Solomon, Taiwan, Tonga, Tuamotu, Vanuatu, and Wake. Figure 216. Approximat e distribution of IWP ESEs. Boundaries separating the ranges of sister taxa -and thus the location of ESEs -are drawn as lines. Numbers indicate how many ESEs occur across each of these zones.

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75 Figure 217. Age distribution (in million years, my) of Calcinus sister species pairs.

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76 CHAPTER 3 PHYLOGENETIC SYSTEMATICS OF CORAL DWELLING BARNACLES (BALANOMORPHA: PYRGOMATIDAE) Introduction The Balanoidea is a large superfamily of both free-living and symbiotic acorn barnacles. Newman and Ross (1976) recognized three balanoid families: the Balanidae, Archaeobalanidae, and Pyrgomatidae. The phylogenetic relationship of these three families are presently not well resolved. Recent molecular and morphological studies (Healy and Anderson 1990; Prez Losada et al. 2004; Prez Losada et al. 2008) have shown that balanids and archaeobalanids are mutually paraphyletic. The placement and status of the Pyrgomatidae is even less understood, because pyrgomatids were not included in these recent phylogenetic studies. Th is study focuses on the phylogenetic systematics within the Pyrgomatidae. The pyrgomatids are the coral dwelling barnacles; a morphologically and ecologically distinctive group whose members are obligatory symbionts of hard corals (mostly scleractinians as well as a few hydrocorals). Pyrgomatid cyprid larvae settle and metamorphose on the coral surface; however, the details of the settlement process are still unknown. Upon settlement, the barnacle exhibits rapid lateral growth (Utinomi 1943), followed later in life by mostly vertical growth at the margin of the basis. This vertical extension allows the barnacle to keep pace with the vertical growth of the coral host (fig. 3 -1). Coral growth over the orifice and wall margins is suppressed through an unknown, possibly chemically mediated process (Anderson 1992). As the barnacle and its host grow vertically, the barnacle develops a distinctive cup-shaped basis that may extend a considerable length into the coral. Aside from the cup-shaped basis, other features c haracterizing this family include a tendency towards the development of a

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77 flattened wall and towards the reduction and fusion of skeletal parts. While most balanoids have 6 wall plates, all extant pyrgomatids either have 4-plated walls or a single, fused w all plate (although one extinct pyrgomatid genus, Eoceratoconcha, had 6 wall plates). The fused, single wall plate is a character unique to the pyrgomatids. The opercular valves (i.e., scutum and tergum) are also fused in many of the pyrgomatid gene ra (fig 3 1). Fusion of skeletal elements has traditionally been one of the most important characters used in delineating pyrgomatid genera and species, and is considered an apomorphic character (e.g., Darwin 1854, Baluk and Radwanski 1967, Ross and Newman 1973) Taxonomy and Global Biogeography of the Pyrgomatids The Pyrgomatidae is currently divided into 3 subfamilies: the Ceratoconchinae, Pyrgomatinae, and Megatreminae; each of these groups is discussed in turn below. The subfamily Ceratoconchinae includes the extinct genus Eoceratoconcha and one extant genus, Ceratoconcha. Eoceratoconcha is known from the early Miocene to the Pliocene in the Caribbean; while Ceratoconcha first appeared in the late Oligocene in the Caribbean and attained its peak diversity and geographic range during the Miocene, with species in the tropical Atlantic, eastern Pacific (EP), and Paratethyan region (Ross and Newman 2002). Only 4 Ceratoconcha species are extant, and all are limited to the tropical West Atlantic (WA; Table 31). The ceratoconchines have never been reported in the present -day Indo West Pacific (IWP). Morphologically, ceratoconchines are distinguished by a 6plated (in Eoceratoconcha) or 4 -plated wall (in Ceratoconcha), typically balanoid and unfused opercular valves, and by the common occurrence of a prominent ridge or plate on the carinal segment of the tergum instead of depressor muscle crests (Table 3-2; Ross and Newman 1973).

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78 The Pyrgomatinae is the largest subfamily with 20 genera and 74 extant species (Table 31) all found exclusively in the IWP. The pyrgomatines show the highest morphological and ecological diversity among coral dwelling barnacles, ranging from 4 plated to single plated taxa, and from typically balanoid opercular valves and cirri to highly deriv ed morphologies (see Table 3-2; reviewed in Ross and Newman 1973; Anderson 1992). Within the pyrgomatines, two tribes were erected for the most distinctive members: Hoekiini (5 genera, 11 species) and Pyrgopsellini (1 genus, 2 species). The hoekines have a bandoned planktotrophy in favor of parasitism -they feed exclusively on coral tissue with enlarged biting mouthparts and have degenerate, non-functional cirri (Ross and Newman 1995). Hoekines are morphologically extremely a pomorphic, with highly modified opercular valves, an irregularly -shaped wall, and a partly membranous basis (e.g., the area of the basis directly below the wall is membranous). The pyrgopsellines, on the other hand, largely resemble other pyrgomatines but have an almost entirely membran ous basis. Previously thought to be sponge dwellers (Rosell 1975), pyrgopsellines are now known to live suspended in the tissue of scleractinian corals (Achituv and Simon-Blecher 2006). The other 14 pyrgomatine genera have been placed in the catchall trib e Pyrgomatini, and range from plesiomorphic taxa that largely resemble free living balanoids in skeletal characters (e.g., Cantellius ) to more apomorphic genera with fused and modified skeletal parts (e.g., Trevathana Nobia etc.; Table 32; fig. 3-1). Th e earliest fossil records for pyrgomatines date from the late Miocene of the IWP. There are also some Pleistocene and Holocene records; however, fossil records for pyrgomatines are relatively scarce.

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79 All reported fossil pyrgomatines have been classed into extant species (Asami and Yamaguchi 1997; Ross and Newman 2002). The Megatrematinae is comprised of the tribes Megatrematini and Pyrgominini; each with 2 genera. This small subfamily (6 extant species; Table 31) is distributed in the WA, IWP, and east At lantic (EA), and includes both shallow water genera on hermatypic corals as well as deeper water taxa on ahermatypic corals. The megatrematines have a fossil record extending to the Pliocene of the Mediterranean and the Pleistocene of the Caribbean, yet de spite their wide distribution have never attained high species diversity (Ross and Newman 2002). All megatrematines possess a fused wall and typically balanoid opercular valves. Members of the tribe Pyrgominini have a tall conical (instead of a flattened) wall, while members of the Megatrematini are distinct in having a trapezoidal beaked tergum (Table 3-2; Ross and Newman 1973; Newman and Ross 1976; Ross and Pitombo 2002). In summary, the oldest records for the Pyrgomatidae date to the late Oligocene (New man and Ladd 1974; Ross and Newman 2002). The family has had 2 areas of radiation: one centered in the Caribbean that attained its peak diversity in the Miocene (e.g., the Ceratoconchinae), and a much larger, extant center of diversification in the IWP (e. g., the Pyrgomatinae). The radiation of pyrgomatines appears to have paralleled the radiation of modern scleractinian corals in the tropical IWP; on the other hand, the decline of the ceratoconchines was roughly concomitant with the decrease in numbers of West Atlantic (WA) and East Pacific (EP) corals and the Messinian salinity crisis in the Mediterranean (Veron 1995; Ross and Newman 2002; Ross and Pitombo 2002). In

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80 contrast, the megatrematines have always had a scattered distribution and relatively few sp ecies. The three subfamilies as well as the tribes, genera, and species constituting them are mostly defined on the basis of only one to a few morphological characters. Many of these characters relate to skeletal morphology, particularly the fusion and form of the wall and opercular valves. A fused and flattened wall, and fused and modified opercular valves, are considered apomorphic; while 6plated or 4plated conical walls, and unfused and typically balanoid opercular valves, are considered plesiomor phic. Given the paucity of characters utilized and diversity of opinion regarding the origins of coral barnacles (see below), it is hardly surprising that the systematics of the pyrgomatids continues to be highly unstable. The numerous new pyrgomatids stil l being described (22 new species since 2000; table 3-1) is further evidence of our imperfect knowledge of the group. Phylogenetic Hypotheses Regarding the Pyrgomatids Earlier barnacle taxonomists essentially espoused non-cladistic views on pyrgomatid evol ution, envisioning that extant basal pyrgomatid taxa evolved into other, more advanced forms. For instance, it has been generally accepted that pyrgomatids evolved from other, less morphologically specialized coral associated balanoids. Several nonpyr gomatid balanoids live obligately on corals (e.g., Megabalanus ajax and M. stultus (Megabalanidae) on the stinging hydrocoral Millepora Ross 1999; Tetraclita sp. (Tetraclitidae) on the blue coral Heliopora Newman and Ladd 1974; Hexacreusia spp. (Archaeob alanidae) on Porites Pitombo and Ross 2002; Armatobalanus spp. (Archaeobalanidae) on various scleractinians, Pilsbry 1913, Anderson 1992). Of these, the archaeobalanid genus Armatobalanus is thought to be closest to the pyrgomatids

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81 (Hiro 1938; Ross and Newman 1973; Healy and Anderson 1990; Anderson 1992; Ross and Newman 2000; Simon-Blecher et al. 2007). Armatobalanus is a relatively small taxon (12 species), and not all members of this genus are coral associated. Characters used to diagnose this genus incl ude 6 wall plates and the presence of teeth on the third or fourth pair of cirri (Pilsbry 1913). Note, however, that at least one Armatobalanus species lacks cirral armature (i.e., A. oryza; Broch 1931). Moreover, teeth on cirri III or IV are not exclusive to Armatobalanus (other examples: the mainly sponge-dwelling family Acastinae, Kolbasov 1993; the gorgonian or antipatharian associated archaeobalanid Conopea cymbiformis Pilsbry 1913). In their review of the Pyrgomatidae, Ross and Newman (1973) suggest ed that the 3 pyrgomatid subfamilies could represent balanoid lineages that independently colonized corals. They based their argument on the subfamilies distributions and also highlighted the plesiomorphic appearance of Ceratoconcha relative to the pyrgom atines. In contrast, monophyly of the family was supported by Healy and Anderson (1990) and Anderson (1992) on the basis of sperm structure and skeletal and cirral morphology, respectively. Ross and Newman (2002) likewise espoused the hypothesis of monophy ly, and proposed that the pyrgomatids may have arisen in the western Tethys during the Palaeogene, thus implying that pyrgomatines and ceratoconchines may both be Tethyan relicts. It has also been suggested that the morphologically plesiomorphic genus Cant ellius represents the ancestral stock from which other pyrgomatine genera evolved, and that several extant Cantellius species independently gave rise to other pyrgomatine genera (e.g., Ross and Newman 1973; Anderson 1992). While the idea of

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82 extant specie s giving rise to other Recent lineages no longer conforms to present day concepts of evolutionary mechanisms, the prevailing belief is that pyrgomatids are derived from a morphologically generalized, 6 -plated ancestor, which evolved into a 4plated form, and then into more morphologically derived forms (e.g., Healy and Anderson 1990, Anderson 1992). These and other evolutionary hypotheses were recently reviewed and tested by Simon -Blecher et al. (2007). Using 3 genes (16S, 12S, and 18S), Simon-Blecher et al (2007) recovered a non -monophyletic Pyrgomatidae: one genus ( Wanella ) fell outside of the pyrgomatid clade; while the presumed closest outgroup ( Armatobalanus ) fell within the pyrgomatid clade and sister to the morphologically plesiomorphic genus Cantell ius However, these relationships were only weakly supported, and the authors were not able to reject the alternative hypothesis of a monophyletic Pyrgomatidae. Overall, the phylogeny of the family is still not well resolved, and this paper attempts to she d greater light on pyrgomatid evolution. Materials and Methods Taxon Selection and Identification Specimens were collected by scuba diving in reef sites spanning the IWP, as well as at locations in the WA and EA. Table 33 lists the samples that were sequenced for this study. I included 64 specimens, including ~16 ingroup genera (80% of all genera), as well as 10 outgroup specimens. I chose outgroups to represent a broad range of both free-living and symbiotic balanoids. The majority of the specimens are de posited in the Invertebrate Zoology collections of the Florida Museum of Natural History, University of Florida (UF). Additional specimens or tissue samples were borrowed from other institutions, and further supplemented by sequences accessed from GenBank (Table 3 -

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83 3). Three samples were identified as Armatobalanus with high confidence by barnacle experts Yair Achituv (Bar -Ilan University, Israel), Gregory Kolbasov (Moscow State University, Russia), and Andrew Hosie (Western Australian Museum, Australia; Achituv pers. comm.; Hosie pers. comm.), while the Pyrgopsella youngi sample was identified by Yair Achituv. I identified most pyrgomatid specimens using the primary taxonomic literature. Because the species level taxonomy of coral -dwelling barnacles is probl ematic, many samples could not be identified to the species level with a high degree of confidence. Thus for the purposes of this study I conservatively only identified specimens to the genus level, and instead confined the analyses to reciprocally monophy letic clades with strong branch support (strong support is herein arbitrarily set at bootstrap values > 60% and/or posterior probabilities > 90%). Genetic analysis started by first sequencing a wide range of pyrgomatids (>350 specimens) for the mitochondrial gene cytochrome oxidase I or COI. Based on these initial results, I selected representatives of each genus to cover as much intrageneric genetic diversity as possible. These representative taxa were sequenced for 4 additional genes (mitochondrial genes 16S ribosomal DNA and 12S ribosomal DNA; nuclear genes 18S ribosomal DNA and Histone 3 or H3; see Table 33). Molecular Methods Two different protocols were employed in two labs for extracting and amplifying DNA. At the Smithsonian Institutions Laboratori es of Analytical Biology (LAB), barnacle tissue was first digested overnight in 150 ul M2 buffer and 150 ul M1 + proteinase K buffer at 56.5C and 50 rpms (revolutions per minute). Extraction was done using an automated phenol -chloroform extraction (Autogen AutoGenprep 965 Automated DNA Isolation System). PCRs were performed on an ABI 2720 Thermal Cycler or MJ

