Bivalvia: Cardiidae) Nathanael D

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2013 Molecular Phylogenetics and Historical Biogeography of Cockles and Giant Clams (Bivalvia: Cardiidae) Nathanael D. Herrera

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COLLEGE OF ARTS AND SCIENCES

MOLECULAR PHYLOGENETICS AND HISTORICAL BIOGEOGRAPHY OF COCKLES

AND GIANT CLAMS (BIVALVIA: CARDIIDAE)

NATHANAEL D HERRERA

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Summer Semester, 2013 Nathanael D. Herrera defended this thesis on June 18, 2013. The members of the supervisory committee were:

Scott Steppan Professor Directing Thesis

Don Levitan Committee Member

Austin Mast Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements.

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Scott Steppan, whose support, guidance, and invaluable expertise made it possible for me to do this research. I would also like to thank my committee members, Dr. Don Levitan and Dr. Austin Mast, whose patience and helpful criticisms were essential to this work. In addition, I am thankful to Dr. John Schenk and Mr. Kenny Wray for providing stimulating discussion, advice, and encouragement while being at FSU. I would also like to thank my fellow colleagues working on the Bivalves in Time and Space project (BiTS), Jan Johan ter Poorten, Rüdiger Bieler, John Hulsenbeck, Nick Matzke, David Jablonski, Paula Mikkelsen, Rafael Robles, and André Sartori, who not only helped make this research possible but taught me an immense amount about bivalves.

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TABLE OF CONTENTS

List of Tables...... v

List of Figures ...... vi

Abstract ...... vii

1. MOLECULAR PHYLOGENETICS AND BIOGEOGRAPHY OF COCKLES AND GIANT CLAMS (BIVALVIA: CARDIIDAE) ...... 1

2. HISTORICAL BIOGEOGRAPHY OF A MARINE BIVALVE (BIVALVIA: CARDIIDAE): GLOBAL PATTERNS OF ORIGINATION AND DISPERSAL ...... 25

APPENDICES...... 44

A.1 SAMPLING AND VOUCHER INFORMATION FOR THE CARDIIDAE USED IN THIS STUDY...... 44

B.1 CARDIID SEQUENCES INCORPORATED INTO THIS STUDY FROM GENBANK. .50

REFERENCES...... 54

BIOGRAPHICAL SKETCH...... 61

LIST OF TABLES

1.1 Primers used in this study...... 16

1.2 Results of the Bayesian inference analysis by gene, the number of generations the chains were run, the number of generations discarded as the burn-in period, and the split frequencies of each run...... 17

2.1 Fossil calibrations used in the BEAST analysis...... 33

2.2 Clade ages estimated by BEAST for 14 focal nodes with upper and lower 95% highest posterior density...... 34

A.1 Sampling and voucher information for Cardiidae representatives used in this study...... 44

B.1 Cardiid sequences incorporated into this study from Genbank...... 50

LIST OF FIGURES

1.1 Phylogenetic relationship of the Cardiidae based on shell morphology and microstructure. Redrawn from Schneider and Carter (2001), Schneider (2002). Crosses indicate extinct taxa...... 17

1.2 Biogeographic regions used in this study of the Cardiidae ...... 18

1.3 16S Maximum-likelihood phylogram...... 19

1.4 28S Maximum-likelihood phylogram...... 21

1.5 Histone 3 Maximum-likelihood phylogram...... 22

1.6 Maximum-likelihood phylogram of concatenated dataset for three genes (His 3, 16S, 28S)...... 23

1.7 Biogeographic reconstruction cladogram...... 24

2.1 Biogeographic regions used in this study of the Cardiidae ...... 35

2.2 Paleogeographical model used in the Cardiidae analysis...... 46

2.3 Chronogram of Cardiidae produced from the BEAST analysis...... 39

2.4 Biogeographical reconstruction of ancestral ranges in the Cardiidae ...... 41

ABSTRACT

This study produces a nearly comprehensive large-scale molecular phylogeny for the marine bivalve family Cardiidae (cockles and giant clams) and uses this topology to examine patterns of diversification in the marine realm. This study uses maximum-likelihood and Bayesian phylogenetic analyses of two nuclear (Histone 3, 28S) and one mitochondrial (16S) gene for 110 species representing 37 of the 44 recognized genera and all eight extant subfamilies. Lineage divergence times were estimated using Bayesian estimation with uncorrelated relaxed rates among lineages (BEAST). To reconstruct ancestral geographic ranges, I used the dispersal- extinction-clodegenesis method (Lagrange) with a stratified paleogeographic model in which dispersal rates were scaled according to area connectivity across three time slices. The resulting topologies are discussed with respect to traditional subfamilial classifications, and previous anatomical and molecular findings. I confirm the monophyly of two subfamilies, Tridacninae and Clinocardiinae as previously defined, but found there to be rampant paraphyly/ polyphyly of the other six subfamilies. The Cardiidae seem to have originated in the tropical Indo-Pacific some time in the early Cretaceous and diversified within the tropical Pacific. The extant diversity seen in the Atlantic is derived from species that dispersed from the tropical Indo-Mediterranean region during the Cenozoic via the Tethys sea.

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CHAPTER ONE

MOLECULAR PHYLOGENETICS AND BIOGEOGRAPHY OF COCKLES AND GIANT CLAMS (BIVALVIA: CARDIIDAE)

Introduction The bivalve family Cardiidae (Cockles and Giant clams) comprises about 250 extant species arranged in ca. 80 genera and eight subfamilies with the oldest fossil representative (Tulongicardiinae) dating back to the Norian (ca. 216 mya) (Lydeard and Lindberg 2003, Ponder and Lindberg 2008). Cardiids inhabit tropical to polar seas worldwide with the bulk of extant taxa distributed throughout tropical-subtropical seas. They are mainly shallowly infaunal to epifaunal in soft sand or mud in water depths up to 500 m. Typically, cardiids are suspensions feeders, but some are highly specialized, such as Tridacna, Corculum, and Fraginae, which form an endosymbiosis with dinoflagellate protists (Maruyama et al. 1998, Schneider 1998b, Kirkendale 2009). The majority of our taxonomic understanding is based upon gross morphological features of the shell and some soft anatomy (Keen 1969, Kafanov 1980, Keen 1980, Voskuil and Onverwagt 1991a, b, Schneider 1992, Vidal 1999, 2000, Schneider 2002, Savazzi and Salgeback 2004), shell microstructure (Carter and Schneider 1997, Schneider and Carter 2001), or phylogenetic studies combining these characters (Schneider 1995, 1998a, b, Nevesskaja et al. 2001, Schneider 2002). However, studies incorporating molecular data are few and restricted to few taxa (Maruyama et al. 1998, Schneider and Foighil 1999, Nikula and Vainola 2003, DeBoer et al. 2008, Kirkendale 2009). Despite the attention the family has received, the phylogeny of the family remains poorly understood, and its classification is still largely based on gross shell morphology and soft anatomy. The classification of cardiids remains incomplete, especially regarding the subfamilial and generic ranking of many groups. Stewart (1930) divided the family Cardiidae into three subfamilies based on shell morphology (Cardiinae, Trachycardiinae, and Fraginae). Kafanov and Popov’s (1977) treatment of Cardiidae divided the family into six subfamilies (Cardiinae, Clinocardiinae, Fraginae, Hemidonacinae, Protocardiinae, and Lymnocardiinae). Keen (1980), who was skeptical of Kafanov and Popov’s (1977) classification because it rested on a single character (microstructure of the shell), divided the Cardiidae into five subfamilies (Cardiinae, Fraginae, Laevicardiinae, Protocardiinae, and Trachycardiinae)

1 based on both shell characteristics and soft anatomy. Further, Keen considered the ‘problematic’ Eurasian brackish-water groups (Cerastoderma, Laevicardium, Parvicardium, Nemocardium, and Cardium s.s.) to be a separate family, the Lymnocardiidae. Hemidonacinae, a family consisting of the monotypic Hemidonax, was added by Iredale and McMichael (1962), and was recognized by Keen (1980) but not by Kafanov and Popov (1974). Further, Ponder et al. (1981) considered Hemidonax sufficiently distinct to justify recognition of the group in a separate family based on morphology and anatomy. Healy et al. (2008) compared sperm ultra structure of Hemidonax with other heterodonts (Cardiidae and Donacidae) and confirmed that Hemidonax should be retained in Hemidonacidae. Despite the evident inconsistencies of cardiid classifications based on traditional shell and soft anatomical character, current recognition of cardiid subfamilies (e.g. Schneider and Carter, 2001) is still largely based on shell and soft anatomical characters. Schneider and Carter (2001) devided the Cardiidae into eleven subfamilies (7 extant and 4 extinct) and erected Orthocardiinae to include Freneixicarda, Afrocardium, and Europicardium only later (Schneider 2002) (Fig. 1.1). There have been few attempts to reconstruct phylogeny based on molecular data and most studies have focused on specific genera or subgroups of cardiids, especially zooxanthellate cardiids (Tridacniinae, Fraginae). Further, most studies suffer from sparse sampling of both taxa and genetic markers. Schneider and Foighil (1999) investigated the generic placement of tridacnids using mitochondrial 16S and found the Tridacninae to be monophyletic with respect to other zooxanthellate cardiids. However, their sampling consisted of a single exemplar of each species. Maruyama et al. (1998) analyzed the phylogenetic relationship of zooxanthellate bivalves belonging to the genera Tridacna, Hippopus, Fragum, and Corculum, as well as the azooxanthellate bivalves (Vasticardium and Fulvia). Again, using only a single representative of each species and a single genetic marker (18S rDNA). Maruyama et al. (1998) found the tridacnids to be more closely related to the azooxanthellate cardiids than to Fragum or Corculum and that Tridacninae is either a member of the Cardiidae (as a subfamily) or the Cardiidae is paraphyletic. Kirkendale (2009) analyzed the relationships of the Fraginae using multiple genetic markers (4 genes; 16S, 28S, COI, CytB) and found them to be paraphyletic with respect to Parvicardium and Papillicardium being nested within a European derived clade composed of three different cardiid subfamily members. This constrasts greatly with previous work (Stewart 1930, Keen 1980, Voskuil and Onverwagt 1991a, Schneider and Carter 2001) based on gross

2 morphology. Thus, it is apparent that incorporating more molecular data with denser sampling may contribute to creating a phylogenetic framework for further investigations of the biology and evolutionary history of the Cardiidae. Additionally, with the incorporation of more data and denser sampling, I may be able to resolve much of the conflict between the morphological and limited molecular data. The major goals of this paper are twofold. First, I present the first comprehensive large- scale molecular phylogeny of the Cardiidae, based on sampling across all eight subfamilies using two nuclear and one mitochondrial genes, which have already been shown to be useful in resolving relationships at the family-level in bivalves (Giribet and Wheeler 2002, Kappner and Bieler 2006, Mikkelsen et al. 2006, Kirkendale 2009). I test the monophyly and composition of proposed subfamilies and discuss the phylogentic position of some controversial taxa such as Dinocardium. Second, I explore the geographic structure of present day marine diversity. I test for the impact of the vicariant break-up of the Tethyan sea and the establishment of present day marine ecoregions.

Materials and Methods Sampling and Gene Sequencing Tissue samples were gathered from museum material or personal collections (Jan Johan ter Poorten). One hundred and ten species representing thirty-seven of the forty four recognized genera and all eight extant subfamilies were sampled, yielding a broad and robust sampling of the family at the genus level (Appendix A). To insure proper identification of sampled specimens, vouchers where examined by Jan Johan ter Poorten when possible. Specimens were sequenced for three genes: mitochondrial large subunit ribosomal RNA gene 16S (~ 400 bp), and portions of two nuclear genes: Histone 3 (~400 bp) and 28S (~1200 bp). Each gene has been shown to resolve family-level questions within bivalves and are relatively easy to sequence with previously designed primers (Giribet and Wheeler 2002, Kappner and Bieler 2006, Mikkelsen et al. 2006, Kirkendale 2009). In addition to our sampled material, I added 101 sequences for 16S and 28S downloaded from genbank for any available cardiids (Appendix B). The addition of previously published sequences will further add resolution at the species level.