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84 Research PTC -225 Peltier Cycler. The PCR profile used for amplifying COI was as follows: an initial denaturation at 95C for 5 minutes, followed by 35 cycles of denaturation at 95C for 30 sec, annealing at 48C for 30 seconds (sec), elongation at 72C for 45 sec, and a terminal elongation at 72C for 5 mins. PCR products were cleaned using ExosapIT (from USB). Inhouse sequencing was done in a 96well format using ABI BigDyeTerminator cycle sequencing reactions. The reactions were cleaned using Sephadex G 50 (Sigma Aldrich), and run on an ABI -3730 XL DNA analyzer. All PCR products were sequenced along both directions. Only COI was sequenced at the LAB. At UF, DNA was extracted from barnacle muscle tissue using DNAzol and proteinase K following the protocol given in Meyer (2003). DNA extracts were cleaned using QIAGEN cleanup kits. Sequence data was collected for three mitochondrial gene fragments (COI, 16S, and 12S) and two nuclear markers (18S and H3). Primers and PCR protocols followed Prez -Losada et al. (2004; for H3 and 18S), Meyer (2003; for COI and 16S), and Simon-Blecher et al. (2007; for 12S). PCR products were cleaned using the exo-sap cleanup protocol and sequenced at the high-throughput sequencing facility of the University of Floridas Interdisciplinary Center for Biotechnology Research (ICBR) in a 96well format using BigDyeTerminator cycle sequencing reactions, employing an ABI 3730 XL for electrophoresis. All PCR products were sequenced along both directions. Phylogenetic Analyses Chromatograms were checked and manually edited using the software Geneious Pro 4.9.2 (Drummond et al. 2009). Sequence alignment was done either: (a) completely m anually for all gene regions, using Se Al v2.0a11 (Rambaut,

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85 http://tree.bio.ed.ac.uk/software/seal/ ); or (b) using MAFFT v.6.717 (Katoh et al. 2002) for the 3 non -coding gene fragments (16S, 18S, and 12S) and manually for the 2 coding genes (COI, H3). In the MAFFT alignments, I used the L-INS -i search strategy and the following parameters: scoring matrix for nucleotide sequences=1PAM/ =2; gap opening penalty=1.53; offset value=0.1. Phylogenies resulti ng from both manually aligned and software aligned sequences were compared for topological congruence. Since no incongruences were found, and automated alignment is more objective, the MAFFT analysis was used for all downstream analyses. In addition, highe r branch support values were obtained from the MAFFT aligned dataset (data not shown) than from the manual alignment. In all of the sequence analyses, all sites were weighted equally, characters were unordered, and gaps were treated as missing data. Both m aximum likelihood (ML) and Bayesian approaches were used for phylogenetic reconstruction. First I determined the simplest model of evolution that best fit the 5 -gene dataset using the Akaike Information Criterion (AIC) as implemented by the program Modelte st 3.6 (for ML analyses; Posada & Crandall 1998). Maximum likelihood analyses were implemented using PAUP* ver. 4.0b10 (Swofford 2002) and RAxML 7.0.4 (Stamatakis 2006). In the PAUP* analyses, heuristic searches started with random addition of taxa replicated 10 times using the tree-bisection-reconnection (TBR) branch-swapping algorithm. For the RAxML analyses, I performed 1,000 rapid bootstrap inferences followed by a thorough ML tree search. A GTRGAMMA model and a random starting tree were utilized. Bayes ian analyses were performed using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) using GTR+I+GAMMA models and flat

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86 priors. For the 5gene concatenated dataset, the dataset was partitioned prior to analysis (the partitions corresp onded to the 5 sequenced gene regions), parameters and models of evolution in each partition were unlinked. I ran 2 independent chains for 1 million generations each; each chain was sampled every 100 generations. All runs reached stationarity before 100,00 0 generations. The initial 25% of the trees was discarded as the burn-in phase, and posterior probabilities were calculated based on the remaining 75% of the trees. To determine the appropriateness of concatenating the 5 gene regions into a single analysi s, I visually compared Bayesian and ML tree topologies from independent searches for each of the 5 gene regions. No strongly supported incongruences were found, and all downstream analyses were performed on the concatenated dataset. Topological Tests The h ighest -scoring ML and Bayesian trees showed uncertainties in the phylogenetic placement of the genus Wanella and in the monophyly of the archaeobalanid Armatobalanus (see Results section). In order to further explore these results, I investigated whether the data were consistent with two alternative phylogenetic hypotheses: (1) that Wanella is monophyletic with the rest of the Pyrgomatidae (hypothesis pyrgomatidae_monophyletic); and (2) that Armatobalanus is monophyletic (hypothesis armato_monophyletic ). To evaluate these alternative hypotheses, I investigated whether the 5 -gene concatenated tree with the highest ML score (obtained using PAUP*) was significantly better than topologies constrained to conform with the pyrgomatidae_monophyletic and armatobalanus_monophyletic hypotheses. This comparison was done using the non -parametric ShimodairaHasegawa (S -H) test (Shimodaira and Hasegawa 1999; Goldman et al. 2002). The S -H

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87 test was implemented in PAUP* by comparing the best -scoring ML tree to an ML t ree where pyrgomatids were constrained to be monophyletic, as well as to additional best scoring trees obtained using the maximum parsimony algorithm. The number of nonparametric bootstrap replicates was set to 1,000 and a RELL approximation was used. I a lso computed the percentage of the post -stationarity Bayesian trees (from the 5gene concatenated analysis) that conformed with each of the two topological constraints tested. This percentage, divided by the total number of post -stationarity trees, gives t he posterior probability of the hypothesis being tested (following http://insects.oeb.harvard.edu/farrell_lab/techniques/pa_hypothesis.html ). Character Tracing and Mo rphological Evolution I analyzed morphological evolution in pyrgomatids by concentrating on phenotypic features related to fusion and simplification of shell structures, as well characters related to the interactions between the barnacle and its coral host The following seven characters were examined: (1) fusion of opercular valves, (2) number of wall plates, (3) height of wall plates, (4) calcification of basis, (5) presence of teeth on the anterior margin of the 3rd pair of cirri, (6) amount of coral overgrowth on the wall, and (7) mechanical vs. nonmechanical prevention of coral overgrowth. Representatives of the different character states are illustrated in fig. 3-2. While most of the phenotypic characters are straightforward, the two characters related to control of coral overgrowth require some explanation. In most pyrgomatid species, the coral overgrows the barnacle wall all the way to the aperture. Coral overgrowth is typically a thin layer coral tissue (t) as well as bits of calcareous coral materi al (c). However, in some individuals the area surrounding the barnacle aperture is free of calcareous material, or free of both

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88 coral tissue and calcareous material. There are also pyrgomatid species whose walls are entirely overgrown by coral tissue but d o not exhibit any deposition of calcareous coral material (see fig. 3-2). To prevent the host from overgrowing the aperture, barnacles can mechanically scrape off the coral using their opercular valves and cirri; alternatively, control of overgrowth can b e by non-mechanical means. The latter is thought to be common in pyrgomatids (Anderson 1992). To determine the mechanism of overgrowth control, I examined the edge of the barnacle orifice microscopically. When the edge of the coral growth on the aperture w as rough, I interpreted that to indicate that coral overgrowth is prevented by the barnacle using mechanical means. When the edge was smooth, or if calcareous deposition by the coral stopped some distance away from the orifice, I interpreted that to indicate that control of coral overgrowth is probably nonmechanical. Potential non -mechanical controls may involve chemically mediated communication or control by the barnacles on the coral margins, as suggested by Anderson (1992), but this remains to be tested. Specimens were removed from the host coral using a hammer and chisel, pliers, or a blunt dental probe, then dissected using fine tungsten needles under a stereomicroscope. All shell characters were scored under a stereomicroscope. To examine cirral armature, cirrus III was excised, mounted on a slide, and examined under a compound microscope. Whenever possible, I examined both the sequenced individual as well as other conspecific barnacles co occurring on the same coral colony. When specimens were unavai lable, observations were supplemented by reports from the published literature (e.g., Pilsbry 1916, Achituv and Simon -Blecher 2006).

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89 The character history was traced onto the pyrgomatid phylogeny using the software package Mesquite 2.72 (Maddison and Madd ison 2009). The parsimony model used to reconstruct the number of wall plates was a weighted step matrix where reversal of wall fusion (i.e., from 1 plate back to 4 plates, and from 4 plates to 6 plates) cost one more step than wall plate fusion. With the exception of wall plates, all other characters were specified as unordered, and character gains and losses were assumed to have equal weights. For wall plate fusion, I decided to apply a step matrix model of evolution because fusion of wall plates is well established as a prevailing and recurring theme in barnacle evolution (e.g., Newman 1987); for instance the Mio-/Pliocene genus Eoceratoconcha was 6 plated (Zullo and Portell in litt.). The phylogram used for tracing characters was obtained from the RaxML analysis. Nodes with <80% bootstrap support and also <95% Bayesian posterior probability were collapsed. To determine whether a phenotypic trait is phylogenetically structured, I calculated the parsimony score (PS; Fitch 1971), and association index (AI, W ang et al. 2001) using the software BaTS v1.0 (Parker et al. 2008). I evaluated the null hypothesis that the trait shows no phylogenetic structure. BaTS explicitly accounts for phylogenetic uncertainty by calculating and averaging phylogeny -trait associati on statistics across a posterior sample of trees (PST) generated by Bayesian Markov chain Monte Carlo (MCMC) programs. For each trait analyzed, a new set of 5 -gene concatenated Bayesian phylogenies were used as input trees; each dataset was first trimmed t o exclude specimens with unknown/unrecorded character states. One hundred replicates of state randomizations were used to calculate the null distributions of the statistics.

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90 Results Sequence Characteristics The length of the gene fragments, number of pars imony -informative sites, number of invariant sites, and the best likelihood models are presented for each gene fragment in Table 34. Parsimony -informative nucleotide sites totaled 173 base pairs (bp) for nuclear genes (H3 and 18S) and 486 bp for mitochondrial genes (COI, 16S, and 12S). Phylogenetic Relationships within the Pyrgomatidae and Congruence of Gene Trees Comparison of phylogenies for each of the 5 sequenced markers showed that individual gene trees were largely congruent (Figs. 3-3 to 3 7). The gene trees were also essentially congruent with the combined mitochondrial only and nuclear only topologies (Fig. 3 -8 to 39), and with analyses of the 5 concatenated markers (Fig. 310). However, some incongruences were noted, notably with respect to the placement of the fire coral associated genus Wanella and of the archaeobalanid outgroup Armatobalanus Instances of topological incongruences are listed in Table 35. The 18S gene tree was responsible for most of the deviations. Note, however, that while high posterior probability values in the 18S tree (Fig. 36) indicate strong topological support, in actuality only 4% of the 18S data is informative (Table 3-4). All analyses identified several well -supported clades (well supported herein defined as > 80% BS and/or > 99% PP; Fig. 3 -10). Most pyrgomatine genera including the most apomorphic groups clustered in Clade I. Adna and Ceratoconcha (representing the subfamilies Megatrematinae and Ceratoconchinae, respectively), were consistently recovered as sis ter taxa (Clade II). Clade III was comprised of the plesiomorphic genus Cantellius and in most cases 2 specimens of the archaeobalanid

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91 outgroup Armatobalanus Lastly, the fire coral associated genus Wanella did not usually cluster with other pyrgomatids in most of the topologies, and was designated Clade IV. Relationships between the different clades were not well resolved. Clades II IV each contained only one or two genera; whereas Clade I contained nine genera. Many pyrgomatid genera and tribes were re covered as reciprocally monophyletic units with high branch support, but 6 were not: Pyrgopsella Hoekiini, Neotrevathana, Trevathana, Galkinia and Hiroa ). Within clade I, the phylogeny recovered a large subclade (herein designated the Trevathana sensu la to subclade) comprised of Trevathana, Neotrevathana Pyrgopsella and the Hoekiini. The genera that make up this subclade were not recovered as reciprocally monophyletic units. Placement of Outgroup Taxa Four different sequences identified as Armatobalanus were included in the analyses. None of the gene trees nor any of the concatenated analyses, recovered a well -supported monophyletic Armatobalanus clade (Figs. 33 to 3-10; Table 3 -5). Armatobalanus allium (KACb154) and Armatobalanus sp. (UF 11887) w ere often recovered as sister to the pyrgomatid genus Cantellius ; A. allium (TAU Ar27835) was resolved as sister to the balanid genus Megabalanus in most trees including the 5 gene concatenated analyses; while Armatobalanus sp. (KACb163) also came out with the outgroup specimens yet did not group with the previous specimen. There are several possible reasons for this unexpected pattern, including DNA contamination, specimen misidentification, or nonmonophyly of Armatobalanus I consider contamination t o be unlikely because none of the Armatobalanus sequences are identical (or even very close to) the other barnacle sequences. Specimen misidentification is possible, particularly since I did not have an opportunity to compare putative Armatobalanus

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92 specime ns side by side. Polyphyly of Armatobalanus is also very possible, particularly since (to my knowledge) there is no publication that comprehensively reviews the genus and compares it to morphologically similar groups, nor am I aware of any synapomorphies t hat unite the genus. A phylogenetic reappraisal of Armatobalanus is beyond the scope of this study. Balanus glandula (Balanoidea: Balanidae) did not group with Megabalanus the other balanid genus represented in the phylogeny. Instead B. glandula was cons istently recovered as sister to the archaeobalanid Semibalanus balanoides This agrees with previous phylogenetic studies suggesting that the taxonomic division between the families Balanidae and Archaeobalanidae may not be valid (Healy and Anderson 1990; Prez Losada et al. 2004; Prez Losada et al. 2008). Topological Tests I used topological tests to investigate whether the Pyrgomatidae is monophyletic and also whether the outgroup Armatobalanus is monophyletic. While the S -H test rejected the 2 most parsimonious trees (MPTs), it was unable to reject the hypothesis of a monophyletic Pyrgomatidae, nor of a monophyletic Armatobalanus (Table 36). In contrast to the S -H test results, I found that of the 15,000 post -stationarity trees obtained from Bayesian analysis, not one of the trees conformed to the hypothesis of a monophyletic Pyrgomatidae. Neither did any of the trees conform to the hypothesis of a monophyletic Armatobalanus Character Tracing and Phylogeny -Trait Correlation Figures 3 11 to 317 show the parsimony reconstructions of phenotypic traits on the pyrgomatid phylogeny. Assuming symmetrical rates of character gains and losses, fusion of the opercular valves is estimated to have occurred approximately 45 times. All