DNA Extraction and PCR All tissues had been preserved in 70-100% ethanol, lysis buffer, or were frozen prior to DNA extraction. However, for older material from museum collections, information on the long- term preservation history was often unavailable. It is possible that some of the tissues were initially preserved in a formalin fixture. Total genomic DNA was extracted by either standard CTAB and phenol-chloroform extraction protocol for molluscan tissue (Brown et al. 1996) or E.Z.N.A. mollusc DNA extraction kit (Omega Bio-Tek. Inc.) following the manufacturer’s instructions. When possible, muscle tissue from the foot or adductor muscle was used; for specimens < 1 cm, the entire body was used. I conducted 25 uL PCR reactions with gene-specific primers (Table 2), which included ca. 25 - 50 µg of DNA template, 5 uL of 10x buffer, 1.5 uL of 2.5 mM dNTPs, 1 uL of 10uM solution of each primer, 0.5 - 2 uL of 25mM magnesium chloride solution, 0.25 uL TAQ, 0.35 uL bovine serum albumin (BSA), 3.5 uL Bataine. Cocktails were then brought up to 25 uL with dDI water. Reactions were run for 35 - 40 cycles for all genes with the following parameters, for 16S rRNA: an initial 2-minute denaturation at 94ºC, further denaturation at 94ºC for 35 seconds, with annealing 40 - 46ºC for 30 seconds. A range was used to fine-tune amplification of problematic specimens. Extension at 72ºC for 1 min. and a final extension for 10 min. at 72ºC, for the nuclear genes: an initial 2 minute denaturation at 94ºC; further denaturation at 94ºC for 32 seconds, with annealing 50 - 58ºC for 45 seconds (28S), 50 - 55ºC for 55 sec. (Histone 3) and extension at 72ºC for 1.5 min, with a final extension for 10 min. at 72ºC. PCR products were visualized and photo documented on an agarose gel with ethidium bromide. Successful amplifications were then prepared for sequencing using enzymatic digestion with ExoSAP-IT (USP, Cleveland, USA). Complementary strands were sequenced using PCR primers by automated sequencing on an ABI 3100 machine using big-dye terminator chemistry (Applied Biosystems).

Gene Alignment Sequences were edited and initially aligned by eye using Sequencher 3.1.1. (Gene Codes). Protein encoding Histone 3 sequences were translated into amino acids and modified by eye using MacClade v4.08 (Maddison 2003) and were easily aligned with no indels present. Multiple sequence alignments for 28S and 16S were performed in CLUSTAL X (Larkin et al. 2007) using default pair-wise alignment parameters of gap opening and gap extension penalties.

Alignments were then manually adjusted by eye in MacClade. To account for the hypervariable regions in the 16S and 28S gene alignments, I conducted two tiers of alignments. Tier 1 (T1) consisted of all data with hypervariable regions left in the alignments. For Tier 2 (T2), I removed the hypervariable sites using GBLOCKS v0.91b (Castresana 2000), which identifies well aligned and conserved regions that are suitable for phylogenetic analyses. In order to retain as much data as possible, I used the least stringent settings in Gblocks to remove ambiguous regions. This resulted in a final data set of 694 bp from 2253 for 28S, and 383 bp from 690 for 16S.

Phylogenetic Analyses All analyses were conducted using Maximum Likelihood (ML) and Bayesian (BI) approaches as implemented in RAxML (Stamatakis 2006) and MrBayes v 3.0 (Huelsenbeck and Ronquist 2003), respectively. All ML and BI analyses were run with partitions. For Histone 3, I partitioned by codon position as well as the first and second position combined. For the concatenated data set, I used five partitions: one for each ribosomal gene and then the codon partitioning used for Histone 3. All Ml analyses were conducted on each gene individually (two tiers for 16S and 28S) and a concatenated dataset. For the concatenated dataset, I combined the three genes using T1 from 16S and 28S. I excluded T2 alignments from the concatenated dataset because I found no significant difference between T1 and T2 gene trees (not shown). 100 replicate ML inferences were performed using a random starting tree and included the GTRGAMMA option (-m). To assess clade support, I conducted 1000 bootstrap replicates with the GTRCAT option (-m). To find the best-fit model of evolution for BI analyses, I used the Akaike Information Criterion in jModelTest (Posada 2008). Partitioned Bayesian analyses were conducted with default priors on individual genes and the concatenated data sets using the GTR + I + Γ model with all parameters estimated for each partition separately using the same partitioning as in ML analyses. All analyses were run with a random starting tree for 35,000,000- 80,000,000 generations (Table 1.2) with tree parameters recorded every 1000 generations. Convergence and stationarity was assessed by comparing the likelihood scores of the MCMC chains in the software Tracer v.1.4 In all analyses, we excluded the first 10% of the MCMC chains as the burn-in generations. The results of BI analyses were summarized with TreeAnnotator v1.6.1 (Drummond and Rambaut 2007) on the maximum clade credibility tree for the ML topology of the concatenated dataset.

Geographic Reconstruction To reconstruct the geographic history of the Cardiidae, I coded each species according to its presence in a marine ecoregion as defined by Spalding et al. (2007). However, I separated the temperate and tropical Atlantic and Pacific into east and west regions (Fig. 1.2). Further, because there are no cardiids found within the Southern ocean or temperate South America, I excluded these regions. Reconstructions were estimated with Mesquite v. 2.72 under a parsimony framework with regions treated as unordered characters. In this case, I used the final ML tree from our concatenated dataset and the character matrix of geographic distributions.

Results Single Gene Analyses Bayesian and maximum likelihood analyses are highly congruent for each locus. I found no significant differences in analyses with and without hypervariable regions (tier 1 and tier 2 analyses; but see 28S below; Not shown). Because there is no strong conflict between ML and BI analyses, only ML analyses of total data sets (tier 1) are shown. Of the three genes, Histone 3 conflicts most with the others; however, many of the conflicting nodes received weak support. 16S and 28S generally agree regarding deeper level relationships (16S, Fig. 1.3; 28S, Fig.1. 4). 16S: The 16S analyses resulted in eight well-supported clades (labeled “1-8” Fig. 1.3). The first clade (clade 1) to diverge consists of Microcardium, Trifaricardium, Frigidocardium, and Lophocardium from the subfamily Laevicardiinae. Following this, clades 2 and 3 diverge and are sister to the remaining cardiids. Within clade 2, it appears the Clinocardiinae first diverged and form a well-supported clade (BP: 11%, PP: 0.99). Fulvia is paraphyletic with Fulvia lineonotata sister to Laevicardium lobulatum (BP: 100%, PP: 1.0). Cardium + Europicardium form a well-supported clade sister to Fulvia, which together are sister to Freneixicardia. Clade 3 contains three distinct clades; the first to diverge contains most of the Caribbean Laevicardium. Dinocardium is sister to Laevicardium senegalense and they are the next clade to have diverged within clade 3. The Trachycardiinae form the rest of clade 3 and are paraphyletic due to Laevicardium biradiatum and Laevicardium attenuatum (Laevicardiinae) being nested within the core trachycardiids. Tridacninae (clade 4) is monophyletic (BP: 100%, PP: 1.0) and is sister to clade 5, a ‘Ctenocardia’ and ‘Trigoniocardia’ groups. Clade 6 is well supported and consists of Lyrocardium + Afrocardium. Clade 7 is sister to clade 8 and is of

European origin and contains Parvicrdium, Acanthocardia, Papillicardium, and the Lymnocardiinae. Clade 8 is Kirkendale’s (2009) ‘Fragum’ group that includes all species in Fragum, Corculum, and Lunulicardia. Within clade 1, none of the genera were monophyletic. Lophocardium (represented by one of two known species) is nested within Frigidocardium and is the sister group to F. helios and F. torresi. Trifaricardium (also represented by one of two known species) is nested within Microcardium. Frigidocardium kirana is also nested within Microcardium and is sister to Microcardium pazianum. Clinocardiinae was monophyletic and is the sister clade to the rest of clade 2. However, the monophyly of most of the genera is untested because only one species for each genus was sampled. The exception is Keenocardium, and it was found to be paraphyletic (with strong support; BP: 100%, PP: 0.99), with C. beullowi sister to the rest of the subfamily. The rest of clade 2 consists of Freneixicardia victor, a paraphyletic Fulvia + Laevicardium lobulatum. The Trachycardiinae (clade 3) is paraphyletic with respect to a Laevicardium group (excluding L. lobulatum), and Dinocardium. Vasticardium is monophyletic with strong support but its placement is uncertain. The ML analysis resulted in a polytomy, while the BI analysis placed Vasticardium sister to a clade of Acrosterigma and Laevicardium with weak support (PP: <0.5). The only other monophyletic genus within clade 3 was Papyridea. The Tridacninae was one of two subfamilies found to be monophyletic with strong support (BP: 100%, PP: 1.0) but is only represented by 16S. The subfamily consists of two monophyletic genera, Hippopus and Tridacna. Within Tridacna, T. derasa is paraphyletic with a Genbank T. gigas being identical to one T. derasa sampled, and the other being the sister clade to a clade containing T. crocea + T. squamosa, and T. maxima. Ctenocardia was paraphyletic. However, Ctenocardia biangulatum and C. media were sister species (both formerly known as Americardia). Apiocardia was sister to Trigoniocardia in the ML analysis but was sister to a clade containing Trigoniocardia + Ctenocardia in the BI analysis. All 16S analyses supported Lyrocardium as sister to Afrocardium with strong support (BP: 98%, PP: 1.0). However, both genera are under sampled and these results were not supported by all genes (see below).

In clade 7 Acanthocardia was recovered as monophyletic by both ML and BI analyses and is sister to Pappillicardium papillosum. Lymnocardiinae is strongly supported as the sister group to the Acanthocardia + Papillicardium clade (BP:69%, PP: n0.95). The Indo-West Pacific ‘Fragum’ group, consisting of Fragum, Lunulicardia, and Corculum, was strongly supported by both BI and ML analyses (BP: 95%, PP: 1.0). Corculum monophyletic while Lunulicardia and Fragum were paraphyletic. 28S: Analysis of 28S (Fig. 1.4) recovered a similar topology to 16S; however, it is our least sampled gene and there are some differences. The Clinocardiinae are monophyletic with strong support (BP:100% PP:1.0) but are sister to a Trachycardiinae + Dinocardium and Laevicardium clade (clade 2 in the 16S tree, Fig. 1.3). The Orthocardiinae are sister to a Fraginae clade (which includes Parvicardium, Acanthocardia and the Lymnocardiinae) but with weak support (BP: < 50% PP: 0.71). This contrasts the 16S tree, that found the Orthocardiinae nested within clade 2. The 16S clade 8 (the ‘Fragum’ group) was recovered with strong support as monophyletic (BP:100% PP:1.0) and is nested within a clade consisting of Acanthocardia, Parvicardium, and the Lymnocardiinae (clade 7 16S, Fig.1.3). Histone 3: Many of the deep relationships were found to be discordant between Histone 3 and the other two gene trees (compare Figs. 1.3, 1.4, 1.5). The most basal clade to the Cardiidae consists of Microcardium, Trifaricardium, and Frigidocardium, which is consistent among all gene trees. However, Scissula similis and Macoma balthica, which belong to the family Tellinidae are nested within the ingroup. Dinocardium robustum is the sister clade to the Fragum group which contrasts both 16S and 28S, were D. robustum was sister to Laevicardium. In addition D. robustum and the Fragum group received weak support as the sister clade to Vasticardium. The Ctenocardia and Trigoniocardia group (clade 5 16S, Fig.1.3) is also paraphyletic. C. fornicata, C. translata, and Microfragum festivum form a strongly supported clade (BP: PP: ) that is sister to Lyrocardium lyratum + ((Lymnocardiinae + Parvicardium) + (Acanthocardia + Papillicardium)). C. media is strongly supported as sister to ((C. biangulatum, + T. granifera) + the Orthocardiinae (excluding Europicardium, which was found to be sister to Cardium). Fulvia was monophyletic with moderately strong support (BP: 75 PP: 100 ) but F. lineonotata, which consistently was sister to Laevicardium lobulatum (16S Fig.1.3, 28S Fig.1.4 ), was not sequenced for this gene. Clinocardiinae was monophyletic with strong support (BP:

100; PP: 92 ) but nested within a clade consisting of Europicardium, Cardium, and Bucardium.

Combined Dataset Six distinct clades of cardiids can be recognized (labeled “A” through “F” in Fig. 1.6) and are well supported by both ML and BI analyses for the concatenated dataset, with three of the six clades (clades A, D, F) exceeding 95% BP and 0.95 PP. One clade (clade B) received moderate support based on the Bayesian posterior probabilities (PP: 0.87), but received low supported by bootstrapping (<50). The remaining two clades (clades C, E) are weakly supported (Fig. 1.6), due to uncertainty in the branching structure of clades C, D, and E. The first named clade to diverge (clade A) is a clade consisting of a Laevicardiinae group. Within this clade, Frigidocardium and Lophocardium is sister to Microcardium and Trifaricardium. Support for these two clades is weak due to the uncertain placement of Lophocardium (BI analyses recovered it as sister to Microcardium and ML analyses recovered it as sister to Frigidocardium). The second named clade to diverge (clade B) consists of species from five subfamilies; all polyphyletic; in two subclades (labeled “1-2”); Freneixicardia and Afrocardium (Orthocardiinae), and Lyrocardium (Laevicardiinae) were sister to Acanthocardia (Cardiinae), Papillicardium and Parvicardium (Fraginae), and Monodacna + Cerastoderma (Lymnocardiinae). Clade 1 was found to have a robust branching structure (all clades received moderate to strong support) while many clades in clade 2 were weakly supported (Fig. 1.6). Freneixicardia is strongly supported as the first to diverge from the rest of subclade 1, with strong support for Afrocardium being sister to Lyrocardium (BP: 66%, PP: 0.96). Within subclade 2, the overall branching structure is robust with the exception of Parvicardium, which was found to be paraphyletic. Parvicardium vroomi, P. scriptum, and P. exiguum form a strongly supported clade, and diverged first from the rest of subclade 2. Following this, Parvicardium minimum diverged. However, because P. minimum is only represented by one gene (16S) the branching order is uncertain. There is also uncertainty of the branching structure of the Lymnocardiinae. ML analyses placed Monodacna as the first to diverge from a clade consisting of Cerastoderma + (Acanthocardia + Papillicardium) where BI analyses (tree not shown) recovered a monophyletic Lymnocardiinae being sister to Acanthocardia + Papillicardium. Acanthocardia is strongly supported as sister to Papillicardium in both BI and ML analyses (BP: 74%, PP: 0.96).

Clade C consists entirely of Fraginae and contains two subclades (3,4). Subclade 3 consists of a strongly supported ‘Ctenocardia’ and ‘Trigoniocardia’ groups and is sister to subclade four, a clade comprised of Kirkendale’s (2009) ‘Fragum’ group, which includes all species in the genera Fragum, Corculum, and Lunulicardia. The Tridacniinae was found to be monophyletic (clade D), with Hippopus and Tridacna reciprocally monophyletic. The placement of the Tridacniinae (clade D) is uncertain and is weakly support to have diverged first from clade E + F (ML analyses). However, BI analyses recovered clade D as sister to clade C. The remaining two clades (clades E and F) form two well-supported sister clades (However, clade E received low BP). Within clade E, the Clinocardiinae diverged first, followed by a split between a clade consisting of Fulvia lineonotata + Laevicardium lobulatum and the rest of Fulvia + (Cardium (Bucardium, + Europicardium)). Within clade F, the Caribbean Laevicardium + Dinocardium diverged from a well-supported Vasticardium (BP: 95%, PP: 1.0) + the Indo-Pacific Laevicardium + Acrosterigma, Phlogocardium, Dallocardia, Mexicardia, and Papyridea.

Biogeographic Analysis The biogeographic reconstruction on the combined data tree suggests a Central Indo- Pacific origin for the Cardiidae (Fig. 1.7). Clade A consists of a “Laevicardium” group, which is primarily found throughout the Central Indo-Pacific (CIP). Lophocardium cumingi and Microcardium pazianum dispersed out of the CIP across the Pacific and are found in the Eastern Tropical Pacific (ETP) and Frigidocardium centumliratum dispersed from the CIP and into the Western Indo-Pacific (WIP). The remaining taxa are either restricted to the CIP or are widespread and occupy the CIP + WIP or the CIP + Western Temperate North Pacific (WTNP). Clade B is of CIP origin with subclade 1 having widespread taxa ranging throughout the WIP, CIP, and the WTempNP. However, Lyrocardium aurantiacum is solely found in one biogeographic region, the CIP. Subclade 2 dispersed from the CIP and is found primarily in the Eastern Temperate Atlantic (ETempA). However, Papillicardium turtoni is restricted to Temperate South Africa (TempSA), while Papillicardium papillosum and Acanthocardia tuberculata are distributed in both the ETempA and Eastern Tropical Atlantic (ETrP). Clade C (Fraginae) is primarily CIP. However, subclade 3 is comprised of an ETrP clade, which includes Trigoniocardia granifera, Apiocardia obovalis and Americardia biangulatum.

Ctenocardia media and A. obovalis + T. granifer, are distributed in the Western Tropical Atlantic (WtrA). The remaining taxa in subclade 3 are primarily CIP/WIP. However, Ctenocardia gustavi is found in the WTrA. The core Fragum group (subclade 4) is primarily CIP and WIP with Fragum aff. mundum and Fragum mundum being co-distributed in the CIP and Eastern Indo- Pacific (EIP). Of the core Fragum group, only Fragum unedo is co-distributed in the CIP and ETempNP. The Tridacninae (clade D) are restricted to the CIP and southern most portions of the WTempNP. Clade E geographically breaks down into three clades. Subclade 5 is made up of the Clinocardiinae, which are broadly distributed throughout the Arctic Ocean. Subclade 6 consists of Europicardium from the ETempA, and Cardium and Bucardium from the ETrA. subclade 6 is sister to the Fulvia group, which is primarily CIP/WTempNP. Clade F is primarily of CIP origin with two primary dispersals from the CIP. The first dispersal is of subclade 7, which comprises the WtrA species of Laevicardium + Dinocardium. Within subclade 7, Laevicardium senegalense and Laevicardium pictum dispersal from the WTrA and are distributed in the ETrA. Further, Laevicardium elenense also dispersal from the WTrA and is found in the ETrP. The remaining Laevicardium (which are shown to be very divergent; Fig.1.4) are distributed in the CIP along with Vasticardium , which are all found to be CIP with the exception of V. insulare, V. vertebratum, and V. lacunosum. Finally, a clade including Papyridea, Mexicardia, Dallocardia, Trachycardium, and Phlogocardium dispersal from the CIP into the ETrP with P. semisulcata, A. magnum, and T. egmontianum further dispersing into the WTrA.

Discussion Our study represents the most comprehensive phylogenetic analysis of the Cardiidae to date and indicates the need for major taxonomic revisions at the subfamilial and generic level. I found little agreement with previous cardiid classifications (Kafanov and Popov 1977, Keen 1980, Schneider and Carter 2001). Of the eight subfamilies, of Schneider & Carter (2001) and Keen (1980), only two (Clinocardiinae and Tridacninae) were recovered as monophyletic (Fig. 1.6) by our study. With the majority of cardiid systematics based on morphological characters and limited taxonomic sampling for molecular data, it is not unexpected to find some discordance due to convergence of characters. It is well known that morphological characters are

11 subject to convergence, especially as a response due to similar selective pressures (i.e. predation, habitat preference, thermal tolerances) and is often observed in bivalves (Vermeij 1980, Schneider and Carter 2001). However, the degree of conflict between our results and previous work is surprising given that systematists work to avoid characters that may be under selection and morphological analyses seems to be in broad agreement across multiple studies using multiple independent datasets. The Laevicardiinae s.l. (e.g. Keen, 1980) is polyphyletic with the majority of genera distributed across three significant clades: Clades A, part of clade 6, and clade 7 of the combined data set (Fig. 1.6). Schneider (1995) placed Fulvia, Frigidocardium, Lophocardium, Microcardium and Trifaricardium and Nemocardium in Laevicardiinae and recovered Laevicardiinae as sister to the rest of the Cardiidae. The first clade to diverge based on our results is clade A and consists of the ‘Frigidocardium group’, which is made up of Frigidocardium, Lophocardium, Microcardium and Trifaricardium but not Fulvia. Fulvia is paraphyletic and forms a large subclade within clade 6. Fulvia lineonotata is not nested within Fulvia and is sister to Laevicardium lobulatum, which together are sister to the core Fulvia + a clade consisting of Europicardium, Bucardium, and Cardium. However, ter Poorten (2009) noted morphological similarities between Fulvia lineonotata, Laevicardium lobulatum, and Pseudofulvia caledonica and further suggests that F. linonotata and L. lobulatum are in fact Pseudofulvia (ter Poorten un pub. data). Laevicardium, the type genus of the subfamily, was also polyphyletic. The Caribbean Laevicardium form a clade that includes Dinocardium robustum, which has long been thought to be a member of Laevicardiinae (Keen 1969, 1980). It was moved into Cardiinae (Schneider 1992, Schneider and Carter 2001) due to its similarity in shell morphology. However, it is clearly closely related to other Caribbean Laevicardium. There has been much contention around the placement of the Indo-Pacific derived taxa that have traditionally been assigned to Laevicardium. Based on gross morphology they share a resemblance with Laevicardium. However, analyses of microstructural characters suggests otherwise (Wilson and Stevenson 1977, ter Poorten 2009). ter Poorten (2009) considered their inclusion into Laevicardium doubtful and more likely a case of morphological convergence rather than resulting from phylogenetic relationship but made no further suggestions. I found both L. attenuatum and L. biradiatum as a clade sister to the Trachycardiinae. Our results indicate that Vidal (1999) was correct in placing both L. attenuatum and L. biradiatum in

Acrosterigma. Further, Lyrocardium is not sister to any other genus belonging to Laevicardiinae but is closely related to Afrocardium and Freneixicardia (Orthocardiinae; Fig 6, clade B-1). Thus, its inclusion in Laevicardiinae is doubtful. Keen (1980) and Kafanov and Popov (1977) considered Europicardium as a subgenus of Bucardium and Cardium, respectively. However, Schneider (2002) elevated Europicardium to full generic rank and placed Europicardium in Orthocardiinae with the newly erected Freneixicardia and Afrocardium and found this clade sister to Cardium and Bucardium. It is evident that Europicardium should be moved to the subfamily Cardiinae as it is sister to Cardium +Bucardium and not sister to any member of Orthocardiinae (Fig. 1.6). Afrocardium, which has been a cause of confusion and little studied (but see ter Poorten 2009), was historically thought to be a member of the Fraginae but Schneider (2002) showed that it shares no derived characters with any member of the Fraginae and is instead closely related to Freneixicardia and a member of Orthocardiinae (due to similarity to the extinct Agnocairia). Our results confirm the findings of Schneider (2002) that Afrocardium is closely related to Freneixicardia (and recommend its inclusion into Orthocardiinae). The Fraginae are not supported as monophyletic, and our data confirm the general findings of Kirkendale’s (2009) molecular study. I recovered a core ‘Fragum’ group, which includes all species in the genera Fragum, Corculum, and Lunulicardia as well as a Ctenocardia + Trigoniocardia group, which are sister to the core ‘Fragum’ group. Of the remaining fragine taxa, there has been disagreement in the placement of and within the earliest diverging fragines, Parvicardium and Papillicardium. Kirkendale (2009) showed both genera to be part of a clade that included other European derived cardiids, which included species from three different subfamilies (Cardiidinae, Fraginae, and Lymnocardiinae). Our analyses consistently recovered a well-supported European clade comprised of both Parvicardium and Papillicardium plus Acanthocardia (Cardiidane), Cerastoderma, and Monodacna (both Lymnocardiinae), further supporting the finding of Kirkendale (2009). However, our results indicate Papillicardium is sister to Acanthocardia and Parvicardium is paraphyletic because Parvicardium minimum is sister to a clade containing Acanthocardia, Papillicardium, and Orthocardiinae. I recovered two morphology-based subfamilies as monophyletic, the Tridacninae and the Clinocardiinae. There is some uncertainty as to the placement of the Tridacninae. ML weakly supported Tridacniinae as sister to clades E + F (Fig. 1.6) while BI analyses weakly supported

13 the Tridacniinae as the sister clade to the core Fragum + Ctenocardia groups. One reason for this weak support may be due to poor sampling of genetic markers, as I only obtained tridacnine sequences for one gene (16S) (Schneider and Foighil 1999). The North Pacific cold water Clinocardiinae, which is composed of four genera and nine species, was monophyletic and is the most basal group of clade 2. I were unable to test and verify the monophyly of most of the genera because only one species for each genus was sampled. The exception is Keenocardium and Serripes. Keenocardium was recovered with weak support as monophyletic in the concatenated dataset but was paraphyletic in the 16S analysis (Fig. 1.3, 1.6). Serripes was recovered as paraphyletic with respect to Ciliatocardium ciliatum. The discordance among the individual gene analyses and the combined data set is likely due to un even sampling of species for each individual gene. Thus illustrating the need for increased species level sampling and genetic markers to further clarify species level relationships within the Clinocardiinae. Membership in Trachycardiinae has long been disputed and there is no consensus among taxonomists about the placement of Vasticardium and Acrosterigma. Both genera were assigned to Cardiinae by Schneider (1992) and Vidal (1999), however Keen (1969, 1980) and Schneider and Carter (2001) placed both genera into Trachycardiinae based on shell morphology and microstructure. Further, Schneider (2002) excluded both genera from the Cardiinae and considered Trachycardiinae to be the sister group to all other living eucardiids: Cardiinae, Clinocardiinae, Fraginae, Trachycardiinae, and Lymnocardiinae (but see ter Poorten 2009). Trachycardiinae is paraphyletic with respect to the Indo-Pacific derived Laevicardium, which are a subclade sister to all Acrosterigma (Fig. 1.6). Our results indicate that Vidal (1999) was correct in placing both L. attenuatum and L. biradiatum in Acrosterigma which would render the Trachycardiinae monophyletic. Biogeography The phylogenetic results I have obtained contrast greatly with the currently held views of cardiid taxonomy and subfamilial relationships. In contrast, many of our results are in better accord with geography. I found many of the subfamilies that are not monophyletic to be geographically widespread and many of the clades I did recover to be geographically restricted (Fig. 1.7). Most strikingly is the European clade (Light blue, Fig. 1.7), which is composed of species from three different subfamilies. Other notable examples are within the Trachycardiidae,

14 which are paraphyletic with respect to the Indo-Pacific Laevicardium being sister to Acrosterigma. All other Laevicardium are located in the Caribbean (Fig. 1.7). Since the Paleozoic, the tropics have been a constant feature of the marine realm but more specifically, the Indo-Pacific has been thought to be the primary center of present day marine diversity (Briggs 2003, 2006). A pattern clearly supported by the results of our biogeographic analysis for bivalves (Fig. 1.7). Our results suggest a Central Indo-Pacific (CIP) origin of the Cardiidae with several expansions into adjacent regions. Most notably, I recovered four expansions from the CIP into the Eastern Tropical Pacific (Fig. 1.7). The eastern Pacific ocean, because of its width and lack of intermediate refuges for shallow water organisms, is a formidable barrier to coastal shelf organisms, which Ekman (1953) named the East Pacific Barrier. Further support for the East Pacific Barrier is illustrated by Grigg and Hey (1992), who studied the biogeographic distribution of corals. They found that corals of the East Pacific are more closely related to those of the West Atlantic than to those of the West Pacific, even for fossil corals dating to the Cretaceous Period. There are exceptions however, Lessios et al. (1998) showed that in the echinoderm Echinothrix diadema there was gene flow across this barrier. They found that populations of Echinothrix from the eastern Pacific islands of Clipperton Atoll and Isla del Coco to be genetically similar to populations found on Hawaii. Further, they suggested that such dispersals must have been possible during years when the El Niño regime was strong as this would decrease the time required to cross the ocean via the northern equatorial counter-current (Lessios et al. 1998). Therefore, it seems the EPB is not so restrictive to marine organisms that broadcast spawn or have pelagic larval life stages. Other patterns that become evident are the close affinities of species within the eastern tropical Pacific to the species of the western tropical Atlantic (Fig. 1.7; yellow and green clades). A well- documented vicariant event is the completion of the Panama Isthmus, which forms a barrier between the western Atlantic and eastern Pacific oceans. Further, the interrelationships of the eastern Pacific, western Atlantic, and the eastern Atlantic were strongly influenced by the continental movements and the closure of the Tethys sea. Evidence of this is the expansion from the CIP to the eastern temperate Atlantic (Fig. 1.7; light blue clade).