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93 instances of valve fusion w ere recovered in Clade I, three in the Trevathana sensu lato subclade alone. There is at least one instance of reversal from fused opercular valves to unfused valves (i.e., Hiroa ; fig. 3 11). Fusion of 4 wall plates to a single plate is estimated to have evolved twice, once in Clade I and another time in Clade IV ( Wanella ). The ancestral state of Clade I was reconstructed as single-plated. Two instances of reversals from 1 plate to 4 wall plates were recovered. In addition, there is one instance of fusion from 6 plates to four plates, in Clade III (fig. 312). The height of the wall (e.g., from high conical to flat) shows multiple state changes within clades, genera, and even single species (fig. 3 13). Nearly all pyrgomatids possess a wholly calcareous ba sis; however, a partly to fully -membranous basis appear to have evolved 34 times, all in Clade I (fig. 3 14). Figure 315 plots the presence or absence of teeth on the anterior margin of cirrus III. All members of Clades II and III had cirral teeth. Thi s character was absent from all other clades. However, cirrus III teeth were present all specimens identified as Armatobalanus spp., including both those included in Clade III and those that fell outside Pyrgomatidae sensu stricto Coral overgrowth in mos t of the specimens examined consisted of both coral tissue and calcareous deposition on the barnacle walls. However, in ~57 instances coral overgrowth was limited to coral tissue only (i.e., without coral skeletal deposition on the barnacle wall), and in several other instances calcareous and/or tissue deposition stopped before reaching the barnacle aperture. While this character is

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94 variable within genera and even species, all instances of coral suppression occurred in Clades I and II (fig. 3-16). Figure 3 17 plots the incidence of apparent mechanical and non mechanical control of coral overgrowth over the barnacle aperture. Clades II and IV showed evidence for mechanical erosion of coral overgrowth, while in all other pyrgomatids control of coral overgrow th was apparently nonmechanical. The BaTS analyses rejected the null hypothesis of no phylogenetic structure for all of the characters investigated (Table 3 7). Discussion Systematics The phylogeny recovered 3 major pyrgomatid clades (clades I -III) that together clearly form a monophyletic unit, i.e., the Pyrgomatidae sensu stricto. However, the genus Wanella (Clade IV) may or may not be the sister taxon of Clades I -III. The new phylogenetic results contradict classical hypotheses of pyrgomatid taxonomy in several fundamental ways. First, the traditional pyrgomatid subfamilies (the Ceratoconchinae, Pyrgomatinae, and Megatrematinae) were not resolved as the basal clades of the family. Second, the division of the Pyrgomatinae into 3 tribes (Pyrgomatini, Pyr gopsellini, and Hoekiini) was likewise not supported by the phylogenetic evidence. The hoekiines and pyrgopsellines, although highly apomorphic morphologically and ecologically, are nested within the large pyrgomatine genus Trevathana. Third, the hydrocoral associate Wanella was not resolved as being part of the pyrgomatid ingroup, and its affinities to the main clade of pyrgomatids are unstable. Fourth, Armatobalanus was not recovered as monophyletic, and although the specimen used in Simon-Blecher et al. (2007) was resolved as an outgroup to the Pyrgomatidae (as expected from the

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95 traditional classification), two other specimens were resolved as sister to Cantellius and well within the main pyrgomatid clade. Some of the shallower portions of the tree corres pond with earlier hypotheses. The tree recovers the Savignium group of Ross and Newman (1973), with the exception of Wanella which was included in the Savignium group by Ross and Newman (1973). While many nominal genera were well resolved phylogenetical ly, 6 were not (i.e., Pyrgopsella, Hoekiini Neotrevathana Trevathana Galkinia and Hiroa ). All the unresolved genera were in Clade I. The following groupings were recovered within Clade I: ( Nobia), (Pyrgoma ,( Galkinia Hiroa ), Darwiniella ), and ( Savignium Trevathana sensu lato). The topology largely agrees with the results of Simon-Blecher et al. (2007), except in the present study Hiroa and Galkinia were strongly resolved as sister taxa ( Galkinia erroneously called Creusia by Simon -Blecher et al. 2007 was resolved as sister to a Hiroa -Darwiniella clade in Simon-Blecher et al. 2007). Within the Trevathana sensu lato subclade, Neotrevathana was resolved as diphyletic (in contrast to the findings of Simon -Blecher et al. 2007), and the tribes Pyrgopsellini and Hoekiini were both nested within Trevathana sensu lato (in agreement with SimonBlecher et al. 2007). Clade II unites the only East Atlantic (EA) genus, Adna (Megatrematinae), with Ceratoconcha (Ceratoconchinae), one of 2 extant WA genera. In the future it will be interesting to sequence the missing megatrematine groups (i.e., the WA/IWP Megatrema and the IWP Memagreta and Pyrgomini ) to test whether the megatrematines truly form a monophyletic clade spanning the WA, EA, and IWP. Such a result would i mply that diversification occurred across these 3 biogeographic regions.

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96 An alternative hypothesis might be that the two WA genera, Ceratoconcha and Megatrema, are sister -taxa, implying that diversification occurred within the WA. Clade III unites Cantellius with 2 of the four sequenced Armatobalanus outgroups. These results have two implications (assuming samples had not been mis -identified) : (1) Armatobalanus as presently defined may be polyphyletic; and (2) the Pyrgomatidae needs to be re -circumscribed. There are also indications that Clade III may be sister to Clade II, and although branch support for this relationship is low, a new morphological character supports such a grouping (see next section). Overall, the results show that the systematic relationships within the Pyrgomatidae need to be thoroughly re examined. Character State Evolution The ancestral character state reconstructions suggest that fusion of shell structures (wall and opercular valves) and reduction in basis calcification evolved multip le times in pyrgomatids, with several instances of reversals from more fused to less fused character states. However, these ancestral state reconstructions should be interpreted with some caution, because the model of evolution used in tracing characters may not be biologically realistic. The state reconstructions (excepting wall plate fusion) are based on the assumption that character gains and losses are equally probable. While this might not be a realistic model, given the absence of fossil information on character states of ancestral pyrgomatids, any model of morphological evolution applied to the barnacles would be mostly conjectural. For the number of wall plates, a step matrix model of evolution was deemed appropriate since there is abundant fossil ev idence indicating that wall fusion is a recurrent theme in barnacle evolution (e.g., Newman 1987).

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97 Even though ancestral character state reconstructions are equivocal, what emerges clearly from data is that changes in shell characters are phylogenetically structured. A tendency towards fusion and reduction of skeletal elements particularly characterizes Clade I pyrgomatids. All known instances of opercular valve fusion and reduction in basis calcification occurred in Clade I. Passageways or membranous zones in the pyrgomatid basis have been proposed to function in barnacle -coral communication (Ross and Newman 1973; Ross and Newman 2000) or perhaps in the direct uptake of dissolved nutrients from the host (Anderson 1992; Ross and Newman 1995). Alternatively, an incompletely calcified basis could be less energetically costly, and may permit more rapid growth (Ross and Newman 1995). A similar reduction in shell calcification has been noted in sponge barnacles (Acastinae), and in this group splits or windows be tween wall plates were also suggested to be involved in barnaclehost communication (Kolbasov 1993). A single wall plate seems to have arisen independently in members of Clade I and in Wanella (Clade IV). Another instance of wall fusion was documented in Clade III. Surprisingly, Clade III contains both 4plated and 6-plated morphs. Previously, the only 6 -plated pyrgomatid known was Eoceratoconcha, from the Miocene to Pliocene in the WA. The fact that Cantellius is sister to another 6 -plated coral -dwelling barnacle (the archaeobalanid Armatobalanus ) implies that the development of 4 wall plates may have occurred more than once. It has been suggested that fusion of wall plates, particularly the development of a singleplated wall, may be an adaptation allow ing pyrgomatids to better withstand the lateral pressure exerted by the coral as it grows,

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98 and that the single plated wall in Wanella and in Clade I pyrgomatids is a result of convergent evolution (SimonBlecher et al. 2007). Characters related to the control of coral overgrowth are also phylogenetically structured. One surprising result in this study was that all specimens from Clades II and III that were examined bore teeth on the anterior margins of the 3rd pair of cirri, while none of the pyrgomatids in the other clades had cirral armature. Cirral teeth are probably used to rasp away coral overgrowing the barnacle aperture (Anderson 1992). Cirral armature was previously believed to be a feature characteristic of Armatobalanus ; its presence in Cantellius was considered by Anderson (1992) to be vestigial and limited to only 2 species. Recent taxonomic papers have often included cirral characters, and yet cirral teeth have not been reported in newly described species of Clade III Cantellius (e.g., Achituv and Hoeksema 2003; Achituv et al. 2009). To my knowledge, this is the first report of cirral armature in the Clade II genera Adna and Ceratoconcha. Interestingly, Clades II and III appear to be sister groups in most phylogenetic reconstructions; however, branch support for this relationship was low. The fact that members of both clades bear teeth on cirrus 3 suggests that cirral armature may be a synapomorphy uniting these two clades. Note, however, that cirral armature is not limited to clades II and III only: among the outgroups there are 2 additional Armatobalanus specimens that also bore teeth on cirrus 3; and the acastine specimen also possessed teeth but on the 4th pair of cirri. Coral overgrowth in most of the pyrgomatids examined consisted of a l ayer of coral tissue and calcareous material extending from the perimeter of the barnacle wall to the edge of the aperture. However, there were also instances where calcareous

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99 deposition stopped some distance away from the barnacle aperture, such that the aperture rim was covered only by coral tissue, or was completely bare of coral derived material. I also observed that in some pyrgomatids the walls were only covered by coral tissue without any calcareous deposition. These variations in coral overgrowth seem to represent different ways by which a pyrgomatid suppresses coral deposition on the wall and aperture. All instances of coral overgrowth suppression clustered in Clades I and II, raising the possibility that suppression of overgrowth m ay have evolved i ndependently in Clades II and III. Incidences of mechanical control of coral overgrowth clustered in Clades II and IV. This is evidenced by abrasions on the portion of the coral skeleton overgrowing the rim of the aperture. Curiously, this character does n ot completely mirror the results on cirral armature: while clade III species bear cirral teeth, they apparently exert nonmechanical control on coral overgrowth. Cirral teeth in Cantellius (Clade III) may indeed be a vestigial non-functional character, as suggested by Anderson 1992. Conversely, Clade IV ( Wanella ) shows evidence of mechanical control, and yet lacks cirral teeth. It is possible that Wanella mechanically abrades coral overgrowth using the occludent margins of the opercular valves instead of ci rral teeth. Only members of Clade II exhibit both cirral armature and mechanical control of coral overgrowth. Evolution of Parasitism The only known pyrgomatid euparasites were resolved as members of Clade I, specifically within the Trevathana sensu lato subclade. The tribe Hoekiini are the coral eating barnacles, a bizarre and highly host -specific taxon with reduced, nonfunctional cirral nets and large biting mouthparts that are used to feed on the coral tissue that completely covers its wall and orifi ce (Ross and Newman 1995). Both the soft parts and

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100 shell characters of hoekiines are extremely apomorphic, hence their placement in the midst of Trevathana sensu lato was one of the more surprising results from phylogenetic analyses (e.g., Simon-Blecher et al. 2007 and this study). The present study also shows that despite their extreme morphological apomorphy and ecological divergence, hoekiines are not a long branch on the phylogeny, suggesting that the group may actually have evolved quite recently from a Trevathana -like ancestor. An alternative explanation for the relatively short hoekine branch may be that the lineage is relatively older, but is somehow evolving at a slower rate than other pyrgomatids; although an evolutionary transition to parasitism i s more commonly associated with an increase, rather than a decrease, in the tempo of molecular evolution (e.g., Dowton and Austin 1995). Conclusions and Recommendations This study recovered 3 major clades within the Pyrgomatidae sensu stricto: Clade I, co ntaining most genera from the subfamily Pyrgomatinae (excepting Wanella and Cantellius ) is exclusively IWP, tends towards fusion and reduction of skeletal elements, and controls coral overgrowth through nonmechanical means. This group includes the only kn own pyrgomatid euparasite, the tribe Hoekiini, which may be a recently evolved lineage within the large Trevathana sensu lato subclade. Clade II is currently known from the WA and EA, possesses unfused opercular valves, 4-plated or 1-plated walls, and uses mechanical means to control coral overgrowth. Clade III is exclusively IWP and possesses unfused opercular valves, 6plated or 4-plated walls, and appears to exert nonmechanical control on the growth of its coral host. Among the pyrgomatids, only Clad es II and III bear teeth on the anterior margins of cirrus 3; although this character has also been noted in other, nonpyrgomatid balanoids.

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101 So what is a pyrgomatid? All pyrgomatids are coral dwellers, and yet not all balanoids living on corals belong to the family Pyrgomatidae (e.g., non-ingroup Armatobalanus spp., Hexacreusia spp., Megabalanus spp., etc.). Some pyrgomatids control coral overgrowth using mechanical means, but most species are able to suppress coral overgrowth through nonmechanical, po ssibly chemical, mechanism(s). While a single, fused wall is believed to be an exclusively pyrgomatid trait, possible non monophyly of the 1-plated Wanella with the Pyrgomatidae sensu stricto suggests that a concrescent wall plate may not be restricted to the pyrgomatids. Previously it was also thought that all of the living pyrgomatid species had no more than 4 wall plates, but the fact that some (but not all) members of the 6plated archaeobalanid genus Armatobalanus are sister to Cantellius proves that 6platedness does occur in the Pyrgomatidae. However, it does appear that fusion of the opercular valves is a character exclusively found in pyrgomatids, in particular among members of Clade I. In the future it would be very interesting to reevaluate the status of the genus Hexacreusia an exclusively coral -dwelling archaeobalanid from the Gulf of California. Zullo (in litt. in Newman 1996) suggested that Hexacreusia be placed in the Pyrgomatidae; however, Ross and Newman (2000) did not agree because Hexac reusia is 6 -plated and uses mechanical means to break coral overgrowth characteristics it shares with Armatobalanus Since (some members of) Armatobalanus have now been shown to belong within the Pyrgomatidae, Hexacreusia might also very well be pyrgomat ids as well. If so, that would mean that the Pyrgomatidae has extant members in the East Pacific as well.