Conclusions Six of the eight subfamilies within the Cardiidae are not monophyletic and significant restructuring is supported at multiple levels. I found broad disagreement with previous analyses of morphological data, with paraphyly/ polyphyly of six of the eight subfamilies. These results suggest a need to re-evaluate the morphological characters traditionally used in cardiid systematics at this level. Further, our results show the importance of integrating molecular data with biogeographic information within a phylogenetic framework and provide a robust framework for future studies on the biology and evolution of the cardiids.

Table 1.1. Primers used in this study. ______NAME SOURCE SEQUENCE GENE ______28S_D2F Park & Ó Foighil, 2000 5’-TCAGTAAGCGGAGGAA-3’ 28S 28S_D24R Park & Ó Foighil, 2000 5’-CACGTACTCTTGAACTCTC-3’ 28S 28S_D23F Park & Ó Foighil, 2000 5’-GAGAGTTCAAGAGTACGTG-3’ 28S 28s-rD1a Park & Ó Foighil, 2000 5’-CCCSCGTAAYTTAAGCATAT-3’ 28S 28s-RD4.6r Park & Ó Foighil, 2000 5’-GAGAATAGGTTGAGGACGTTCG-3’ 28S 28sb Whiting, M. F. 2002 5’-TCGGAAGGAACCAGCTAC-3’ 28S 16Sa Xiong and Kocher 1991 5’-CGCCTGTTTATCAAAAACAT-3’ 16s 16Sb Xiong and Kocher 1991 5’-CTCCGGTTTGAACTCAGATCA-3’ 16s 16Sbr Xiong and Kocher 1991 5’-CCGGTCTGAACTCAGATCAGGT-3’ 16s H3F Mikkelsen et al. 2006 5’-ATGGCTCGTACCAAGCAGACVGC-3’ His 3 H3R Mikkelsen et al. 2006 5’-ATATCCTTRGGCATRATRGTGAC-3’ His 3

Table 1.2. Results of the Bayesian inference analyses by gene, the number of generations the chains were run, the number of generations discarded as the burn-in period, and the split frequencies of each run. ______Gene # Generations Burn-in Split Freq. ______Histone 3 80000000 8000000 0.010395 16S Tier 1 30000000 3000000 0.008855 16S Tier 2 30000000 3000000 0.007615 28S Tier 1 40000000 4000000 0.002333 28S Tier 2 40000000 4000000 0.006534 Concatenated 50000000 5000000 0.006677

Corculum cardissa Lunulicardia retusa Fraginae Trigoniocardia granifera Ctenocardia imbricata Americardia media Parvicardium pinnulatum Clinocardiinae Clinocardium Tridacninae + Cerastoderma edule Lymnocardiinae Cerastoderma glaucum Tridacna crocea Cardium Cardiinae Vepricardium Dinocardium Acanthocardium Orthocardiinae Freneixicardia Loxocardium Agnocardia (Agnocardia) † Agnocardia (Afrocardium) Trachycardium elongatum Trachycardium leucostomum Papyridea Trachycardiinae Trachycardium eversum † Trachycardium isocardia Profraginae Profragum † Nemocardium (Keenaea) Nemocardium (Microcardium) Nemocardium turgidum † Nemocardium nicoletti † Nemocardium diversum † Laevicardiinae Nemocardium cooperi † Fulvia Laevicardium biradiatum Laevicardium laevigatum Protocardia (Pachycardium) † Protocardiinae + Lahillinae Protocardia † Lahillia † Pleurocardiinae Pleuriocardia †

Figure 1.1. Phylogenetic relationship of the Cardiidae based on shell morphology and Microstructure. Redrawn from Schneider and Carter (2001), Schneider (2002). Crosses indicate extinct taxa.

G G G

J F L I

A D H K C E E

Figure 1.2. Biogeographic regions used in this study of the Cardiidae. Key: A, western tropical Atlantic; B, temperate south Africa; C, Western Indo-Pacific; D, Central Indo-Pacific; E, Eastern Indo-Pacific; F, western temperate north Pacific; G, Arctic, H, eastern tropical Pacific; I, western temperate north Atlantic; J, eastern temperate north Atlantic; K, eastern tropical Atlantic; L, eastern temperate north Pacific

Figure 1.3. 16S Maximum-likelihood phylogram. Numbers above branches indicate ML bootstrap and Bayesian posterior probabilities, respectively, and in percentages.

79/66 Fragum mundum EU733102 96/71 Fragum mundum EU733077 100/99 Fragum mundum EU733076 96/65 Fragum mundum EU733075 100/99 Lunulicardia retusa EU733096 98/98 69/66 Lunulicardia retusa BMNH20060464 100/58 Lunulicardia retusa EU733097 EU733098 84/0 Lunulicardia retusa Lunulicardia hemicardia EU733099 100/100 Fragum aff. mundum EU733063 Fragum aff. mundum EU733062 Fragum fragum EU733093 95/0 95/64 Fragum fragum EU733094 95/92 Fragum fragum EU733095 100/100 Fragum fragum IM200730138 Fragum fragum IM200730139 Fragum fragum EU733072 99/84 Fragum unedo EU733074 100/100 Fragum unedo EU733073 Corculum cardissa JJTP3938 Corculum cardissa RMNH41472 100/100 Corculum cardissa JJTP3978 Corculum cardissa EU733078 Fragum loochooanum EU733067 Fragum loochooanum EU733068 Fragum loochooanum EU733071 Fragum scruposum EU733066 100/100 Fragum scruposum EU733065 Fragum loochooanum EU733070 Fragum loochooanum EU733069 8 100/98 85/0 Fragum scruposum IM200730199 100/100 Fragum carinatum EU733064 Fragum scruposum IM200730190 85/100 Fragum erugatum EU733101 100/100 Fragum erugatum EU733100 Fragum sueziense EU733060 Fragum sueziense EU733061 94/63 34/100 Cerastoderma edule EU733081 Cerastoderma edule EU733080 BMNH20030333 80/- 100/100 Cerastoderma edule Cerastoderma edule BMNH20070234 AF122971 100/100 Cerastoderma edule 100/100 Cerastoderma glaucum lamarcki EU733087 100/96 Cerastoderma glaucum lamarcki EU733086 100/99 Cerastoderma glaucum FMNH318977 AF122972 96/69 Cerastoderma glaucum 100/100 Acanthocardia echinata BMNH20070236 89/74 Acanthocardia echinata EU733105 86/- 100/90 Acanthocardia tuberculata JJTP1827 7 100/98 Acanthocardia aculeata JJTP1825 73/- JJTP1396 79/71 Acanthocardia paucicostata 100/100 Papillicardium papillosum EU733051 Papillicardium papillosum UF374115 100/100 Parvicardium minimum EU733049 Parvicardium minimum EU733048 Afrocardium exochum IM20099796 100/100 Afrocardium exochum IM20099789 6 100/98 Afrocardium exochum IM20099780 Lyrocardium lyratum IM200730001 83/63 Lyrocardium aurantiacum UF298808 Ctenocardia biangulata UF351609 Ctenocardia biangulata ANSPA15529 100/100 Ctenocardia biangulata EU733091 90/- Ctenocardia biangulata EU733090 Ctenocardia media UF380487 87/- 100/100 Ctenocardia media EU733059 Ctenocardia media EU733058 100/- Ctenocardia festivum EU733092 100/95 Trigoniocardia obovalis EU733088 100/100 Apiocardia obovalis BMNH20080641 Trigoniocardia obovalis EU733089 5 100/100 Trigoniocardia granifera EU733057 100/100 Trigoniocardia granifera EU733056 100/90 Microfragum subfestivum IM200732254 100/95 Ctenocardia translata IM200730162 51/- Microfragum festivum IM200730232 100/100 Ctenocardia gustavi EU733055 Ctenocardia gustavi IM200730164 82/64 Ctenocardia fornicata EU733053 96/93 Ctenocardia fornicata EU733107 100/100 Ctenocardia fornicata IM20099775 55/53 Ctenocardia fornicata IM200730155 100/99 Tridacna crocea AF122980 100/79 Tridacna crocea NDH02 100/92 Tridacna squamosa AF122978 100/70 Tridacna maxima AF122979 83/65 Tridacna maxima NDH01 100/61 Tridacna derasa AF122976 100/91 Tridacna derasa RMNH 40948 4 AF122975 100/100 Tridacna gigas Tridacna tevoroa AF122977 100/98 Hippopus hippopus AF122973 Hippopus porcellanus AF122974 100/99 Dallocardia senticosum UF351612 99/65 Dallocardia senticostum UF359633 100/87 Dallocardia senticosum UF351587 90/- Acrosterigma magnum FMNH315294 100/70 100/100 Trachycardium egmontianum UF433948 Trachycardium egmontianum UF433840 Trachycardium belcheri UF391879 Papyridea aspersa UF371942

Papyridea aspersa UF371942 100/100 Papyridea sp EU733104 100/70 96/78 100/60 Papyridea aspersa EU733085 85/- Papyridea crockeri UF371362 Papyridea semisulcata UF286647 Acrosterigma simplex UF425951 100/94 Laevicardium biradiatum UF304138 99/56 Laevicardium biradiatum UF285613 Laevicardium attenuatum IM20099777 100/100 Vasticardium angulatum IM20099834 92/57 Vasticardium angulatum IM20099835 100/66 Vasticardium flavum UF323694 Vasticardium elongatum IM200630157 94/80 Vasticardium enode UF299284 100/100 Vasticardium pectiniforme IM200741443 Vasticardium pectiniforme IM200740245 93/- Vasticardium lacunosum UF292823 100/- 60/- Vasticardium insulare RMNH41367 Vasticardium kenyanum IM20099785 98/98 Acrosterigma transcendens IM20099779 100/100 Acrosterigma transcendens IM20099805 99/60 Acrosterigma transcendens IM20099795 3 97/- Acrosterigma punctolineatum IM200730197 86/- Laevicardium senegalense RMNH119658 Dinocardium robustum BV22 100/84 Laevicardium elenense UF371589 99/75 Laevicardium pictum UF286690 100/81 Laevicardium laevigatum UF322245 87/- Laevicardium pristis JJTP2139 Laevicardium laevigatum UF348860 100/89 Fulvia australis EU733083 98/50 Fulvia sp IM200730235 100/96 Fulvia nsp1 IM200743103 Fulvia nsp1 IM200743104 100/97 Fulvia aperta IM200730141 95/70 Fulvia aperta RMNH41468 Fulvia boholensis UF304156 100/100 Fulvia colorata IM20099793 100/100 Fulvia colorata IM20099791 100/64 Fulvia colorata IM200730156 -/84 Fulvia dulcis IM200730168 100/- 100/100 Fulvia dulcis IM200730192 Fulvia dulcis IM200730225 Fulvia undatopicta BV31 100/100 Fulvia undatopicta BV30 100/100 Fulvia undatopica BV32 97/61 Fulvia mutica BV19 Fulvia australis IM200730159 70/60 Fulvia subquadrata IM200730142 100/100 Fulvia hungerfordi IM200730231 100/96 Fulvia hungerfordi IM200730237 68/- Fulvia hungerfordi BV18 100/85 100/100 Laevicardium lobulatum IM200730151 Fulvia lineonotata UF305206 100/100 Cardium maxicostatum RMNH119660 99/- 91/70 Cardium costatum RMNH119661 92/- Cardium indicum RMNH119659 100/100 Europicardium caparti RMNH13183 Europicardium caparti RMNH13182 2 100/100 Freneixicardia victor IM20099774 100/88 Freneixicardia victor EU733054 100/91 Serripes groenlandicus FMNH326762 100/74 Ciliatocardium ciliatum FMNH326761 100/74 Clinocardium nutallii BV27 100/99 Keenocardium blandum BV26 Clinocardium buellowi BV33 Microcardium pazianum UF372357 100/100 Microcardium pazianum UF351595 99/51 Microcardium pazianum EU733082 Frigidocardium kirana UF294008 EU733106 100/100 Microcardium velatum IM200732278 98/72 Microcardium velatum IM200732277 Microcardium tenuilamellosum IM200730196 100/94 Microcardium n. sp. IM200732269 100/100 Microcardium n. sp. IM200732250 Microcardium trapezoidale IM20099765 Microcardium trapezoidale IM200732259 84/66 Microcardium trapezoidale IM20099767 Microcardium sakuraii IM200732245 100/100 Microcardium sakuraii IM200732260 Trifaricardium nomurai IM200732271 100/100 Microcardium aequiliratum IM200732265 100/99 Frigidocardium torresi IM200730234 100/92 Frigidocardium torresi IM200730191 Frigidocardium helios IM200730161 73/0 Lophocardium cumingi BMNH20080645 Frigidocardium eos BV29 1 100/100 Frigidocardium centumliratum IM20099803 Frigidocardium centumliratum IM20099768 Frigidocardium centumliratum IM20099786 Circe plicatina AMNH311616 Circe nummulina AMNH311617 Katelysia scalarina AMNH311620 Katelysia rhytiphora FMNH302051 Tresus capax Rangia cuneata Macoma balthica rubra Scissula similis

0.3

95/90 Dallocardia senticosum UF351612 88/86 Trigoniocardia granifera EU733025 80/78 Dallocardia senticosum UF351587 100/91 Acrosterigma magnum FMNH315294 100/88 100/100 Trachycardium egmontianum UF433948 S260 71/- Trachycardium egmontianum UF433840 Trachycardium belcheri UF391879 100/91 100/99Papyridea aspersa EU733046 Papyridea aspersa UF371942 98/60 Papyridea semisulcata EU733045 100/100 Acrosterigma transcendens IM20099779 Acrosterigma transcendens IM20099805 Acrosterigma simplex UF425951 100/ Laevicardium attenuatum IM20099777 82 100/85 Vasticardium enode UF299284 100/ Vasticardium elongatum IM200630157 100 Vasticardium kenyanum IM20099785 85/100 100/ Vasticardium angulatum IM20099835 97 Vasticardium angulatum IM20099834 100/100 Vasticardium pectiniforme IM200740245 IM200741443 100/100 Vasticardium pectiniforme 100/ Vasticardium flavum UF323694 99 Vasticardium philippinense CASIZ114334 RMNH41367 100/97 Vasticardium insulare 99/68 Dinocardium robustum BV22 88/ Laevicardium sengalense RMNH119658 56/- 65 Laevicardium laevigatum UF348860 S260 99/100 Laevicardium elenense ANSPA15494 100/98 Laevicardium elenense UF371589 86/100 Laevicardium laevigatum UF322245 80/57 Serripes groenlandicus FMNH326762 Keenocardium blandum BV26 100/ Ciliatocardium ciliatum FMNH326761 100 Serripes laperousii CASIZ168221 98/54 Fulvia n sp 1 IM200743103 98/59 Fulvia australis EU733044 100/99 100/88 Fulvia aperta IM200730141 100/76 Fulvia boholensis UF304156 42/79 Fulvia undatopica BV32 100/77 100/100 Fulvia undatopicta BV30 Fulvia undatopicta BV31 Fulvia mutica BV19 100/90 Fulvia hungerfordi BV18 Fulvia colorata IM200730156 100/96 Fulvia colorata IM20099791 100/ Fulvia colorata IM20099793 100 Cardium maxicostatum RMNH119660 81/54 Fulvia lineonotata UF305206 100/100 Fragum scruposum EU733032 94/57 Fragum scruposum EU733031 64/40 Fragum carinatum EU733030 100/100 Fragum sueziense EU733028 99/79 100/ Fragum sueziense EU733029 94 Fragum fragum EU733033 Fragum fragum IM200730139 100/69 100/99 Fragum unedo EU733035 EU733034 75/- 100/99 Fragum unedo 81/51 Corculum cardissa JJTP3978 100/100 Corculum cardissa EU733039 Corculum cardissa EU733040 100/100 100/100 Fragum mundum EU733037 100/100 Fragum mundum EU733038 96/- Fragum mundum EU733036 54/- Lunulicardia hemicardia EU733047 Ctenocardia fornicata EU733021 Parvicardium scriptum UF374117A Parvicardium scriptum EU733018 Parvicardium vroomi EU733017 Parvicardium vroomi EU733016 Parvicardium exigum UF374117B Parvicardium sp. EU733015 100/100 93/- Cerastoderma glaucum FMNH318977 78/ Cerastoderma edule EU733041 50 Cerastoderma edule EU733042 96/- Monodacna colorata FMNH312499 95/72 Acanthocardia tuberculata BMNH20070236 100/99 Parvicardium papillosum EU733019 Parvicardium papillosum EU733020 74/96 Afrocardium exochum IM20099796 100/100 Afrocardium exochum IM20099780 Afrocardium exochum IM20099782 97/63 100/100 Freneixicardia victor EU733022 Freneixicardia victor IM20099774 69/72 Ctenocardia media EU733027 100/90 74/53 Ctenocardia media EU733026 100/93 Trigoniocardia granifera EU733024 100/100 Ctenocardia biangulata UF351609 100/100 Ctenocardia fornicata IM20099775 Ctenocardia fornicata IM200730155 S260 100/96 Ctenocardia gustavi EU733023 100/85 Microcardium velatum IM200732278 100/72 Trifaricardium nomurai IM200732271 94/70 Microcardium sakuraii IM200732260 100/72 Microcardium n sp. IM200732269 Microcardium pazianum UF372357 Microcardium pazianum UF351595 Microcardium pazianum EU733043 Frigidocardiumcentumliratum IM20099786 100/100 Frigidocardiumcentumliratum IM20099768 100/100 Frigidocardiumcentumliratum IM20099803 Circe nummulina AMNH311617 Circe plicatina AMNH311616 Katelysia scalarina AMNH311620 Katelysia rhytiphora FMNH302051 Rangia cuneata Tresus capax Scissula similis Macoma balthica rubra

0.3 Figure 1.4. 28S Maximum-likelihood phylogram. Numbers above branches indicate ML bootstrap and Bayesian posterior probabilities, respectively, and in percentages.

99/77 Fulvia aperta RMNH 41468 Fulvia hungerfordi IM 200730195 99/99 Fulvia_colorataIM20099791 Fulvia colorata IM 200730156 Fulvia n. sp.1 IM 200743104 Fulvia n. sp.1 IM 200743103 100/ Fulvia dulcis IM 200730168 92 82/87 Fulvia dulcis IM 200730192 100/99 Fulvia dulcis IM 200730168 S260 Fulvia dulcis IM 200730225 Fulvia mutica BV19 Fulvia undatopicta BV30 98/90 Fulvia undatopica BV32 Fulvia undatopicta BV31 Fulvia boholensis UF 304156 Fulvia hungerfordi BV18 100/75 Fulvia australis IM 200730159 Fulvia sp. IM 200730235 -/88 Fulvia hungerfordi IM 200730231 99/93 Fulvia hungerfordi IM 200730237 Fulvia hungerfordi IM 200730142 Fragum sueziense IM 200730189 S260 Fragum sueziense IM 200730167 Fragum sueziense IM 200730189 100/83 Keenocardium blandum BV26 Clinocardium buellowi BV33 94/63 Ciliatocardium ciliatum FMNH 326761 Serripes laperousii CASIZ 168221 100/92 Serripes groenlandicus FMNH 326762 88/77 Clinocardium nutallii BV27 100/100 Ciliatocardium ciliatum FMNH 278012 Clinocardium nuttallii FMNH 170895 100/100 Europicardium caparti RMNH 13183 Europicardium caparti RMNH 13182 Bucardium ringens RMNH 119662 Cardium indicum RMNH 119659 96/95 Parvicardium scriptum UF 374117A 100/96 Parvicardium scrizptum JJTP 271 Parvicardium exigum UF 374117B 100/99 Cerastoderma edule BMNH 20030333 98/63 Cerastoderma edule BMNH 20070234 Cerastoderma glaucum FMNH 318977 Acanthocardia tuberculata JJTP 91/66 Acanthocardia aculeata JJTP 1825 100/93 Acanthocardia echinata BMNH 20070236 99/27 98/65 Acanthocardia paucicostata JJTP 1396 100/98 Papillicardium papillosum UF 374115 Papillicardium turtoni NMSA W2602 Monodacna colorata FMNH 312499 Lyrocardium lyratum IM 200730001 98/89 Ctenocardia translata IM 200730162 Microfragum festivum IM 200730232 Ctenocardia fornicata IM 200730155 Ctenocardia fornicata IM 20099775 100/100 Afrocardium exochum IM 20099796 99/90 Afrocardium exochum IM 20099789 Afrocardium exochum IM 20099782 100/93 Afrocardium exochum IM 20099780 100/96 Afrocardium richardi IM 200730236 Afrocardium richardi IM 200730229 100/100 Ctenocardia biangulatum ANSP A15529 Ctenocardia biangulatum UF 351609 Trigoniocardia granifera SBMNH 149259 Ctenocardia media UF 380487 Laevicardium lobulatum IM 200730151 Dallocardia senticostum UF 359633 97/- Acrosterigma magnum FMNH 315294 Dallocardia senticosum UF 351612 Dallocardia senticosum UF 351587 99/76 Trachycardium panamense SBMNH 149262 Papyridea crockeri UF 371362 Trachycardium belcheri UF 391879 100/96 Trachycardium egmontianum UF 433948 Trachycardium egmontianum UF 433840 Laevicardium attenuatum IM 20099777 Acrosterigma simplex UF 425951 100/89 Vasticardium angulatum IM 20099835 Vasticardium angulatum IM 20099834 Vasticardium elongatum enode IM 200630157 Vasticardium elongatum enode UF 299284 Vasticardium flavum UF 323694 Vasticardium pectiniforme IM 200740245 Vasticardium pectiniforme IM 200741443 Vasticardium flavum RMNH 41477 Vasticardium lacunosa UF 292823 98/44 Vasticardium vertebratum NTM 43909 Vasticardium kenyanum IM 20099785 98/61 Vasticardium insulare RMNH 41367 Vasticardium phlippinense CASIZ 114334 -/88 Corculum cardissa JJTP 3938 -/88 Corculum cardissa RMNH 41473 93/73 Corculum cardissa RMNH 41472 Corculum cardissa JJTP 3978 99/80 100/100 Fragum scruposum IM 200730199 Fragum scruposum IM 200730158 Fragum scruposum IM 200730190 Dinocardium robustum BV22 Acrosterigma transcendens IM 20099805 Acrosterigma transcendens IM 20099779 100/98 Acrosterigma transcendens IM 20099780 Acrosterigma punctolineatum IM 200730197 96/69 Laevicardium elenense ANSP A15494 100/73 Laevicardium elenense UF 371589 99/66 Laevicardium pictum UF 286690 100/90 Laevicardium laevigatum UF 322245 Laevicardium laevigatum UF 348860 99/85 Microcardium velatum IM 200732278 Microcardium velatum IM 200732277 99/64 Microcardium sakuraii IM 200732260 Microcardium n. sp. IM 200732269 Trifaricardium nomurai IM 200732271 98/95 Frigidocardium centumliratum IM 20099803 99/90 100/100 Frigidocardium centumliratum IM 20099786 Frigidocardium eos BV29 86/- Microcardium pazianum UF 372357 Scissula similis Macoma balthica Katelysia scalarina AMNH 311620 Katelysia rhytiphora FMNH 302051 Antigona lamellaris FLMNH 281662 Anomalocardia auberiana FMNH 305976 Circe rivularis FMNH 306188 Circe plicatina AMNH 311616 Petricola lapicida AMNH 305124 Circe_nummulina AMNH 311617 Rangia cuneata Petricolaria pholadiformis AMNH 311609

0.05

Figure 1.5. Histone 3 Maximum-likelihood phylogram. Numbers above branches indicate ML bootstrap and Bayesian posterior probabilities, respectively, and in percentages.