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102 Table 3 1. List of recent pyrgomatid species. Type species are marked with an asterisk. Family Pyrgomatidae Gray 1825 Subfamily Ceratoconchinae New man and Ross 1976 Ceratoconcha Kramberger Gorganovic 1889 domingensis (Des Moulins 1866) floridana (Pilsbry 1931) paucicostata Young 1989 quarta (Kolosvary 1947) Subfamily Pyrgomatinae (Gray 1825) Tribe Pyrgomatini Gray 1825 Arossella (Anderson 1993) lynnae* Ross 2000 Cantellius Ross and Newman 1973 acutum (Hiro 1938) albus Ren 1986 alphonsei Achituv 2001 arcuatum (Hiro 1938) brevitergum (Hiro 1938) cardenae Achituv and Hoeksema 2003 euspinulosa (Broch 1931) gregarius (Sowerby 1823) hiroi Galkin 1982 hoegi Achituv, Tsang, and Chan 2009 iwayama (Hiro 1938) madreporae (Borradaile 1903) maldiviensis Galkin 1982 octavus Ross and Newman 1973 pallid us (Broch 1931) preobrazhenskyi Galkin 1982 pseudopallium (Kolosvary 1947) quintus Ross and Newman 1973 secundus (Broch 1931) septimus (Hiro 1938) sextus (Hiro 1938) sinensis Ren 1986 sumbawae (Hoek 1913) transv ersalis* (Nilsson Cantell 1938) tredecimus (Kolosvary 1947) Cionophorus Ross and Newman 1999 guillaumae Achituv and Newman 2002 soongi* Ross and Newman 1999 Creusia Leach 1817 spinulosa* Leach 1818

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103 Table 3 1. Continued Family P yrgomatidae Gray 1825 Darwiniella Anderson 1992 conjugatum* (Darwin 1854) Galkinia Ross and Newman 1995 angustiradiata (Broch 1931) decima (Ross and Newman 1973) indica* (Annandale 1924) supraspinulosa Ogawa 2000 Hi roa Ross and Newman 1973 stubbingsi* Ross and Newman 1973 Neopyrgoma Ross and Newman 2002 lobata* (Gray 1825) Neotrevathana Ross 1999 elongatum (Hiro 1931) Nobia Sowerby 1839 grandis* Sowerby 1839 halomitrae (Kolosvary 1 947) orbicellae (Hiro 1934) Pyrgoma Leach 1817 cancellatum* Leach 1818 japonica Weltner 1897 kuri Hoek 1913 projectum Nilsson Cantell 1938 sinica (Ren 1986) Savignium Leach 1825 crenatum* (Sowerby 1823) tuamo tum Achituv and Langsam 2005 Trevathana Anderson 1992 dentata* (Darwin 1854) isfae Achituv and Langsam 2009 margaretae Brickner et al. 2010 jensi Brickner et al. 2010 mizrachae Brickner et al. 2010 niuea Achituv 2004 orientale (Ren 1986) paulayi Asami and Yamaguchi 2001 sarae Brickner et al. 2010 synthesysae Achituv and Langsam 2009 tureiae Achituv and Langsam 2005 Wanella Anderson in Ross 1999 andersonorum (Ross 1999) milleporae* (Dar win 1854)

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104 Table 3 1. Continued Family Pyrgomatidae Gray 1825 snelliusi (Kolosvary 1950) Tribe Pyrgopsellini Ross and Newman 1995 Pyrgopsella Zullo 1967 annandalei* (Gruvel 1907) youngi Achituv and Simon Blecher 2006 T ribe Hoekiini Ross and Newman 1995 Ahoekia Ross and Newman 1995 chuangi Ross and Newman 1995 microtrema Ross 2000 tanabensis* Ross and Newman 1995 Australhoekia Ross and Newman 2000 cardenae* Ross and Newman 2000 Eohoekia Ro ss and Newman 1995 chaos* Ross and Newman 1995 nyx Ross and Newman 1995 Hoekia Ross and Newman 1973 fornix Ross and Newman 1995 monticulariae* (Gray 1831) mortensi Ross and Newman 1995 philippinensis Ross 2000 Parahoek ia Ross and Newman 1995 aster* Ross and Newman 1995 Subfamily Megatrematinae Holthuis 1982 Tribe Megatrematini Holthuis 1982 Megatrema Sowerby 1823 madreporarum* (Bosc 1801) youngi Ross and Pitombo 2002 Memagreta Ross and Pitombo 2002 pandorae* Ross and Pitombo 2002 Tribe Pyrgominini Ross and Pitombo 2002 Adna Sowerby 1823 anglica* Sowerby 1823 Pyrgomina Baluk and Radwanski 1967 djanae Ross and Pitombo 2002 oulastreae (Utinomi 1962)

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105 Table 3 2. Morphological and ecological characters of the genera, as synthesized from the taxonomic literature Current biogeog. region No. wall plates Wall externally conical (c), low conical (lc), or flat (f) Operc. valves unfused (uf) or fused (f) Operc. v alves balanoid (b) or modified (m) Basis fully calc. (ca), w/ passageways (cp), w/ narrow membranous zone (mz), mostly memb. (me) Planktotrophic (pk) or parasitic (pr) Host zooxanthellate scleractinian (zx), azoox. scleractinian (az), or hydrocoral (h) F amily Pyrgomatidae Gray 1825 Subfamily Ceratoconchinae Newman and Ross 1976 Ceratoconcha Kramberger Gorganovic 1889 WA 4 c uf b ca pk zx Subfamily Pyrgomatinae (Gray 1825) Tribe Pyrgomatini Gray 1825 Arossella ( Anderson 1993) IWP 4 f uf m ca pk zx Cantellius Ross and Newman 1973 IWP 4 c / f uf m ca pk zx / h Cionophorus Ross and Newman 1999 IWP 1 f f m ca pk zx Creusia Leach 1817 IWP 4 lc f b ca pk zx Darwiniella Anderson 1992 IWP 1 f f m ca pk zx Galkinia Ross and Newman 1995 IWP 4 f f b ca pk zx Hiroa Ross and Newman 1973 IWP 4 lc / f uf m ca pk zx Neopyrgoma Ross and Newman 2002 IWP 1 lc unk. unk. cp pk zx Neotrevathana Ross 1999 IWP 1 lc? f m ca pk zx Nobia Sowerby 1839 IWP 1 f f m ca pk zx Pyrgoma Leach 1817 IWP 1 lc / f uf m cp pk zx / az Savignium Leach 1825 IWP 1 f uf m ca pk zx Trevathana Anderson 1992 IWP 1 f uf m ca pk zx Wanella Anderson in Ross 1999 IWP 1 f uf m ca pk h

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106 Table 3 2. Continued Cu rrent biogeog. region No. wall plates Wall externally conical (c), low conical (lc), or flat (f) Operc. valves unfused (uf) or fused (f) Operc. valves balanoid (b) or modified (m) Basis fully calc. (ca), w/ passageways (cp), w/ narrow membranous zone (mz) mostly memb. (me) Planktotrophic (pk) or parasitic (pr) Host zooxanthellate scleractinian (zx), azoox. scleractinian (az), or hydrocoral (h) Tribe Pyrgopsellini Ross and Newman 1995 Pyrgopsella Zullo 1967 IWP 1 lc uf m me pk zx Tribe Hoek iini Ross and Newman 1995 Ahoekia Ross and Newman 1995 IWP 1 f f m mz pr zx Australhoekia Ross and Newman 2000 IWP 1 f f m mz pr zx Eohoekia Ross and Newman 1995 IWP 1 f f m mz pr zx Hoekia Ross and Newman 1973 IWP 1 f f m mz pr zx Parahoekia Ross and Newman 1995 IWP 1 f f m mz pr zx Subfamily Megatrematinae Holthuis 1982 Tribe Megatrematini Holthuis 1982 Megatrema Sowerby 1823 WA, IWP 1 f uf b ca pk zx Memagreta Ross and Pitombo 2002 IWP 1 f uf b ca pk zx Tribe Pyrgominini Ross and Pitombo 2002 Adna Sowerby 1823 EA 1 c uf b ca pk zx / az Pyrgomina Baluk and Radwanski 1967 IWP 1 c uf b ca pk zx / az

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107 Table 3 3. List of sequenced specimens. Co unt Extr# Accessi on# Genus Host Specimen pro venance H3 CO I 16 S 18 Sa 18 Sb 12 S s 1 H326 Adna Oculina patagonica Spain 1 1 1 1 1 1 2 H329 Adna Oculina patagonica Spain 1 1 1 1 1 1 3 H331 Adna Oculina patagonica Spain 1 1 1 1 1 1 4 H167 UF 8664 Cantellius Montipora Philippines 1 1 1 1 1 1 5 H213 Cantellius Porites Philippines 1 1 1 1 1 1 6 H169 Cantellius Montipora Philippines 1 1 1 1 1 1 7 UF 6541 Cantellius Acropora Philippines 1 1 1 1 1 1 8 H173 UF 8676 Cantellius Acropora Philippines 1 1 1 1 1 1 9 H218 Cantellius Acropora Philippi nes 1 1 1 1 1 1 10 H160 UF 8634 Cantellius Pachyseris rugosa Philippines 1 1 1 1 1 1 11 H172 UF 8636 Cantellius Pachyseris rugosa? Philippines 1 1 1 1 1 1 12 H209 Cantellius Porites rus Philippines 1 1 1 1 1 1 13 UF 13225 Ceratoconch a Siderastre a Flo rida, USA 1 1 1 1 1 0 14 UF 13227 Ceratoconch a Siderastre a Florida, USA 1 1 1 1 1 0 15 UF 13228 Ceratoconch a Madracis Florida, USA 1 1 1 1 1 1 16 H158 UF 13125 Darwiniella Goniastrea pectinata complex Philippines 1 1 1 1 1 1 17 H212 UF 8635 Darwiniel la Goniastrea pectinata complex Philippines 1 1 1 1 1 1 18 H238 Darwiniella Hydnophor a aff microcono s Oman 1 1 1 1 1 1 19 UF 13136 Darwiniella Hydnophor a exesa Philippines 1 1 1 1 1 1 20 H219 Darwiniella Montastrea curta Fiji 1 1 1 1 1 1 21 H161 Ga lkinia Leptastrea purpurea? Philippines 1 1 1 1 1 1 22 H244 UF 7460 Galkinia cf. Cyphastre a Oman 1 1 1 1 1 1 23 UF 11796 Galkinia cf. Cyphastre a Taiwan 1 1 1 1 1 1 24 UF 8966 Hiroa Astreopora Papua New Guinea 1 1 1 1 1 1

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108 Table 3 3. Continued Co unt Extr# Accessi on# Genus Host Specimen provenance H3 CO I 16 S 18 Sa 18 Sb 12 S s 25 UF 14102 Hiroa Astreopora Madagasca r 1 1 1 0 1 1 26 H240 Hoekiini Hydnophor a Philippines 1 1 1 1 1 1 27 H243 Hoekiini Hydnophor a microcono s Philippines 1 1 1 1 1 1 28 H274 UF 9273 Neotrevathan a Cyphastre a Society Ids. 1 1 1 1 1 1 29 H252 UF 8690 Neotrevathan a Favia Philippines 1 1 1 1 1 1 30 H265 UF 9277 Neotrevathan a Favia rotumana Tuamotu Ids. 1 1 1 1 1 1 31 H214 Nobia Coeloseris mayeri Philippines 1 1 1 1 1 1 32 H157 UF 10361 Nobia Galaxea Philippines 1 1 1 1 1 1 33 H211 Nobia Goniopora Philippines 1 1 1 1 1 1 34 H156 UF 10364 Nobia Euphyllia Philippines 1 1 1 1 1 1 35 H150 UF 9278 Pyrgoma Tubastrea Philippines 1 1 1 1 1 1 36 UF 13133 Pyrgoma Turbinaria Philippines 1 1 1 1 1 1 37 h ORI 11A Pyrgoma Turbinaria South Africa 1 1 1 1 1 1 38 from GenBa nk Pyrgopsella Symphyllia radians Indonesia* 1 1 1 1 1 1 39 H171 Savignium Echinophyl lia aspera Philippines 1 1 1 1 1 1 40 H237 Savignium Acant hastr ea lordhowen sis Oman 1 1 1 1 1 1 41 H202 Savignium Oxypora lacera Philippines 1 1 1 1 1 1 42 H181 UF 1327 Trevathana Acanthastr ea echinata Tuamotu Ids. 1 1 1 1 1 1 43 H255 UF 9271 Trevathana Acanthastr ea echinata Society Ids. 1 1 1 1 1 1 44 H253 Trevathana Echinopora lamellosa Philippines 1 1 1 1 1 1 45 H227 Trevathana Favia stelligera Cook Ids. 1 1 1 1 1 1

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109 Table 3 3. Continued Co unt Extr# Accessi on# Genus Host Specimen provenance H3 CO I 16 S 18 Sa 18 Sb 12 S s 46 H225 UF 15304 Trevathana Fa via stelligera Fiji 1 1 1 1 1 1 47 UF 10408a Trevathana Favia stelligera Guam 1 1 1 1 1 1 48 H221 UF153 03 Trevathana Goniastrea Fiji 1 1 1 1 1 1 49 H226 UF 10353 Trevathana Montastrea curta Cook Ids. 1 1 1 1 1 1 50 H166 UF 8645 Wanella branching Mille pora Philippines 1 1 1 1 1 1 51 9099A Wanella Millepora Papua New Guinea 1 1 1 1 1 1 52 12617A Wanella Millepora Mascarene Ids. 1 1 1 1 1 1 53 H184 UF 8030 Wanella Millepora Vanuatu 1 1 1 1 1 1 54 UF 13193 Acastinae Philippines 1 1 1 1 1 1 55 KACb 154 Armatobalan us 1 0 1 1 1 1 56 TAU Ar2783 5 Armatobalan us 0 0 1 1 1 1 57 KACb163 Armatobalan us 1 0 1 1 1 1 58 UF 11887 Armatobalan us Montipora Taiwan 1 1 1 1 1 1 59 H241 Tetraclita Heliopora Palau 1 1 1 1 1 1 60 H159 unknown Leptogorgi a Flo rida, USA 1 1 1 1 1 1 61 UF 11767 Conopea Eunicea flexuosa Panama 1 1 1 1 1 1 62 UF 13338 Megabalanus Panama 1 1 1 1 1 1 63 from GenBa nk Semibalanus 1 1 1 1 1 1 64 from GenBa nk Balanus 1 1 1 1 1 1

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110 Table 3 4. Sequence attributes for the 5 gene fragments. Gene Sequence length (bp) #Parsimony informative sites (bp) #Invariable sites (bp) A T bias Best fit model COI 599 228 360 66% GTR+I+G 16S 458 126 296 72% TVM+I+G 12S 361 132 193 69% TVM+I+G 18S 1766 74 1633 47% GTR+I+G H3 324 9 6 212 37% TrN+I+G GTR=general time -reversible model; TVM=transversional model; TrN=Tamura -Nei model; I=invariant sites; G=gamma shape parameter for rate variation among sites.