Papyridea semisulcata UF 286647 -/.87 Papyridea aspersa UF 371942 Papyridea crockeri UF 371362 78/1 Mexicardia panamensis SBMNH 149262 97/1 Dallocardia senticosum UF 351612 Acrosterigma magnum FMNH 315294 Trachycardium egmontianum UF 433840 -/.76 51/.92 64/.97 Phlogocardium belcheri UF391879 -/.6 Acrosterigma punctolineatum IM 200730197 Acrosterigma transcendens IM 20099805 Acrosterigma simplex UF 425951 -/1 Laevicardium biradiatum UF 304138 8 Laevicardium attenuatum IM 20099777 75/1 58/.97 69/1 Vasticardium angulatum IM 20099835 Vasticardium flavum UF 323694 53/.67 Vasticardium elongatum IM 200630157 54/.63 Vasticardium pectiniforme IM 200740245 Vasticardium kenyanum IM 20099785 F Vasticardium philippinense CASIZ 114334 90/1 99/1 Vasticardium insulare RMNH 41367 Vasticardium vertebratum NTM 43909 95/1 Vasticardium lacunosum UF 292823 100/.99 73/1 Laevicardium elenense UF 371589 80/1 Laevicardium pictum UF 286690 Laevicardium serratum UF 348860 83/1 Laevicardium pristis JJTP 2139 Dinocardium robustum BV22 7 64/.99 Laevicardium senegalense RMNH 119658 76/.38 Fulvia australis IM 200730159 Fulvia sp. IM 200730235 Fulvia australis 110 Fulvia n. sp. 1 IM 200743103 94/.99 Fulvia aperta IM 200730141 100/1 Fulvia boholensis UF 304156 -/.8 Fulvia aperta RMNH 41468 Fulvia undatopica BV32 Fulvia mutica BV19 -/1 Fulvia dulcis IM 200730192 Fulvia colorata IM 20099791 -/.92 Fulvia hungerfordi IM 200730237 Fulvia hungerfordi IM 200730142 54/1 Fulvia hungerfordi BV18 100/.95 Cardium costatum RMNH 119661 6 60/.99 Cardium maxicostatum RMNH 119660 67/1 Bucardium ringens RMNH 119662 Cardium indicum RMNH 119659 Europicardium caparti RMNH 13182 100/1 Fulvia lineonotata UF 305206 56/1 Laevicardium lobulatum IM 200730151 79/1 Ciliatocardium ciliatum FMNH 326761 E 88/1 Serripes laperousii CASIZ 168221 5 61/- Serripes groenlandicus FMNH 326762 99/1 Keenocardium blandum BV26 Keenocardium buellowi BV33 Clinocardium nuttallii BV27 85/.97 Tridacna squamosa AF122978 98/.95 Tridacna crocea NDH02 -/.61 Tridacna maxima NDH01 D -/.50 99/1 Tridacna gigas AF122975 100/.72 Tridacna derasa RMNH 40948 Tridacna mbalavuana AF122977 Hippopus hippopus F122973 90/.98 -/.51 100/.93 Hippopus porcellanus AF122974 89/.68 Lunulicardia retusa BMNH 20060464 Fragum mundum 78 Lunulicardia hemicardia 136 -/.79 Corculum cardissa JJTP 3978 77/.26 Fragum whitleyi IM 200730199 95/.93 Fragum unedo 131 Fragum fragum IM 200730139 Fragum loochooanum 121 Fragum whitleyi 78 -/.91 93/1 Fragum whitleyi IM 200730190 -/.86 Fragum carinatum 318 4 Fragum aff. mundum 375 87/.58 100/.97 Fragum erugatum 133 Fragum sueziense 31 Fragum sueziense IM 200730189 72/.98 Ctenocardia translata IM 200730162 82/1 Microfragum subfestivum IM 200732254 66/.98 Microfragum festivum IM 200730232 C Ctenocardia gustavi 311 3 98/1 Ctenocardia fornicata IM 20099775 -/.78 -/.62 Trigoniocardia granifera 333 92/.98 Apiocardia obovalis BMNH 20080641 Ctenocardia media UF 380487 72/.84 Americardia biangulatum UF 351609 80/.99 Acanthocardia aculeata JJTP 1825 99/1 Acanthocardia tuberculata JJTP 1827 74/.96 Acanthocardia echinata BMNH 20070236 Acanthocardia paucicostata JJTP 1396 99/.99 Papillicardium turtoni NMSA W2602 Papillicardium papillosum UF 374115 100/1 Cerastoderma edule 01 Cerastoderma glaucum FMNH 318977 Monodacna colorata FMNH 312499 2 91/.99 84/1 Parvicardium minimum 194 100/.91 Parvicardium vroomi 294 Parvicardium scriptum UF 374117A B Parvicardium exiguum UF 374117B 62/.79 -/.87 1 Lyrocardium aurantiacum UF 298808 78/.97 Lyrocardium lyratum IM 200730001 66/.96 96/1 Afrocardium richardi IM 200730236 Afrocardium exochum IM 20099780 Freneixicardia victor IM 20099774 97/1 Microcardium tenuilamellosum IM 200730196 52/.79 Microcardium velatum IM 200732278 -/.79 Trifaricardium nomurai IM 200732271 Microcardium aequiliratum IM 200732265 -/.92 Microcardium sakuraii IM 200732260 Microcardium trapezoidale IM 20099767 66/.99 Microcardium pazianum UF 351595 A 100/1 Microcardium n. sp. IM 200732269 86/1 Frigidocardium torresi IM 200730234 Frigidocardium helios IM 200730161 -/.75 Frigidocardium kirana UF 294008 Frigidocardium eos BV29 Frigidocardium centumliratum IM 20099803 Lophocardium cumingi BMNH 20080645 Anomalocardia auberiana FMNH 305976 Antigona lamellaris FLMNH 281662 Katelysia scalarina AMNH 311620 Katelysia rhytiphora FMNH 302051 Circe rivularis FMNH 306188 Circe plicatina AMNH 311616 Circe nummulina AMNH 311617 Petricola lapicida AMNH 305124 Petricolaria pholadiformis AMNH 311609 Rangia cuneata Tresus capax Macoma balthica Scissula similis !"#

Figure 1.6. Maximum-likelihood phylogram of concatenated dataset for three genes (His 3, 16S, 28S). Numbers above branches indicate ML bootstrap and Bayesian posterior probabilities, respectively, and in percentages. Colors indicate recognized subfamilies.

Eastern Temperate Atlantic Western Temperate Atlantic Papyridea semisulcata Eastern Tropical Atlantic Papyridea aspersa Papyridea crockeri Cardiinae Western Tropical Atlantic Mexicardia panamensis Dallocardia senticosum Clinocardiinae Temperate South Africa Acrosterigma magnum Fraginae Trachycardium egmontianum Western Indo-Pacific Phlogocardium belcheri Laevicardiinae Acrosterigma punctolineatum Central Indo-Pacific Acrosterigma transcendens Lymnocardiinae Eastern Indo-Pacific Acrosterigma simplex Orthocardiinae Laevicardium biradiatum Eastern Temperate North Pacific 8 Laevicardium attenuatum Trachycardiinae Vasticardium angulatum Western Temperate North Pacific Vasticardium flavum Tridacninae Vasticardium elongatum Eastern Tropical Pacific Vasticardium pectiniforme Vasticardium kenyanum Arctic F Vasticardium philippinense Vasticardium insulare Vasticardium vertebratum Vasticardium lacunosum Laevicardium elenense Laevicardium pictum 7 Laevicardium serratum Laevicardium pristis Dinocardium robustum Laevicardium senegalense Fulvia australis Fulvia sp. Fulvia australis Fulvia n. sp. 1 Fulvia aperta Fulvia boholensis Fulvia aperta Fulvia undatopica Fulvia mutica Fulvia dulcis Fulvia colorata Fulvia hungerfordi Fulvia hungerfordi Fulvia hungerfordi Cardium costatum 6 Cardium maxicostatum Bucardium ringens Cardium indicum Europicardium caparti E Fulvia lineonotata Laevicardium lobulatum Ciliatocardium ciliatum Serripes laperousii Serripes groenlandicus 5 Keenocardium blandum Keenocardium buellowi Clinocardium nuttallii Tridacna squamosa Tridacna crocea Tridacna maxima Tridacna gigas D Tridacna derasa Tridacna mbalavuana Hippopus hippopus Hippopus porcellanus Lunulicardia retusa Fragum mundum Lunulicardia hemicardia Corculum cardissa Fragum whitleyi Fragum unedo Fragum fragum Fragum loochooanum Fragum whitleyi Fragum whitleyi Fragum carinatum 4 Fragum aff mundum Fragum erugatum Fragum sueziense Fragum sueziense C Ctenocardia translata Microfragum subfestivum Microfragum festivum Ctenocardia gustavi 3 Ctenocardia fornicata Trigoniocardia granifera Apiocardia obovalis Ctenocardia media Americardia biangulatum Acanthocardia aculeata Acanthocardia tuberculata Acanthocardia echinata Acanthocardia paucicostata Papillicardium turtoni Papillicardium papillosum Cerastoderma edule Cerastoderma glaucum 2 Monodacna colorata Parvicardium minimum Parvicardium vroomi B Parvicardium scriptum Parvicardium exiguum Lyrocardium aurantiacum Lyrocardium lyratum 1 Afrocardium richardi Afrocardium exochum Freneixicardia victor Microcardium tenuilamellosum Microcardium velatum Trifaricardium nomurai Microcardium aequiliratum Microcardium sakuraii Microcardium trapezoidale Microcardium pazianum A Microcardium n. sp. Frigidocardium eos Frigidocardium centumliratum Frigidocardium torresi Frigidocardium helios Frigidocardium kirana Lophocardium cumingi Anomalocardia auberiana Antigona lamellaris Katelysia scalarina Katelysia rhytiphora Circe rivularis Circe plicatina Circe nummulina Petricola lapicida Petricolaria pholadiformis Rangia cuneata Tresus capax Macoma balthica Scissula similis

Figure 1.7. Biogeographic reconstruction cladogram. Branch colors indicate geographic region and taxon names colored by subfamily. Labels correspond to the labeling in Concatenated Tree (Fig. 1.6).

CHAPTER TWO

HISTORICAL BIOGEOGRAPHY OF A MARINE BIVALVE (BIVALVIA: CARDIIDAE): GLOBAL PATTERNS OF ORIGINATION AND DISPERSAL

Introduction Understanding the historical forces shaping biodiversity in an important aspect of marine biogeography. One way to do this is to use phylogenetic trees to make inferences about the evolutionary dynamics of geographic ranges. More specifically, one may begin to estimate ancestral distributions, the mode and tempo of speciation, and the effects of range expansion on lineage diversification (Wiens and Donoghue 2004, Ree et al. 2005, Ree and Smith 2008, Sanmartín et al. 2008, Ree and Sanmartín 2009). Among the major biogeographical regions of the oceans, the Central Indo-Pacific (CIP) has the greatest amount of marine biodiversity among macrofauna groups and taxa such as fish, corals, and mollusks. This diversity ‘hotspot’ has both latitudinal and longitudinal gradients in diversity. The latitudinal diversity gradient, with high species diversity in the tropics and a decrease towards the poles, is one of the fundamental patterns of biological diversity on the planet (Roy and Witman 2009),and is found in many groups such as marine mollusks and reef associated organisms (Crame 2000, Willig et al. 2003, Roy et al. 2009). In addition, the longitudinal decline in marine species richness has spurred much debate (Briggs 2003, Cox and Moore 2010). Central to the debate are hypotheses describing the origin and maintenance of faunal diversity throughout the marine realm. Much of the debate has focused on whether the tropics, especially the CIP, are a center of origin, overlap, or accumulation of diversity. While a lot of attention has been focused on the Indo-Pacific, there has been little attempt to explore global patterns of origination, dispersal, and accumulation between the major marine biogeographic regions (Cowman et al. 2013). Several challenges exist for these studies, such as the lack of obvious physical barriers within the marine environment. Further, dispersal via planktonic larvae has confounded the patterns of connectivity and origin of marine species (Cowman et al. 2013). Therefore, it is evident that a detailed assessment of global patterns of dispersal and origination among the major biogeographic is needed. The circum-tropical belt can be divided into five major realms: the Western Indo-Pacific, Central Indo-Pacific, Eastern Indo-Pacific, tropical Atlantic, and the tropical East Pacific (Briggs 2003, Spalding et al. 2007). These realms are distinguished by a taxonomic makeup influenced