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111 Table 3 5. Topological incongruencies in ML (computed using RAxML) and Baye sian (computed using MrBayes) gene trees. Strongly supported (arbitrarily set at >60% BS and >90% PP) incongruencies with the 5gene concatenated analysis & with majority of the gene trees are italicized COI 16S 12S 18S ML Bayesia n ML Bayesian ML Bayesi a n ML Bayesian UF 11887 Armatobalanus unresolv ed unresolv ed sister to Cantelliu s (76% BS) sister to Cantelliu s (95% PP) unresolv ed w/ Cantelli us (93% PP) w/ main (non Wanella) pyrgomatid clade (90% BS) w/ clades I & II + 2 Cantellius sequences (93% PP) K ACb154 Armatobalanus allium no data no data unresolv ed unresolv ed unresolv ed unresolv ed w/ TAU Ar27835 (81% BS), w/in main (nonWanella) pyrgomatid clade (90% BS) sister to TAU Ar27835 (94% PP) w/ clades I & II (93% PP) TAU Ar27835 Armatobalanus allium no data no data w/ Megabal anus (97% BS) w/ Megabal anus (100% PP) w/ Megabal anus (99% BS) sister to Megabal anus (99% PP) w/ KACb154 (81%), w/in main (nonWanella) pyrgomatid clade (90%) sister to KACb154 (94% PP) w/ clades I & II (93% PP) KACb163 Armatobalan us sp. no data no data unresolv ed unresolv ed unresolv ed w/ H159"C onopea" (93% PP) w/ H159"Conopea" (77% BS) w/ H159"Conopea" (99% PP) Wanella clade no sister clade no sister clade no sister clade no sister clade no sister clade no sister clade w/ outgrou p seqs Acastinae+Conopea+M egabalanus (61% BS) w/ outgroup seqs Acastinae+Conopea+ Megabalanus (96% BS) Others UF 11887+2 Cantellius sequences not monophyletic w/ rest of Cantellius (93% PP)

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112 Table 3 5. Continued H3 MT NUC 5GENE PART ML Bayes ia n ML Bayesian ML Bayesian ML Bayesian UF 11887 Armatobalanus unre solve d unres olved sister to Cantelliu s (94% BS) sister to Cantelliu s (99% PP) unresolved w/in main (non Wanella) pyrgomatid clade (88% BS) unresolved w/in main (non Wanella) pyrgomatid clad e (100% PP) sister to Cantellius (91% BS) sister to Cantelliu s (100% PP) KACb154 Armatobalanus allium unre solve d unres olved sister to 11887 and Cantelliu s (76% BS) sister to 11887 and Cantelliu s (99% PP) w/ TAU Ar27835 (89% BS), w/in main (nonWanella) py rgomatid clade (88% BS) sister to TAU Ar27835 (97% PP) w/ clades I, II, & III (100% PP) sister to UF11887 and Cantellius (80% BS) sister to UF11887 and Cantelliu s (99% PP) TAU Ar27835 Armatobalanus allium no data no data w/ Megabal anus (100% BS) w/ Megabal anus (100% PP) w/ KACb154 (89% BS), w/in main (nonWanella) pyrgomatid clade (88% BS) sister to KACb154 (97% PP) w/ clades I, II, & III (100% PP) w/ Megabalanus (69% BS) w/ Megabal anus (100% PP) KACb163 Armatobalanus sp. unre solve d unres olved w/ H159"Co nopea" (91% BS) unresolv ed w/ H159"Conopea" (91% BS) w/ H159"Conopea" (100% PP) w/ H159"Conop ea" (93% BS) w/ H159"Co nopea" (100% PP) Wanella clade no sister clade no sister clade no sister clade no sister clade no sister clade no sister clade no sister cl ade no sister clade

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113 Table 3 6. P values obtained from topological tests. Hypothesis tested S H test Pyrgomatidae (incl. Wanella ) monophyletic 0.187 Armatobalanus monophyletic 0.057 MPT 1 9.038* MPT 2 0.007* *Significant at P < 0.05

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114 Table 3 7. Results of BaTS analyses of seven phenotypic characters. Observed value (95% CI) Expected value (95% CI) Obs.& exp. overlap? P value Opercular valve fusion AI 0.246 (0.153 0.351) 2.778 (1.871 3.651) n 0.000 PS 6.360 (6.000 7.000) 15.1 22 (12.997 16.998) n 0.000 No. wall plates AI 0.189 (0.123 0.269) 4.428 (3.573 5.381) n 0.000 PS 7.000 (7.000 7.000) 26.499 (23.033 29.230) n 0.000 Wall height AI 0.7541 (0.6967 0.8832) 3.3272 (2.4736 4.2729) n 0.000 PS 11.8032 (11 .000 12.000) 19.1661 (17.0089 20.9992) n 0.000 Basis calcareousness AI 0.3204 (0.2958 0.3823) 1.5993 (1.1363 2.1020) n 0.000 PS 5.0083 (5.000 5.000) 7.9273 (7.9365 8.000) n 0.000 Cirrus III teeth AI 0.2776 (0.2501 0.5001) 1.3096 (0.62 23 1.900) n 0.000 PS 2.6542 (2.000 3.000) 7.5975 (5.0933 9.4393) n 0.000 Coral overgrowth AI 1.0293 (0.9756 1.1005) 2.0907 (1.4787 2.6407) n 0.000 PS 8.7616 (8.000 9.000) 10.3453 (9.0595 11.000) n 0.050 Overgrowth control AI 0.3003 (0.2500 0.3650) 2.6583 (1.9177 3.4918) y 0.000 PS 4.2861 (3.000 5.000) 12.8256 (11.3960 14.000) n 0.000 Significant at P < 0.05

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115 Figure 31. Schematic illustrations of pyrgomatid anatomy and growth process, and diversity in shell morphology.

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116 Figure 32. Phenotypic characters traced onto the pyrgomatid phylogeny, with the various character states illustrated.

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117 Figure 33. Maximum likelihood phylogram for COI computed using RAxML. Values above the branches are bootstrap support values (RAxML) values below the branches are Bayesian posterior probabilities (MrBayes).

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118 Figure 34. Maximum likelihood phylogram for 16S computed using RAxML. Notation follows fig. 3 -3.

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119 Figure 35. Maximum likelihood phylogram for 12S computed using RAxML. Notat ion follows fig. 3-3.

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120 Figure 36. Maximum likelihood phylogram for 12S computed using RAxML. Notation follows fig. 3 -3.

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121 Figure 37. Maximum likelihood phylogram for H3 computed using RAxML. Notation follows fig. 3 -3.

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122 Figure 38. Maximum likelihood ph ylogram for all mt genes computed using RAxML. Notation follows fig. 3-3.

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123 Figure 39. Maximum likelihood phylogram for all nuc genes computed using RAxML. Notation follows fig. 33.

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124 Figure 310. Maximum likelihood phylogram for all 5 sequenced genes c omputed using RAxML. Nodes with <60% bootstrap support and <90% posterior probability were collapsed. Taxa and branches are colored according to their different nominal genera; taxa in black represent non -pyrgomatid samples. Values above the branches are b ootstrap support values (RAxML), values below the branches are Bayesian posterior probabilities (MrBayes).

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125 Figure 311. Reconstruction of ancestral character states for opercular valve fusion. The topology follows fig. 3 -10.

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126 Figure 312. Reconstructi on of ancestral character states for number of wall plates. The topology follows fig. 3 -10.

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127 Figure 313. Reconstruction of ancestral character states for wall height. The topology follows fig. 3 10.

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128 Figure 314. Reconstruction of ancestral character states for basis calcareousness. The topology follows fig. 3 -10.

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129 Figure 315. Reconstruction of ancestral character states for cirrus 3 armature. The topology follows fig. 3 -10.

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130 Figure 316. Reconstruction of ancestral character states for degree of coral overgrowth. The topology follows fig. 3-10.

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131 Figure 317. Reconstruction of ancestral character states for mechanism of overgrowth suppression. The topology follows fig. 3-10.

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132 CHAPTER 4 GEOGRAPHY AND HOST -SPECIFICITY BOTH INFLUENCE SPECIATIO N OF CORAL BARNACLES IN THE CLADE Trevathana SENSU LATO Introduction Speciation in the sea is still not well understood. While recent phylogeographic work has revealed that the marine realm is far more geographically structured than previously thought (e.g., Lessios et al. 2001, Meyer et al. 2005, Barber et al. 2006), other potential mechanisms of speciation have not yet been fully evaluated. For instance, while the role of host -specificity has been well studied in several terrestrial organisms (e.g., in th e apple maggot fly, Berlocher and Feder 2002; pea aphids, Hawthorne and Via 2001; fig wasps, Weiblen and Bush 2002), similar systems in the sea have only begun to be explored (e.g., in alpheid shrimp, Duffy 1996; coral -dwelling gobies, Munday et al. 2004; phestillid nudibranchs, Faucci et al. 2006; coral -dwelling barnacles, Mokady et al. 1999, Mokady and Brickner 2001, Tsang et al. 2009). The limited studies available have shown that cryptic or incipient species can be highly specialized on different hosts, suggesting that host shifts are involved in reproductive isolation and speciation. Such a mechanism of speciation may be especially important in marine systems characterized by widespread symbiotic relationships, such as c oral reefs. In other words, symbi oses could be one reason for high species diversity in reefs: new taxa could arise through ecological speciation (Schluter 2001) and species diversity itself may beget more species (Emerson & Kolm 2005) through the mechanism of host race speciation Studie s of host shifts in marine systems have so far had limited geographic coverage, making it impossible to assess the relative contributions of host specificity and geographic isolation to genetic structuring. In this chapter I am interested in

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133 superimposing two layers of information, e.g., geographic structure and host specificity, onto phylogenetic data, in order to evaluate their interactions in the diversification and speciation of marine organisms. My study organisms are the coral -dwelling pyrgomatid barn acles in the clade Trevathana sensu lato (s.l.). Pyrgomatids are a common but often overlooked group of balanomorph barnacles that obligately dwell on living corals. The genus Trevathana Anderson 1992 is the second largest genus of coral dwelling barnacles Together with its sister -group Savignium Trevathana has the widest geographic range among the pyrgomatids, extending from the coast of E Africa to the Tuamotus and Marquesas in the central Pacific. In Chapter 3 I showed that Neotrevathana Pyrgopsella, and the parasitic Hoekiini are nested within a paraphyletic Trevathana I call this entire assemblage Trevathana s. l .. The 25 n ominal species of this clade, their reported hosts, and the known geographic occurrences are listed in Table 4 -1. Earlie r studie s have shown highly host differentiated species complexes in Trevathana from the Red Sea and Great Barrier Reef (GBR; Mokady et al. 1999; Brickner et al. 2010). Results from this chapter extend earlier results, and demonstrate that both host specialization as well as geographic isolation contribute to the diversity of the clade, albeit at different timescales. Materials and Methods Specimens and Morphological Examinations Specimens were collected by scuba diving on reefs across the IndoWest Pacific (Table 4 2 ). The following areas were well sampled for pyrgomatid barnacles: the Red Sea, Oman, Taiwan, NW Philippine Ids., Guam, Fiji, Rarotonga in the Cook Ids., Moorea in the Society Ids., and the Tuamotu Ids. I considered localities to be well

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134 sampled when they were visited specifically to find pyrgomatid barnacles, and where divers spent a minimum of 2 weeks in low coral diversity localities, and 4 weeks in high coral diversity localities, searching for barnacles. A dditional locations were sampled less tho roughly as opportunities permitted. Coral specimens infested by barnacles were collected, and the barnacles removed from the coral using pliers or dental tools. Anatomical dissections were done under a dissecting microscope using fine tungsten needles. A t issue subsample was also dissected out for genetic analyses. Skeletal structures (wall and opercular valves) were briefly soaked in a weak bleach solution to remove adhering membranes, dried, and mounted on stubs. The skeletal structures were sputter -coated and imaged using a Field Emission-Scanning Election Microscope (SEM) at the University of Floridas Interdisciplinary Center for Biotechnology Research (ICBR) All specimens are housed in the Division of Invertebrate Zoology at the Florida Museum of Natural History (FLMNH). A total of 81 FLMNH specimens were sequenced. Identifications of coral hosts were done by G. Paulay of the FLMNH. Molecular M ethods All specimens were sequenced for the mitochondrial barcoding gene COI. DNA was extracted from the tis sue subsamples using DNAzol and proteinase K, following the protocol given in Meyer (2003). DNA extracts were purified using QIAGEN cleanup kits. The PCR primers and amplification profile followed protocols in Meyer (2003). PCR products were cleaned and sequenced at UFs high -throughput sequencing facility (at the ICBR) following protocols detailed in Chapter 3. All PCR products were sequenced along both directions. Some of the tissue subsamples were extracted and sequenced at the Smithsonian Institutions Laboratories of Analytical Biology, following the same

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135 methodology as in Chapter 3. The 81 newly generated sequences were supplemented with published Trevathana sequences originating from specimens collected in the Red Sea and a few other localities for a total of 120 sequences (Table 42) Data Analyses Chromatogram s were checked, manually edited, and assembled into contigs using Geneious Pro 4.9.2 (Drummond et al. 2009). All sequences lacked insertions and deletions, thus sequence alignment was a trivial task. Codon positions were verified using Macclade 4.08 (Maddison and Maddison 2005). The best -fit model of sequence evolution was determined using the Akaike Information Criterion (Akaike 1974) as implemented in Modeltest 3.7 (Posada and Crandall 1998). The data were analyzed using RAxML 7.0.4 (Stamatakis 2006) using a GTR+GAMMA model and a random starting tree, and with the analysis partitioned into the 3 codon positions A thousand bootstrap replicates were also performed. The tree was rooted on the di stantly -related genera Cantellius and Armatobalanus I also used a Bayesian approach to analyze the data, using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). Flat priors and a GTR+I+GAMMA model were employed. I ran 2 independent chains for 1 million generations each; each chain was sampled every 100 generations. All runs reached stationarity before 100,000 generations. The initial 25% of the trees was discarded as the burnin phase, and posterior probabilities were calculated based on the remaining 75% of the trees. T he sequence s were run either (1) unpartitioned, or (2) divided into the 3 codon positions with all parameters unlinked between partitions Prior to partitioning the appropriate model of evolution was selected for each codon position using Modeltest.

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136 In analyzing the topologies, I focused on clades with high support values (arbitrarily set at > 60% bootstrap values and > 90% posterior probabilities). These well supported clades were then used to defin e Evolutionary S ignificant Units (ESUs; sensu Moritz 1994). ESUs are reciprocally monophyletic populations that have at least one other independent character such as a distinct morphology, distribution, ecological niche (e.g., host occupancy), or that show reciprocal monophyly in another, independent marker. ESUs are clades with an evolutionary history separate from other ESUs. ESUs are thus species -level units (phylogenetic species) which can be readily defined in allopatric as well as in sympatric settings, unlike biological species. ESUs proved difficult to define in instances where only a single sequence was available for a particular locality and host. In those cases, I defined putative ESUs as singletons with a distinct locality or host, and having >2% sequence dive rgence from the nearest ESU. I analyzed pairs of sister ESUs in order to study patterns among the most recent evolutionary divergences. I conservatively defined pairs of sister -ESUs according to the following criteria: (1) each ESU must have robust branch support, (2) only ESUs with >1 sequence were considered (i.e., putative ESUs were excluded), (3) when there was more than one potential sister -ESU, the selected sister -ESU should not have a disjoint geographic distribution. I called the divergence of sis ter ESUs evolutionarily significant events or ESEs (following Chapter 2). Results Figure 41 presents the results from the ML and Bayesian analyses. Twelve strongly supported clades were identified, each with between one to approximately 9 ESUs (Table 4-3 ). Some presumed groups contained only a single specimen, and thus

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137 were assigned putative ESU status. The phylogeny is structured both by host and geography. These 2 factors are discussed in turn below. Host -S p ecificity Patterns Most (but not all) Trevatha na species inhabit the traditional coral family Faviidae, a tangled paraphyletic/polyphyletic set of lineages, most members of which form a well defined clade together with several other traditional coral families (Mussidae, Pectiniidae, Merulinidae, T rachyphyliidae; Fukami et al. 2004; Fukami et al. 2008). Most Trevathana s.l species live on corals of Fukami et al.s (2007) Clade XVII in the latest scleractinian phylogeny, with a few on related corals outside this focal clade: Leptastrea Plesiastrea, and Acanthastrea. Thus Tr e vathana s.l. appears to be predominantly specialized to a single coral clade. Of the 10 Trevathana s.l. clades represented by more than 1 sequence, eight (80%) are restricted to a single coral genus (Fig. 41; Table 4 -3). The two exceptions (clades IX and XII) were both found on the genera Cyphastrea and Plesiastrea. However, closer examination showed clear morphological differences between the Cyphastrea colonies hosting the 2 different barnacle clades. It seems likely that the 2 morphs (both potentially undescribed species) are not actually members of Cyphastrea, but rather derived forms of Plesiastrea (Paulay, pers. comm.). Regardless of the status of Clades IX and XII, the overall pattern shows that Trevathana s.l. clades are specific to a single coral genus. Host specificity is also clear at the ESU level. Of 23 ESUs containing >1 sequenced specimen, at least eleven (or up to 15, depending on incomplete host identifications; i.e., about 50%) were found on a single host specie s (Fig. 4 1; Table 43).

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138 Eight evolutionarily significant events or ESEs were identified (Table 4 -4), of which five appeared to involve a switch to a different coral host species, while 3 did not involve host -switching. However, since the same host coral s pecies were not consistently sampled across the entire range of Trevathana s.l. it is also possible that apparent instances of host switching are artefacts of incomplete coral sampling. While most coral genera were inhabited by a single Trevathana s.l. cl ade, three coral genera hosted more than one clade, i.e., Favia (occupied by clades I, IV, VIII, and XI), Plesiastrea (clades IX and XII), and Cyphastrea (clades VI, IX and XII; but see above for discussion of Cyphastrea identification problems). However Favia is a polyphyletic genus (Fukami et al. 2008), while the phylogenetic status of Plesiastrea is presently not known because only 1 species has been sequenced for that genus. Therefore, in general a single coral taxon only hosts a single barnacle clade. An apparent exception is the well defined species Favia stelligera which is present in 3 clades (IV, VIII, and XI). Two of the ESUs inhabiting F. stelligera are sympatric (VIII -A and XI B, on Guam). These 2 clades showed no obvious differences in skeletal characters (not illustrated). The two clades may represent cryptic species, or the genetic difference may be due to DNA contamination or the presence of pseu dogenes. Increasing the number of sequenced specimens will hopefully resolve this question. Bioge ographic Patterns The geographic distributions of Trevathana s.l. clades a re shown in figures 42 to 4 -11. Clades I and IV range across the entire IWP, encompassing three subregions, i.e., the Western Indian Ocean (WIO; including Madagascar, the Iles Epars es surrounding Madagascar, the Seychelles, Oman, and the Red Sea), Western Pacific (WP; including the Philippines, Indonesia, Taiwan, Palau, Guam, the Northern Marianas/CNMI, Papua

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139 New Guinea/PNG, and Australia), and Central Pacific (CP; including Fiji, Vanuatu, the Cook Ids., Society Ids., and Tuamotus). Clades II, III VI, and VII extend from the WIO to the WP; while Clades X and XI span the WP to CP. Clade VIII was only found in Oman, Clade XII is known only from the WIO, and Clade IX is only known from the CP. Two clades are only known from single specimens: Clade V from Indonesia, and Clade VIII from Guam (not illustrated). Most clades are divided into multiple ESUs following the subregional boundaries, and show genetic cohesiveness within each subregi on. ESUs are generally restricted to a single subregion, with three singleton exceptions, i.e., ESU IV -C (all from CP except 1 Great Barrier Reef/GBR sequence; fig. 45), IV -D (all from the WIO except 1 GBR sample; fig. 45), and VI -A (all from the WIO exc ept 1 Philippine sample; fig. 47). Most clades have only 1 ESU per subregion. The exceptions are Clades III, IV, and VII. Clade III may have 2 ESUs in the WIO (tentative assignments; see fig. 4 -4). Clade VII has 2 sympatric ESUs in one locality in the Phi lippines (fig. 4-8). Perhaps the most atypical patterns are exhibited by Clade IV (fig. 4 -5). This clade contains many more ESUs than all the other clades (at least 5 and possibly up to 9 ES Us). Several localities (e.g., the Red Sea, Guam, Great Barrier Reef, Fiji, and the Tuamotus) harbor 2 sympatric Clade IV ESUs. Of the eight E SEs (Table 4 -4), 7 were geographically structured and one was not. Among the geographically structured ESEs, four were located between the WIO and WP, and 3 were between the WP and CP. The single non -geographically structured ESE involved Clade VII in the Philippines. Note, however, that this ESE would actually be geographically structured if the putative ESU VII -B (from the WIO) were included in

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140 the analysis. ESEs were difficult to determine for the Clade IV ESUs because of a lack of branch support, thus this clade was excluded pending additional genetic data. Discussion Host Specificity Researchers have been interested in patterns of host specificity in pyrgomatid barnacles for ove r 70 years (e.g., Hiro 1935). However, studies of host specificity in pyrgomatids have been hampered by a number of factors: (1) some pyrgomatid material in collections lack host records; (2) barnacle taxonomists typically lack expertise in coral taxonomy, thus coral mis -identifications are relatively common in published records; and (3) corals are themselves highly polyphyletic at multiple levels (e.g., Romano and Cairns 2000; Fukami et al. 2008). The results show that Trevathana s.l. is host specific at multiple levels. The entire group is a specialist on faviids or Clade XVII (sensu Fukami et al. 2008) corals. At the clade level, 80 100% occupy only 1 host coral genus; while at the ESU level, about half are known from only single coral host species. For the most part, each coral group hosts only 1 clade of barnacles. The only coral species hosting more than one Trevathana s.l. clade is Favia stelligera (Fig. 4 -1; Table 4 3). The results imply that Trevathana s.l. shows phylogenetic conservatism in hos t use. How host specificity is accomplished is not yet known, but may involve specificity at the larval settlement stage. Biogeography In Trevathana s.l. the ESEs (corresponding to speciation events) are located between the WIO and WP, and between the WP and CP. The exact location of the speciation events cannot yet be ascertained because of the disjunct distribution of

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141 sampling localities. In general ESUs are restricted to a single geographic region. Genetic structuring of sister -ESUs largely corresponds with geographic separation, and all of the sister -ESUs are allopatrically distributed. Temporal Difference Between Host Shifts and Geographical Isolation Both geography and host specificity have affected the diversification of Trevathana s.l The deeper, clade -level divergences are structured by host (80100% of all clade level splits), implying that early diversification of the group was accomplished by radiating over a range of coral hosts. The more recent, ESU -level divergences are structured by geograp hy (in seven out of 8 ESEs). This implies that while both factors are acting on diversification, they operate on different timescales. Geographic separation works more quickly in creating genetic diversity than does host switching; however, given time the geographic signal is lost, probably as a result of shifts and extensions of geographic ranges. A shift in host use is a slower evolutionary process, thus its signature is retained in the phylogeny for a longer period of time. This rate difference between t he two evolutionary forces is compatible with the idea that ecological niche, e.g. host choice, tends to be conserved over evolutionary time (i.e., phylogenetic niche conservatism; Peterson et al. 1999; Wiens and Graham 2005). Species Diversity Patterns T he ESU richness in Trevathana s.l. does not peak in any one area (Fig. 412). Among the well sampled localities, diversity was highest in the Southwest Indian Ocean (SWIO; n=4 6; data combined from the Iles Eparses, Seychelles, and Madagascar), Red Sea (n= 6 ESUs), Guam (n=5 7), and the Tuamotus (n=6). This is followed by the Philippines (n=5) and the Society Ids. (n=4), while ESU richness was relatively low in Oman, Taiwan, Fiji, and the Cook Ids. (all n=2 ESUs). Surprisingly, diversity in

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142 Trevathana s.l. d oes not parallel the pattern for scleractinian corals, which show a diversity hotspot in the IndoMalayan region and progressively decreasing diversity in all directions, especially towards the eastern Pacific (Stehli et al. 1967). Diversity at the edges o f the distribution (e.g., the Red Sea, SWIO, Tuamotus) is just as high as in the IndoMalayan coral tr iangle, if not slightly higher. While Trevathana s.l. does not exhibit a biodiversity hotspot, the overall diversity across all pyrgomatid barnacles may still peak in the Indo Malayan coral triangle. What could account for this disparity in patterns? As with Calcinus hermit crabs (discussed in Chapter 2), the overall hotspot may result from the accumulation of different cladespecific patterns, each ref lecting a separate evolutionary history. The diversity pattern in an individual clade may not necessarily parallel the overall pattern at a higher taxonomic level. What Are Species? In an earlier section I explained that ESUs satisfy the conditions of the phylogenetic species concept (see materials and methods). However, some may argue that the clades themselves could be considered species, and that the ESUs merely represent geographic variation within a species. Since sister -ESUs are all allopatrically dis tributed, it is not possible to determine whether sister -ESU pairs have intrinsic barriers to reproduction (i.e., the biological species concept cannot be applied). Given the absence of information on reproductive isolation, the decision of where to draw t he line between different species is dependent on how much variation (genetic, morphological, ecological, etc.) the taxonomist is willing to include within a species. Nonetheless, the species question is not merely academic, because the assignment of a u nique species name to a phylogenetic unit influences our perception of whether

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143 species are geographically restricted or wide-ranging. In the case of Trevathana s.l. assigning a clade only one species name means that a single species can range from the Red Sea to the Tuamotus, in accordance with early views that marine species are able to disperse across vast ocean distances. On the other hand, assigning separate species names to each ESU restricts the range of species to a single geographic region; i.e., t he WIO, WP, or CP. Moreover, in the case of Trevathana s.l. lumping multiple ESUs under a single species name will give the impression that species are not highly host specific, when clearly host specificity is highly prevalent in these barnacles. Restr icting the geographic range and host range of species by delimiting smaller species units may have management and conservation implications as well, since biodiversity is (typically) managed and conserved at the species level. There are also clear morphological differences between the different ESUs, particularly in the tergal region (see fig. 413). Indeed, previous to this study, Brickner et al. (2010) assigned separate species names to two ESUs in a single clade (e.g., T. jensi and T. dentata, both in Clade IV). This is further evidence that taxonomists recognize the ESUs as natural units. For all these reasons, I believe that the ESUs recovered in these studies all need to be described as separate species. New species descriptions will be forthcoming in a future paper. Status of Neotrevathana The genus Neotrevathana Ross 1999 currently contains a single species, N. elongatum (see Table 41), described by Hiro (1931) from Seto, Honshu Is., Japan; from Madrepora (the ho st identification is very likel y incor rect, as t he name Madrepora was formerly applied to the staghorn coral Acropora, which does not host Trevathana s.l. ). Hiro (1931) assigned his new species to the genus Pyrgoma Leach 1817, which at