25 by evolutionary history, patterns of dispersal, and isolation (Spalding et al. 2007). Major barriers among these realms include: (1) the East Pacific Barrier (EPB), an open expanse of ocean separating the Indo-Pacific from the East Pacific; (2) the Terminal Tethian event (TTE), which cut off dispersal between the Indo-Pacific and the Atlantic via an Indo-Mediterranean waterway; and (3) the Isthmus of Panama (IOP), which separates the Atlantic from the East Pacific. Within the Indo-Pacific, species have been able to maintain widespread geographic ranges spanning the entire Western Indo-Pacific region to the islands making up the central Pacific, and in some cases, the Pacific coast of the Americas (Lessios et al. 1998, Hughes et al. 2002, Reece et al. 2011). This is primarily due to a lack of obvious barriers (e.g. land bridges or vast expanses of open ocean). However, the complex distribution of taxa that generally characterizes the western, central, and Eastern Indo-Pacific is a result of a combination of tectonic activity and multiple semi-permeable hydrological barriers (Barber et al. 2000, Barber et al. 2002, Bellwood and Wainwright 2002, Santini and Winterbottom 2002, Jones et al. 2006, Cox and Moore 2010). With such a complex history and a lack of salient barriers, it is difficult to identify the mode and tempo of species evolution within the marine realm. To explore the complex evolution of the marine realm, I examine inferred patterns of origination and dispersal in the marine bivalve family Cardiidae under a dispersal, extinction, and cladogenesis (DEC) model (Ree and Smith 2008). The Cardiidae are a diverse marine bivalve family of about 250 extant species arranged in ca. 80 genera in eight subfamilies with the oldest representative (Tulongicardiinae) dating back to the Norian (ca. 216 mya) (Lydeard and Lindberg 2003, Ponder and Lindberg 2008). Cardiids are found inhabiting worldwide tropical to polar seas with the bulk of extant taxa distributed throughout tropical-subtropical seas. They are mainly shallowly infaunal to epifaunal in soft sand or mud in water depths up to 500 m. Typically, cardiids are suspensions feeders, but some are highly specialized, such as Tridacna, Corculum, and Fraginae, which form an endosymbiosis with dinoflagellate protists (Maruyama et al. 1998, Schneider 1998b, Kirkendale 2009). The family contains endemic species in most of the major regions, as well as many widespread species (Wilson and Stevenson 1977, Vidal 1999, 2000, Kafanov 2001, 2002, Harzhauser et al. 2008, ter Poorten 2009). The Cardiidae have planktotrophic larvae however, little work has been done on the duration the larvae spend in the water column. Within Corculum, larval development is very rapid with the veliger larvae settling out of the water column within 24 hours (Kawaguti 1949). However, Coscia et al. (2013) showed

26 that in Cerastoderma, the planktotrophic larvae remain in the water column for up to five weeks. Further, the Cardiidae have a rich fossil record. Therefore, the family offers an ideal opportunity to explore ancestral range reconstruction and patterns of origination within the marine realm. In this study, I aim to examine patterns of origination and dispersal of the biogeographic history of a diverse, nearly globally distributed bivalve family. Thus, providing an opportunity to evaluate possible sources of current marine biodiversity and the relationship between the major biogeographic regions over the past 135 mya. The goal of this study is to assess patterns of origination and dispersal in the biogeographic history of the Cardiidae. This will provide a framework for evaluating possible sources of bivalve diversity, directionality of dispersal and regional connectivity within the major biogeographic regions. Specific questions I aim to be answered are: 1. Is the CIP a center of origination and diversification for marine bivalve diversity? 2. How has the changing paleogeogrpahy, such as the closure of Indo-Mediterranean, shaped present day marine diversity?

Materials and Methods Dataset For this study, I used the maximum likelihood, ultrametric phylogeny from Herrera et al. (In prep.), which included 110 species representing 37 of the 44 recognized genera and all eight extant subfamilies of the Cardiidae. Specimens were sequenced for three genes: mitochondrial large subunit ribosomal RNA gene 16S (~ 400 bp), and portions of two nuclear genes: Histone 3 (~400 bp) and 28S (~1200 bp). Additionally, previously published sequences were downloaded from Genbank and combined with our dataset to increase our resolution at the species level. Analyses were conducted using Maximum Likelihood (ML) as implemented in RAxML (Stamatakis 2006).

Divergence Time Estimation Bayesian estimation of divergence times was estimated using BEAST 1.7.5 (Drummond et al. 2012) for the combined nDNA + mtDNA data set. I implemented a GTR+I+Γ model of DNA substitution with four rate categories and base frequencies set to be estimated for all partitions. I used an uncorrelated lognormal relaxed molecular clock model to estimate

27 substitution rates with the Yule process of speciation as the tree prior. I used the ML, ultrametric phylogeny from Herrera et al. as a starting tree for all runs. I ran two independent analyses, sampling every 1000 generations. Tracer 1.5 was used to determine convergence, measure of effective sample size of each parameter and calculate the mean 95% highest posterior density interval for divergence times. The results of the two runs were combined with LogCombiner 1.7.5, and the consensus tree was compiled with TreeAnnotator 1.7.5 (Drummond et al. 2012). The two analyses were run for 200,000,000 generations, with the initial 10% discarded as burn- in. I first ran the analysis without data to determine whether fossil calibration priors were being implemented properly and not interacting unexpectedly by checking to see if I recovered posterior distribution that were similar to the prior distributions (Drummond et al. 2006). I then rejected calibrations that yielded a posterior distribution that largely differed from the prior distribution. I incorporated 13 fossil calibrations to calibrate the chronogram in the Beast analysis (Table 1). I applied a lognormal prior distribution to all calibrations, with the means and standard deviations of the distributions set to represent the 95% estimated confidence interval for the actual origination of a taxon based on first occurrences of genera. Fossil selection, identification, and verification of placement was verified by David Jablonski and is a subset of a larger database.

Biogeographic Analysis Biogeographic regions were defined following Spalding et al’s. (2007) “Marine Ecoregions of the World”. However, I excluded three regions from our analysis: (1) the Southern ocean because no known cardiid ranges into the southern Antarctic range; (2) Temperate Australasia, and (3) Temperate South America because I did not sample any taxa from those regions. I further divided the northern temperate and tropical Atlantic and Pacific into east and west components based on current known cardiid geographic distributions in which most are restricted to one region the other. Within the Indo-Pacific I combined the Central Indo-Pacific and Eastern Indo-Pacific into a single region. Thus, I had a total of 11 biogeographic regions (Fig. 2.1). I also analyzed a 10 geographic model (not shown), were I lumped the eastern, central, and western Indo Pacific into one region. However, our results were very similar to the 11 state analyses.

Ancestral range estimation based on the time-calibrated phylogeny was implemented in the program RASP v 2.1 beta (http://mnh.scu.edu.cn/soft/blog/RASP) using the DEC model of geographic range evolution with the Lagrange module (which uses source code from the c++ version of Lagrange developed by Smith (2010)). Lagrange implements a ML approach based on a stochastic model of geographic range evolution involving dispersal, extinction, and cladogenesis (DEC model; (Ree et al. 2005, Ree and Smith 2008). In the DEC model, anagenic range evolution is governed by a Q matrix of instantaneous transition rates, whose parameters are dispersal (range expansion) between geographic regions and local extinction (range contraction) within a region along branches of a time calibrated phylogeny (Ree and Smith 2008). The DEC model assumes that only one event (a single dispersal or local extinction event) can occur at any one moment in time. Therefore, transitions that imply more than one event are given a rate of zero in the Q matrix. I restricted dispersal to only occur between adjacent regions. Cladogenetic evolution is modeled as three alternative inheritance scenarios (Ree et al. 2005): (1) For ancestors whose range comprises a single area, the two daughter lineages identically inherit the entire ancestral range (sympatric speciation). For widespread ancestors, lineage divergence can arise either (2) between a single area and the rest of the range, so that both lineages have different ranges from the ancestor and that are mutually exclusive from each other (vicariance or allopatric speciation), or (3) within a single area were one descendant lineage inherits a range of only where the divergence occurred, while the other lineage inherits the entire ancestral range (peripheral isolate speciation). Therefore, scenario 3 allows non-identical range inheritance and for a widespread ancestral range to be inherited by a single descendant lineage. The DEC method uses ML to integrate over all possible ancestral states at internal nodes of the phylogeny and estimates global rates of dispersal and extinction. It then uses these rates to estimate node-by- node relative probabilities for each range inheritance scenario without conditioning on assumptions of other range inheritance scenarios elsewhere in the tree (Ree and Sanmartín 2009). Lagrange also allows for constraint to be placed on the DEC model, which reflects past geological events. I stratified the phylogeny into different time slices (TS) reflecting major changes in the continental configuration over time that are applicable to cardiid evolution. When constructing a stratified biogeographic model, it is important not to divide it too finely so that there are multiple phylogenetic events in each TS (Ree & Sanmartín, 2009). I modeled our TS to retain multiple phylogenetic events and based on major geological events (e.g. closing of the

Tethys sea). I used three separate TS: between the root age of 135 and 60 mya (TS1), between 60 and 18 mya (TS2), and between 18 and 3 mya (TS3; Fig. 2.2). Further, I ran two models, an adjacency model (M0), which allows equal probability of dispersal between adjacent areas at any time and an adjacency with constraints model (M1), where I assigned a reduced probability of dispersal from the Indo-Pacific to the tropical eastern Pacific to 0.05 to account for the east Pacific barrier in all time slices. From TS 1 to TS 2, I decreased the probability of dispersal from 1 to .5 between the Western Indo-Pacific and eastern north temperate Atlantic (transition between regions C <--> I; Fig. 2.1) and the eastern and western Atlantic (transitions between regions H <--> I; A <--> J Fig. 2.2). This is to account for the slowly closing Indo-Mediterranean region and the expanding Atlantic ocean. From TS 2- TS 3, I decreased the probability of crossing the Atlantic to 0.1 and decreased the probability of crossing through the Tethys sea via the Indo-Mediterranean region from the CIP to the northeastern Atlantic from 0.5 to 0 to account for the closure of the Tethys and continual expansion of the Atlantic Ocean (Fig. 2.2).

Results I observed large ESS values (>200) and convergence for all parameters, including the prior, posterior, and date estimates for the combined runs for the Bayesian divergence time analysis. The results sampled from the prior yielded a root age dating to the upper Triassic, 200.52 mya (95% highest posterior density [HPD]= 200.00-208.11 mya; Fig. 2.3). This is consistent with previous estimates based on fossil occurrences (Schneider 1995, 1998b, Schneider and Carter 2001). ML estimation of geographical ranges for the Cardiidae is presented in Fig. 2.4 (adjacency with constraints model). The adjacency with constraints model (M1) was favored over the adjacency model (M0) with significantly better log-likelihood ratios scores (M0: -lnL = 275.38; M1: -lnL = 269.15). A biogeographical and dating analysis inferred that the crown group of the extant Cardiidae (node W) began to diversify in the tropical Pacific (regions DG; Fig. 2.3) and dates to the early Cretaceous, ca. 134 mya (95% HPD= 101.62-175.05 mya; Fig.2.3 and 2.4). Clade A diverged from all other cardiids around 34.5 mya (95% HPD = 33.72-36.53 mya) and further diversified within the Indo-Pacific/ eastern tropical Pacific. Movement to the eastern temperate north Atlantic via the Tethys Sea was estimated in the lineage leading to clade X around the early/late Cretaceous boundary (ca. 103.5 mya; 95% HPD = 83.17-127.51 mya). Two major

30 lineages diverged from clade X, clade B and clade Y. Clade B inherited the widespread ancestor from clade X, while clade Y was estimated as being found in only the CIP (region D). Within clade B, the major split- clade 1 resulted in the formation of a European clade (clade 2) sometime around the late Paleocene (57.28 mya; HPD = 40.66-76.78 mya). The split between the primarily CIP derived clades, C and D, occurred around the late Cretaceous. The split between clade E and clade F occurred between 60 and 97 mya with a most likely distribution of just the CIP (Clade Z, Fig. 2.4). Clade E is comprised of the widespread temperate derived Clinocardiinae (clade 5), that diverged from clade 6 around the Paleocene/Eocene between 48 and 81 mya. The split between the two main lineages in clade F occurred in the Eocene between 40 and 67 mya. Clade 8 was estimated as being widespread throughout the tropical Pacific while clade 7 is primarily restricted to the tropical Atlantic.