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144 that time encompassed all pyrgomatids with a single wall p late, following the precedent set by Darwin (1854). Pyrgoma was divided into multiple genera in the revis i on of Ross and Newman (1973). Anderson (1992) later transferred P. elongatum to Newmania n. gen., together with Newmania milleporae Later recognizi ng that Newmania was preoccupied, Anderson (1993) proposed the genus Wanella for these 2 species However Ross (1999) noted that based on illustrations, the specimen described by Anderson (1992) could not be the same species that Hiro (1931) described. Ross (1999) concluded that the specimen described by Hiro (1931) was not closely related to W. milleporae and he correctly recognized the affinity of elongatum to Trevathana dentata (Darwin 1854). At that time Trevathana was a monospecific genus. However, i nstead of including elongatum in Trevathana Ross erected a new genus, Neotrevathana, to accommodate it. In comparing N. elongatum to T. dentata, Ross (1999, p. 835) stated that Neotrevathana differs from Trevathana in having broad, low ridges on the shel l surface, coalescent opercular plates, a broad occludent ledge, and by lacking a depression for insertion of the lateral depressor muscle. The tergal spur in Neotrevathana is reduced to a knoblike projection, whereas the tergal tooth in Trevathana appears to be an elaboration of the tergal spur. Of these characters, the fusion of the opercular valves is the most distinct and readily recognizable character. The absence of the lateral depressor muscle insertion is merely a secondary condition related to v alve fusion, as the muscle insertion is located on the basi -tergal angle of the scutum and would be obliterated by the fusion of the scutum and tergum. The other differences between Neotrevathana and Trevathana have disappeared with the discovery of additi onal

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145 Trevathana species. For instance, widely spaced, low radiating ridges on the external surface of the wall have since been observed in the newly described species T. mizrachae, T. jensi and T. margaretae (see illustrations in Brickner et al. 2010). Likewise, the broad occludent ledge and appearance of the tergal spur are no longer unique to Neotrevathana. The only remaining character differentiating Neotrevathana is fusion of the scutal and tergal valves. I foun d 5 different ESUs with fused (thus Ne otrevathana -like) opercular valves in Trevathana s.l. (fig. 4 13). In addition, clade VII (the parasitic hoekiines) also possesses fused valves. Fusion of opercular valves may have occurred at least 3 times in the evolution of Trevathana s.l. (fig. 4-13). Moreover, I found that valve fusion can even vary within a single ESU, notably in II -A. Mokady et al. (1999) suggested that fusion of valves develops ontogenetically in Neotrevathana and that the articular ridge on the fused valve represents the site of valve fusion. Indeed, a superficial suture on the external surface of the valves (more rarely on the internal surface) is visible in most of the Trevathana s.l. ESUs with fused valves. However in clade X, there is a complete absence of a suture demarcating the scutal and tergal regions, and it is difficult to imagine valve fusion occurring ontogenetically in this ESU, nor in the highly apomorphic parasitic hoekiines (Clade VII), which also lack a demarcating suture. Clearly valve fusion is a highly labile character, and cannot be sufficient basis for differentiating genera. Therefore I propose that Neotrevathana Ross 1999 be synonymized with Trevathana Anderson 1992. Which of the five ESUs with fused opercular valves corresponds to the N. elongatum of Hiro (1931)? Unfortunately, the type specimen appears to have been lost

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146 and no neotype has been designated (Ross 1999), thus we only have the illustrations of Hiro (1931) to refer to. Two other published illustrations for N. elongata by Ross (1999) and Mokady et al. (1999), appear to correspond to different taxa. The specimen illustrated in Ross (1999; fig.1) was collected from Goniastrea aspera in the Ryukyu Ids.; while the specimen illustrated by Mokady et al. (fig. 3c in Mokady et al. 1999; also ESU II A in fig. 4 1) was collected from the Red Sea on Echinopora gemmacea Given the high host specificity and geographic structuring of Trevathana s.l ., the fact that host corals and geographic locations were different is already a strong indication that the two s tudies dealt with 2 different species. The specimen of Mokady et al. (1999; ESU II -A in fig. 413) is most likely not the same as the original N. elongatum because the former specimen has a pronounced tergal tooth not visible in the latter. In the illustr ation of Ross (1999), the tergal tooth is unfortunately not clearly visible, thus it is difficult to determine if his specimen was the same as Hiros. Hiros specimen most closely resembl es specimens from Clade I (fig. 4-13). There are 3 ESUs in Clade I, one each in the WIO, WP, and CP. Logically I would assume that Hiros specimen from Japan would fall in the WP ESU, I -B. Indeed the opercular valve appears to closely match Hiros illustration. How ever, given that geographical structuring and substantial cr yptic differentiation are rampant in this group, Hiros (1931) specimen could also represent a different species at the northern limits of pyrgomatid distribution. Only by sampling in Hiros original collection in Seto can this puzzle be resolved completel y. Conclusions Speciation in these highly specialized coral symbionts is influenced both by geographic separation and by host switching; however, these two factors operate at

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147 different timescales. Geographic structuring occurs relatively rapidly, thus the most recently diverged ESUs show allopatric distributions. Host switching is an evolutionarily slower process, thus more anciently diverged lineages inhabit different host coral genera. Despite the extensive evolutionary diversification of Trevathana s.l. in the IWP, there was no observable gradient in diversity across its range. This surprising result does not seem to be evident at higher taxonomic scales, because large -scale patterns of species richness reflect the accumulation of many different biodivers ity patterns resulting from different evolutionary histories. Thus, larger patterns can obscure biologically relevant processes. In order to unravel patterns that are biologically meaningful, we need to look at phylogenetically cohesive units of diversity, that is, reciprocally monophyletic groups. This bottom up approach offers an interesting contrast to the patterns obtained through more traditional top-down studies of biodiversity. My results also show that the genus Neotrevathana is polyphyletic, and ne eds to be synonymized with Trevathana. Multiple new ESUs were discovered in the course of this study, all of which will need to be taxonomically described. Molecular phylogenetic data is facilitating both revisionary systematics and species discovery in py rgomatid barnacles

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148 Table 4 1. All nominal species within the Trevathana sensu lato subclade. No attempt was made to reevaluate barnacle and coral identifications. Count Localities Host(s) References Tribe Pyrgomatini Gray 1825 Trevathana Ander son 1992 1 dentata (Darwin 1854) Red Sea, Mauritius, Gulf of Thailand, Persian Gulf, Andaman Sea, Bay of Bengal, Singapore, Japan, Hongkong, Taiwan, Indonesia, Philippines, Guam, New Guinea, Great Barrier Reef, Palau, Fiji, Gambiers, Niue, French Poly nesia, Tuamotus. Also known from Pleistocene deposits in Japan. Goniastrea sp., G. retiformis, G. edwardsi, Meandrina spongiosa, Favia russelli, F. stelligera, F. cf. laxa, F. favus, Cyphastrea sp. C. serailia, C. microphthalma, Echinophyllia lamellosa, Montastrea sp., M. curta, M. valenciennesi, Plesiastrea versipora, Favites sp., F. cf. abdita, F. russelli, Platygyra lamellina, Leptastrea transversa Ross and Newman 1973; Newman and Ross 1976; Foster 1980; Mimoto 1991; Ogawa et al. 1998; Ogawa 2000; Asa mi and Yamaguchi 2001; Achituv 2004; Achituv and Langsam 2005; Brickner et al. 2010 2 isfae Achituv and Langsam 2009 Gambiers, Tuamotus Favia stelligera Achituv and Langsam 2009 3 margaretae Brickner et al. 2010 Red Sea Favia favus Brickner et al. 2010 4 jensi Brickner et al. 2010 Red Sea Favites abdita Brickner et al. 2010 5 mizrachae Brickner et al. 2010 Red Sea Platygyra lamellina Brickner et al. 2010 6 niuea Achituv 2004 Niue Id. Goniopora sp. Achituv 2004 7 orientale (Ren 1986) Japan, Guang dong, China Favia stelligera, Cyphastrea serailia, Goniastrea sp. Ren 1986; Asami and Yamaguchi 1997 8 paulayi Asami and Yamaguchi 2001 Guam, Gambiers Acanthastrea sp., A. echinata Asami and Yamaguchi 2001; Achituv and Langsam 2005 9 sarae Brickner et al. 2010 Seychelles, Red Sea Cyphastrea chalcidium Brickner et al. 2010 10 synthesysae Achituv and Langsam 2009 Reunion Is. Plesiastrea versipora Achituv and Langsam 2009 11 tureiae Achituv and Langsam 2005 Tuamotu Ids. Goniastrea sp. Achituv and Lang sam 2005

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149 Table 4 1. Continued. Count Localities Host(s) References Neotrevathana Ross 1999 12 elongatum (Hiro 1931) Mauritius, Red Sea, Gulf of Thailand, Japan, Hongkong, Timor Sea, Palau, Guam, Heron Is., Tuamotus Echinopora sp., E. lamello sa, E. gemmacea, Madrepora sp., Favia mathaii/pallida, Goniastrea sp., G. aspera Hiro 1931; Newman and Ross 1976; Foster 1980; Galkin 1983; Ogawa et al. 1998; Ogawa 2000; Mokady et al. 1999; Achituv & Langsam 2005; SimonBlecher et al. 2007 Tribe Pyrgops ellini Ross and Newman 1995 Pyrgopsella Zullo 1967 13 annandalei (Gruvel 1907) Andaman Ids. ? Gruvel 1907 14 youngi Achituv and Simon Blecher 2006 Sulawesi, Indonesia Symphyllia radians Achituv and Simon Blecher 2006 Tribe Hoekiini Ross and New man 1995 Ahoekia Ross and Newman 1995 15 chuangi Ross and Newman 1995 Java Sea, Indonesia Hydnophora rigida Ross and Newman 1995 16 microtrema Ross 2000 Hydnophora 17 tanabensis Ross and Newman 1995 Japan Hydnophora ?exesa, H. bonsai Ross and Newman 1995 Australhoekia Ross and Newman 2000 18 cardenae Ross and Newman 2000 Hydnophora Eohoekia Ross and Newman 1995 19 chaos Ross and Newman 1995 Red Sea Hydnophora sp. Ross and Newman 1995 20 nyx Ross and Newman 1995 Red Sea Hydnoph ora exesa Ross and Newman 1995 Hoekia Ross and Newman 1973 21 fornix Ross and Newman 1995 Moluccas, Indonesia Hydnophora exesa Ross and Newman 1995 22 monticulariae (Gray 1831) Singapore Hydnophora exesa Ross and Newman 1995 23 mortensi Ross and Newman 1995 Mauritius Hydnophora exesa Ross and Newman 1995 24 philippinensis Ross 2000 Hydnophora Parahoekia Ross and Newman 1995 25 aster Ross and Newman 1995 New Caledonia Hydnophora microconos Ross and Newman 1995

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150 Table 4 2. List of sequenced specimens Accession# Extraction# ID (if available) ESU Host ID Provenance UF 18608 I A Favia sp. Eparses UF 18651 I A F. mathaei Eparses Red1 T. margaretae I A Favia favus Red Sea Red2 T. margaretae I A F. favus Red Sea Red21 T. margareta e I A F. favus Red Sea Red22a T. margaretae I A F. favus Red Sea Red22b T. margaretae I A F. favus Red Sea Red22c T. margaretae I A F. favus Red Sea Red23 T. margaretae I A F. favus Red Sea Red3 T. margaretae I A F. favus Red Sea Red4 T. margar etae I A F. favus Red Sea Red5 T. margaretae I A F. favus Red Sea UF 11834 I B Favia sp. Taiwan UF 8690 H235 I B Favia sp. Philippines UF 8690 H252 I B Favia sp. Philippines UF 9274 H266 I C Favia rotumana Tuamotus UF 9274 H267 I C F. rotumana Tuamotus UF 9274 H268 I C F. rotumana Tuamotus UF 9277 H265 I C F. rotumana Tuamotus UF 18657 II A Echinopora gemmacea Eparses UF 18657 II A E. gemmacea Eparses UF 18658 II A Echinopora hirsutissima Eparses UF 18658 II A E. hirsutissima Ep arses Red15 "N. elongata" II A Echinopora sp. Red Sea UF 11868 II B Echinopora sp. Taiwan UF 8632 II B Favia? Philippines UF 8633 H162 II B Echinopora lamellosa Philippines UF 8633 H253 II B E. lamellosa Philippines UF 8633 H254 II B E. lame llosa Philippines MOM5 4a III A ? Oman

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151 Table 4 2. Continued. Accession# Extraction# ID (if available) ESU Host ID Provenance UF 20402 III A Platygyra sp. Oman Red1 T. mizrachae III A Platygyra lamellina Red Sea Red11 T. mizrachae III A P. lamel lina Red Sea Red17 T. mizrachae III A P. lamellina Red Sea Red25 T. mizrachae III A P. lamellina Red Sea UF 10396 III B Platygyra pini Guam UF 10397 III B P. pini Guam UF 10398 III B P. pini Guam UF 14544 III C? ? Madagascar UF 10331 IV A Montastrea curta Cook Ids. UF 10350 IV A M. curta Cook Ids. UF 1324 H182 IV A M. curta Tuamotus UF 15302 H224 IV A M. curta Fiji UF 15305 H223 IV A M. aff. curta Fiji UF 9428 IV A M. curta Society Ids. UF 9431 IV A M. curta Society Ids. UF 9458 IV A M. curta Tuamotus UF 9522 IV A M. curta Society Ids. UF 10399 IV B? Favites russelli Guam UF 15304 H225 IV C Favia stelligera? Fiji UF 15503 H221 IV C Goniastrea sp. Fiji UF 9459 IV C F. stelligera Tuamotus GBR14c "T. dentat a" IV C Leptastrea sp. GBR GBR28 T. jensi IV D Favites halicora GBR Red abd16 T. jensi IV D Favites abdita Red Sea Red2 T. jensi IV D F. abdita Red Sea Red21 T. jensi IV D F. abdita Red Sea Red2b T. jensi IV D F. abdita Red Sea Red3 T. jen si IV D F. abdita Red Sea Red5 T. jensi IV D F. abdita Red Sea Red71 T. jensi IV D F. abdita Red Sea

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152 Table 4 2. Continued. Accession# Extraction# ID (if available) ESU Host ID Provenance Red72 T. jensi IV D F. abdita Red Sea Red1 "T. dentata" IV E Leptastrea transversa Red Sea Red3 "T. dentata" IV E L. transversa Red Sea Red4 "T. dentata" IV E L. transversa Red Sea UF 6053 IV F? Goniastrea edwardsi CNMI UF 8536 IV G? F. halicora Vanuatu UF 10395 IV H G. edwardsi Guam UF 6055 IV H G. edwardsi CNMI UF 8978 IV I? G. edwardsi Palau TAU Ar27804 P. youngi V A Symphyllia radians Indonesia UF 6601 VI A Cyphastrea serailia Philippines Red13a T. sarae VI A Cyphastrea chalcidium Red Sea Red13c T. sarae VI A C. serailia Red Sea RedB1 T. sarae VI A C. chalcidium Red Sea RedB2 T. sarae VI A C. serailia Red Sea RedB3 T. sarae VI A C. chalcidium Red Sea RedE T. sarae VI A C. chalcidium Red Sea Sey1 T. sarae VI A Cyphastrea sp. Seychelles Sey2 T. sarae VI A Cyphas trea sp. Seychelles Sey3 T. sarae VI A Cyphastrea sp. Seychelles UF 10406 VI B C. serailia Guam UF 13693 VI B Cyphastrea sp. Guam UF 10423 Hoekiini VII A Hydnophora microconos Philippines UF 8679 H243 Hoekiini VII A H. microconos Philippines H234 Hoekiini VII A H. microconos Philippines UF 18617 Hoekiini VII B? Hydnophora sp. Eparses UF 6564 H240 Hoekiini VII C Hydnophora exesa Philippines UF 6564 Hoekiini VII C H. exesa Philippines UF 10400 VIII F. stelligera Guam UF 9268 H269 IX A Plesiastrea sp. Tuamotus UF 9268 H270 IX A Plesiastrea sp. Tuamotus

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153 Table 4 2. Continued. Accession# Extraction# ID (if available) ESU Host ID Provenance UF 9273 H273 IX A aff. Cyphastrea sp. 1 Society Ids. UF 9273 H274 IX A aff. Cyphastrea sp. 1 S ociety Ids. UF 9276 H271 IX A aff. Cyphastrea sp. 1 Tuamotus UF 9276 H272 IX A aff. Cyphastrea sp. 1 Tuamotus UF 10919 T. aff. paulayi X A Acanthastrea echinata Tuamotus UF 1327 H181 T. aff. paulayi X A A. echinata Tuamotus UF 9260 T. aff. paulayi X A A. echinata Tuamotus UF 9265 H258 T. aff. paulayi X A A. echinata Society Ids. UF 9271 H255 T. aff. paulayi X A A. echinata Society Ids. UF 10381 T. paulayi X B A. echinata Guam UF 10384 T. paulayi X B A. echinata Guam UF 10359 H227 XI A F. st elligera Cook Ids. UF 10912 XI A F. stelligera Cook Ids. UF 9267 H262 XI A F. stelligera Society Ids. UF 9275 H263 XI A F. stelligera Tuamotus UF 9275 H264 XI A F. stelligera Tuamotus UF 10402 XI B F. stelligera Guam UF 6052 XI B F. stellige ra CNMI UF 18634 XII A aff. Cyphastrea sp. 2 Eparses UF 20280 XII A Plesiastrea versipora Oman UF 20280 XII A P. versipora Oman UF 20351 XII A P. versipora Oman UF 20351 XII A P. versipora Oman UF 20425 XII A P. versipora Oman UF 20 425 XII A P. versipora Oman TAUAr27836 C. pallidus outgroup Porites sp. Thailand UF 11887 Armatobalanus outgroup Montipora sp. Taiwan UF 8664 H167 Cantellius sp. outgroup Montipora sp. Philippines

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154 Table 4 3. List of ESUs. Putative ESUs are mark ed with a ?. Clade ESU Barnacle ID (if available) Localities Host IDs I A T. margaretae Red Sea; Iles Eparses Favia sp., F. favus, F. mathaei B Philippines; Taiwan Favia sp. C Tuamotus F. rotumana II A "Neotrevathana elongata" Red Sea; Iles Ep arses Echinopora gemmacea, E. hirsutissima B Philippines; Taiwan Echinopora sp., E. lamellosa III A T. mizrachae Red Sea; Oman Platygyra sp., P. lamellina B Guam P. pini C? Madagascar Platygyra sp. IV A Fiji; Cook Ids.; Society Ids.; Tua motus Montastrea curta B? Guam Favites russelli C "T. dentata" GBR; Fiji; Tuamotus Favia stelligera?, Leptastrea, Goniastrea D T. jensi Red Sea; GBR Favites abdita; F. halicora E "T. dentata" Red Sea Leptastrea transversa F? CNMI Goniastrea edw ardsi G? Vanuatu F. halicora H Guam; CNMI G. edwardsi I? Palau G. edwardsi V A? Pyrgopsella youngi Indonesia Symphyllia radians VI A T. sarae Red Sea; Seychelles; Philippines Cyphastrea sp., C. serailia, C. chalcidia B Guam Cyphastrea s p., C. serailia, VII A Hoekiini Philippines Hydnophora microconos B? Hoekiini Iles Eparses Hydnophora sp. C Hoekiini Philippines Hydnophora exesa VIII A? Guam F. stelligera IX A Society Ids.; Tuamotus "Cyphastrea sp."; Plesiastrea sp. X A So ciety Ids.; Tuamotus Acanthastrea echinata B T. paulayi Guam A. echinata XI A Cook Ids.; Society Ids.; Tuamotus F. stelligera B Guam; CNMI F. stelligera XII A Iles Eparses; Oman Plesiastrea sp., P. versipora, "Cyphastrea sp."

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155 Table 4 4. List of analyzed ESEs. Clade ESU1 ESU2 Geog. break Host switch? I I A I B WIO WP y I I B I C WP CP y II II A II B WIO WP y III III A III B WIO WP y VI VI A VI B WIO* WP n VII VII A VII C NA y X X A X B WP CP n XI XI A XI B WP CP n *VI -A predominantly from the WIO except for 1 Philippine sample

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156 Figure 41 RAxML phylogram for Trevathana s.l. Values above branches are ML bootstraps, values below branches are Bayesian posterior probabilities .

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157 Fig. 4 2. Geographic distribution of Clade I ESUs.

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158 Fig. 4 3. Geographic distribution of Clade II ESUs.

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159 Fig. 4 4. Geographic distribution of Clade III ESUs.

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160 Fig. 4 5. Geographic distribution of Clade IV ESUs.

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161 Fig. 4 6. Geographic distribution of Clade VI ESUs.

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162 Fig. 4 7. Geographic distribution of Clade VII ESUs.

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16 3 Fig. 4 8. Geographic distribution of Clade IX ESUs.

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164 Fig. 4 9. Geographic distribution of Clade X ESUs.

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165 Fig. 4 10. Geographic distribution of Clade XI ESUs.

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166 Fig. 4 11. Geographic distribution of Clade XII ESUs.

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167 Fig. 4 12. ESU ric hness in sampled localities. Well sampled locales are in red.

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168 Fig. 4 13. Occurrences of unfused (open circle) and fused (filled circle) opercular valves in the phylogeny. SEMs depict the inner surface of the tergum.

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169 CHAPTER 5 CONCLUSIONS The species r ichness of coral reefs has long captivated observers and has focused attention on the sources of this diversity My dissertation was motivated by a desire to use a molecular phylogenetic and phylogeographic approach to better understand factors promoting d iversification in IWP coral reefs. In order for this approach to be successful, two conditions are necessary: (1) the systematics of the group of interest must be well known, so that all lineages are represented in the study; and (2) sampling must be as comprehensive as possible across all lineages and across the entire geographic range of the group, so that boundaries between sister -ESUs can be accurately identified. These conditions were quite feasible for the first study (Chapter 2), because Calcinus is a taxonomically well studied group with relatively well delineated species boundaries; in addition, these hermit crabs are often conspicuous and locally abundant and hence easy to sample. However, applying the same approach to coral dwelling pyrgomatid bar nacles proved much more of a challenge since the taxonomy of the group is still in a state of confusion and many species remain undescribed; moreover, pyrgomatids are cryptic and more difficult to sample. Thus it was necessary to first understand the phylogenetic relationships of the family Pyrgomatidae before attempting a phylogeographic study. The systematics of the Pyrgomatidae were tackled in Chapter 3, and this in itself proved to be a very fruitful study. I discovered that morphological characters traditionally used to define the family and to delimit genera and species within the family (e.g., number of wall plates, opercular valve fusion) are highly homoplasious and therefore misleading indicators of phylogenetic relationships. I found that other mor phological

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170 characters that need to be considered in pyrgomatid evolution include the presence (or absence) of teeth on the anterior margins of the cirri, and the means by which the barnacle controls coral overgrowth. The pyrgomatids sensu stricto fell into three major clades that do not correspond to the traditional taxonomic groupings; one genus (Wanella ) fell outside of the pyrgomatids, and a non -pyrgomatid (the archaeobalanid Armatobalanus ) was resolved in Clade III of the Pyrgomatidae sensu stricto N otably, the genus I was studying phylogeographically ( Trevathana ) was not recovered as reciprocally monophyletic, and instead included genera with membranous bases (Pyrgopsella Hoekiini) and even euparasitic and highly apomorphic genera (Hoekiini). Had I not conducted this study, taxon sampling for the Trevathana study (Chapter 4) would have been less complete, and therefore the conclusions less robust. Studying the speciation patterns of Calcinus hermit crabs (Chapter 2) and Trevathana sensu lato pyrgoma tids (Chapter 4) offered interesting parallels and contrasts. In both systems, the mode of speciation was overwhelmingly allopatric (an allopatric mode of speciation was deemed most likely in all except one of the Calcinus ESEs, and in all of the Trevathan a s.l. ESEs). Young pairs of sister -ESUs are allopatric (shown by Calcinus and Trevathana s.l. data), and it appears that older sister -ESUs regain sympatry only after >2 my (shown by the Calcinus data). This supports the idea that speciation is mostly thro ugh geographic isolation, and secondary sympatry can only develop once sufficient time has passed for incipient species to develop barriers to interbreeding. The location of speciation events differed in the two systems. In Calcinus most of the recent spe ciation events occurred in remote CP archipelagos (Hawaii, Marquesas,

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171 etc.), or (less frequently) at the tropical -subtropical latitudinal boundary. The IO WP boundary was a relatively unimportant region for speciation in Calcinus In contrast, in Trevath ana s.l. all speciation events occurred somewhere between the WIO and WP, or between the WP and the CP. Thus ESU boundaries were completely non-concordant in the two systems studied. The reasons for this difference are currently unknown, but may be related to factors such as dispersal ability (the planktonic larval duration of Calcinus is not known; while in Trevathana s.l. the planktonic phase lasts approximately 2 weeks in the Society Ids., Malay unpub. data), ecological niches ( Calcinus appears to prefer oceanic reefs over continental settings; no such preference was observed in Trevathana s.l. ), the timing of speciation events (may not be concordant in the 2 systems), etc. Studying speciation in pyrgomatid barnacles allowed the examination of an extra l ayer of information: the role of host -switching in diversification. In Trevathana s.l. I found that more recent divergences are structured geographically, while older divergences are structured by host use. This suggests that both host switching and geogr aphic isolation cause diversification, but that these 2 forces act at different rates. Geographic isolation is a relatively rapid engine of diversification, thus recently diverged pairs of sister ESUs bear a geographic signature; however, this signature be comes erased through evolutionary time as species ranges change. In contrast host -switching is a much slower engine of speciation; switching events occur relatively rarely and thus the host signature is retained in the phylogeny for a longer time. It is a well known fact that the center of tropical marine biodiversity is located in the so -called Indo -Malayan coral triangle, bordered by Indonesia, the Philippines and New Guinea. In multiple taxa (reef fish, scleractinian corals, gastropods, seagrasses, etc .;

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172 reviewed in Hoeksema 2007) diversity steeply declines in all directions away from this hotspot. And yet I did not find a diversity hotspot in the coral triangle for either Calcinus nor Trevathana s.l This shows that the coral triangle biodiversity peak is not consistent across all groups or across all taxonomic scales. I propose that if one were to compile species occurrence data for all diogenid hermit crabs, or for all pyrgomatid barnacles, one may still observe a hotspot in the coral triangle (such d ata are not yet available for those groups). Such an observation would be further demonstration of heterogeneity in patterns of diversification in the IWP. Overall, studies are showing that divergence patterns of individual lineages do not match well worn models put forth to explain the origin of Indo West Pacific diversity (such as those discussed in Chapter 1). Rather, the overall patterns are simply summations of disparate evolutionary histories, and serve to underscore the complexity of marine diversifi cation

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186 BIOGRAPHICAL SKETCH Maria Celia (Machel) Defrance Malay was born and (mostly raised) in the Philippines, to her French mother, Odile Defrance, and her Filipino father, Badi Malay. She is the youngest of three children. Fascinated with nature from an early age, Machel complet ed a Bachelor of Science in biology from the University of the Philippines Diliman in 1998. At that point she knew she wanted to pursue research in the biological sciences, but still had no clue what particular field to specialize in. However, an opportunity to take scuba-diving lessons opened her eyes to the beauties and mysteries of life in the oceans, and she quickly decided to take the plunge into marine biology. Machel worked as a research assistant at the University of the Philippines Marine Scien ce Institute for 3 happy years, an experience that let her to discover the astounding diversity and heterogeneity of coastal ecosystems in the Philippines. Eventually, this led her to undertake a PhD in Zoology with Dr. Gustav Paulay at the Florida Museum of Natural History, specializing in the systematics, speciation, biodiversity, and biogeography of coral reef associated invertebrates. Machel completed her doctorate in August 2010.