Discussion Our results suggest that the Cardiidae originated sometime in the early Cretaceous with an initial area of diversification within the IWP. From the IWP, there were multiple expansions into the temperate north Atlantic, which was part of a tropical Indo-Mediterranean region within the Tethys Sea until the Oligocene (ca. 35 mya). The Indo-Mediterranean region that existed between Africa and Eurasia since the early Cretaceous and acted as a major source for most present day marine diversity (Kaufman 1973, Briggs 2006). Our results suggest the Indo- Mediterranean region acted as a source for much of the present day cardiid diversity in the eastern Atlantic and Arctic regions, evidenced by vicariant events that gave rise to four cardiid clades (Fig. 2.4; clades 1,2,5,6). At the start of the Cenozoic (ca. 65 mya) the tropics began to undergo a significant decrease due to dropping sea levels and more importantly, a decrease in global temperature (Briggs 2003, 2006). The decrease in sea surface temperatures during the Paleogene is suggested as the reason for the evolution and diversity of many extant families and genera of marine organisms. Examples of the Indo-Mediterranean as a source include many of the earliest coral reef fish assemblages (Bellwood and Meyer 2009), parrot fishes (Streelman et al. 2002), the fish family Triacanthidae (Santini and Tyler 2003), the gastropod family Cypraeidae (Kay 1990), and many bivalve and echinoderm taxa (Roy and Witman 2009). The connection of the IWP to the Atlantic during the Paleocene/Eocene is also evidenced by the fossil record of Monte Bolca in

Italy were the majority of the fish families represented in deposits are present in both the Atlantic and Indo-Pacific (Bellwood and Wainwright 2002). Further, Kaufman (1973) recognized an Indo-Mediterranean region within the early Cretaceous Tethys, between Africa and Eurasia based on fossil mollusk assemblages. Our reconstructions suggest that species in the eastern temperate/tropical Atlantic are the result of unidirectional range expansions out of the Indo- Mediterranean region. This unidirectional expansion is also concordant with past paleoceanographic reconstructions of currents which suggest that during the Mid- Eocene, there was a westerly transport of warm Indian ocean water into the Atlantic via the Tethys (Stille et al. 1996, Bush 1997, Thomas et al. 2003, von der Heydt and Dijkstra 2006, Allen and Armstrong 2008). A striking pattern in our results is the lack of influence the Indo-Mediterranean region had on the western Atlantic. It is evident that the present cardiid diversity in the western Atlantic, more particularly, the Caribbean diversity, is more closely tied to the Indo-Pacific via connection to the tropical east Pacific through the Isthmus of Panama. This contrasts the recent works of Rocha et al. (2005), Joyeux et al. (2001), and Floeter et al.(2008), who found the eastern tropical Pacific and Atlantic have been largely isolated from the Indo-Pacific for some time. However, there is some evidence for trans Atlantic dispersal in the east to west direction seen in the Clinocardiinae (Fig. 2.4; clade 5), a wide-ranging subfamily found throughout the Arctic and northern temperate oceans. The height of lineage diversification in the western tropical Atlantic (Caribbean) is not until the Miocene. I recovered three dispersals into the Caribbean via the tropical east Pacific (Fig. 2.4; cladesC,7,8) all around ca. 30 mya. During the Eocene and into the Oligocene, warm tropical water flowed in an easterly direction from the tropical Eastern Pacific into the Atlantic prior to the closure of the Panamanian Isthmus (Stille et al. 1996, Bush 1997, Thomas et al. 2003, von der Heydt and Dijkstra 2006). The Central Indo-Pacific (CIP) is a major source of regional cardiid diversity. Our reconstruction resulted in most of the major lineages being of Indo-Pacific origin. The Western Indo-Pacific (WIP) and western temperate north Pacific are both macroevolutionary sinks, were species originating in the CIP either disperse or expand their ranges into. Our reconstruction shows prolonged movement from the CIP into the WIP and western temperate north Pacific beginning in the Miocene and escalating during the Pliocene to recent.

Table 2.1. Fossil calibrations used in the BEAST analysis. ______Genus Age (period) Date (myr) Source ______1 Root Triassic 200.00-250.0 Gradstein et al. 2012 2 Vasticardium upper Eocene 35.85-37.80 Oppenheim, 1903 3 Dinocardium middle Eocene 41.20-47.80 Harbison, 1944 4 Fulvia lower Oligocene 28.10-33.90 Jung, 1974 5 Europicardium lower Miocene 20.40-23.00 Cossmann & Peyrot, 1911 6 Clinocardium lower Oligocene 28.10-33.90 Kafanov, 1998 7 Hippopus lower Miocene 20.40-23.00 Mansfield, 1937 8 Lunulicardia Pliocene 2.59-5.33 Fischer, 1927 9 Americardia lower Oligocene 28.10-33.90 Olsson, 1932 10 Acanthocardia lower Oligocene 28.10-33.90 Wolff, 1897 11 Parvicardium lower Eocene 51.90-56.00 Cossmann, 1882 12 Afrocardium middle Eocene 44.50-47.80 Glibert, 1936 13 Lophocardium upper Eocene 33.90-37.80 Clark & Durham, 1946

Table 2.2. Clade ages estimated by Beast for 14 focal nodes with upper and lower 95% highest posterior density. ______Node Age Estimate ______A 34.47 (33.72,36.53) B 83.78 (62.88,107.77) B 1 56.57 (44.37,75.01) B 2 57.28 (40.66,76.78) C 81.26 ( no HPD) C 3 45.41 (33.68,60.77) C 4 39.87 (27.11,54.67) D 21.11 (20.21,23.18) E 63.26 (48.26, 80.97) E5 28.67 (27.95, 31.29) E6 51.77 (38.54, 67.47) F 51.97 (40.28, 67.03) F7 40.04 (27.9, 54.11) F8 36.6 (35.68, 38.37) W 134.19 (101.62, 175.05) X 103.46 (83.17, 127.51) Y 96.66 (no HPD) Z 77.24 (60.12, 96.9

I E K H

A D G J C

Figure 2.1. Biogeographic regions used in this study of the Cardiidae. Key: A, western tropical Atlantic; B, temperate south Africa; C, Western Indo-Pacific; D, Central Indo-Pacific; E, western temperate north Pacific; F, Arctic, G, eastern tropical Pacific; H, western temperate north Atlantic; I, eastern temperate north Atlantic; J, eastern tropical Atlantic; K, eastern temperate north Pacific

Figure 2.2: Paleogeographical model used in the Cardiidae analysis. Three time slices were used to reflect the probability of area connectivity through time using a Q matrix of instantaneous transition rates. Bold indicates a change in probability between time slices. Key: A- western tropical Atlantic; B- temperate South Africa; C- Western Indo-Pacific; D- Central Indo-Pacific; E- western temperate north Pacific; F- Arctic; G- eastern tropical Pacific; H- western temperate north Pacific; I-eastern temperate north Atlantic; J- Eastern tropical Atlantic; K- eastern temperate north Pacific. Maps produced by Blakey, R. Global Paleogeography. http://www2.nau.edu/rcb7/globaltext2.html.

L-E Cretaceous ~105 ma

(a) Time slice 1: before 60 my A B C D E F G H I J K A - 0 0 0 0 0 1 1 0 1 0 B 0 - 1 0 0 0 0 0 0 1 0 C 0 1 - 1 0 0 0 0 1 0 0 D 0 0 1 - 1 0 0.05 0 0 0 0 E 0 0 0 1 - 1 0 0 0 0 0.05 F 0 0 0 0 1 - 0 1 1 0 1 G 1 0 0 0.05 0 0 - 0 0 0 1 H 1 0 0 0 0 1 0 - 1 0 0 I 0 0 1 0 0 1 0 1 - 1 0 J 1 1 0 0 0 0 0 0 1 - 0 K 0 0 0 0 0.05 1 1 0 0 0 - 36

Oligocene ~35 ma

(b) Time slice 2: 60-25 ma A B C D E F G H I J K A - 0 0 0 0 0 1 1 0 0.5 0 B 0 - 1 0 0 0 0 0 0 1 0 C 0 1 - 1 0 0 0 0 0.5 0 0 D 0 0 1 - 1 0 0.05 0 0 0 0 E 0 0 0 1 - 1 0 0 0 0 0.05 F 0 0 0 0 1 - 0 1 1 0 1 G 1 0 0 0.05 0 0 - 0 0 0 1 H 1 0 0 0 0 1 0 - 0.5 0 0 I 0 0 0.5 0 0 1 0 0.5 - 1 0 J 0.5 1 0 0 0 0 0 0 1 - 0 K 0 0 0 0 0.05 1 1 0 0 0 -

Miocene ~20 ma

A B C D E F G H I J K A - 0 0 0 0 0 1 1 0 0.1 0 B 0 - 1 0 0 0 0 0 0 1 0 C 0 1 - 1 0 0 0 0 0 0 0 D 0 0 1 - 1 0 0.05 0 0 0 0 E 0 0 0 1 - 1 0 0 0 0 0.05 F 0 0 0 0 1 - 0 1 1 0 1 G 1 0 0 0.05 0 0 - 0 0 0 1 H 1 0 0 0 0 1 0 - 0.1 0 0 I 0 0 0 0 0 1 0 0.1 - 1 0 J 0.1 1 0 0 0 0 0 0 1 - 0 K 0 0 0 0 0.05 1 1 0 0 0 -

Figure 2.3. Chronogram of Cardiidae produced from the BEAST analysis. Maximum clade credibility tree with mean nodal ages and 95% highest posterior density (HPD) intervals indicated by bars. The time-scale in Mya (million years ago) and geological time periods are shown at the bottom. Black squares represent the 13 fossil calibrations as listed in table 1.

Vasticardium angulatum IM 20099835 Vasticardium flavum UF 323694 Vasticardium elongatum IM 200630157 Vasticardium vertebratum NTM 43909 Vasticardium pectiniforme IM 200740245 Vasticardium lacunosum UF 292823 Vasticardium kenyanum IM 20099785 Vasticardium philippinense CASIZ 114334 Vasticardium insulare RMNH 41367 Acrosterigma punctolineatum IM 200730197 Acrosterigma transcendens IM 20099805 Acrosterigma simplex UF 425951 8 Laevicardium attenuatum IM 20099777 Laevicardium biradiatum UF 304138 Papyridea crockeri UF 371362 Mexicardia panamensis SBMNH 149262 Papyridea aspersa UF 371942 Papyridea semisulcata UF 286647 F Trachycardium egmontianum UF 433840 Phlogocardium belcheri UF 391879 Dallocardia senticosum UF 351612 Acrosterigma magnum FMNH 315294 Laevicardium elenense UF 371589 Laevicardium pictum UF 286690 Laevicardium serratum UF 348860 7 Laevicardium pristis JJTP 2139 Dinocardium robustum BV22 Laevicardium senegalense RMNH 119658 Fulvia australis 110 Fulvia sp. IM 200730235 Fulvia1 n. sp. IM 200743103 Fulvia aperta IM 200730141 Fulvia aperta RMNH 41468 Fulvia boholensis UF 304156 Fulvia australis IM 200730159 Fulvia undatopica BV32 Fulvia mutica BV19 Fulvia dulcis IM 200730192 Fulvia colorata IM 20099791 Fulvia hungerfordi IM 200730237 Fulvia hungerfordi IM 200730142 Fulvia hungerfordi BV18 Cardium maxicostatum RMNH 119660 Cardium costatum RMNH119661 6 Bucardium ringens RMNH 119662 Cardium indicum RMNH 119659 Europicardium caparti RMNH 13182 E Fulvia lineonotata UF 305206 Laevicardium lobulatum IM 200730151 Serripes laperousii CASIZ 168221 Ciliatocardium ciliatum FMNH 326761 Serripes groenlandicus FMNH 326762 5 Keenocardium buellowi BV33 Keenocardium blandum BV26 Clinocardium nuttallii BV27 Lunulicardia retusa BMNH 20060464 Fragum mundum 78 Fragum whitleyi IM 200730199 Lunulicardia hemicardia 136 Corculum cardissa JJTP 3978 Fragum fragum IM 200730139 Fragum unedo 131 Fragum loochooanum 121 Fragum whitleyi 78 4 Fragum carinatum 318 Fragum whitleyi IM 200730190 Fragum aff. mundum 375 Fragum erugatum 133 Fragum sueziense 31 Ctenocardia translata IM 200730162

Fragum sueziense 31 Ctenocardia translata IM 200730162 Microfragum subfestivum IM 200732254 C Microfragum festivum IM 200730232 Ctenocardia fornicata IM 20099775 3 Ctenocardia gustavi 311 Americardia biangulatum UF 351609 Ctenocardia media UF 380487 Trigoniocardia granifera 333 Apiocardia obovalis BMNH 20080641 Tridacna squamosa AF122978 Tridacna crocea NDH02 Tridacna maxima NDH01 Tridacna derasa RMNH 40948 Tridacna gigas AF122978 Tridacna mbalavuana AF122977 D Hippopus hippopus F122973 Hippopus porcellanus AF122974 Fragum sueziense IM 200730189 Acanthocardia aculeata JJTP 1825 Acanthocardia tuberculata JJTP 1827 Acanthocardia echinata BMNH 20070236 Acanthocardia paucicostata JJTP 1396 Papillicardium turtoni NMSA W2602 Papillicardium papillosum UF 374115 Cerastoderma edule 01 2 Cerastoderma glaucum FMNH 318977 Monodacna colorata FMNH312499 Parvicardium scriptum UF 374117A Parvicardium vroomi 294 B Parvicardium exiguum UF 374117B Parvicardium minimum 194 Afrocardium richardi IM 200730236 Afrocardium exochum IM 20099780 1 Lyrocardium lyratum IM 200730001 Lyrocardium aurantiacum UF 298808 Freneixicardia victor IM 20099774 Microcardium pazianum UF 351595 Microcardium n. sp. IM 200732269 Microcardium tenuilamellosum IM200730196 Microcardium velatum IM 200732278 Microcardium trapezoidale IM 20099767 Microcardium sakuraii IM 200732260 Trifaricardium nomurai IM 200732271 Microcardium aequiliratum IM 200732265 Frigidocardium centumliratum IM 20099803 A Frigidocardium eos BV29 Lophocardium cumingi BMNH 20080645 Frigidocardium helios IM 200730161 Frigidocardium torresi IM 200730234 Frigidocardium kirana UF 294008 Katelysia rhytiphora FMNH 302051 Katelysia scalarina AMNH 311620 Anomalocardia auberiana FMNH 305976 Antigona lamellaris FLMNH 281662 Circe rivularis FMNH 306188 Circe plicatina AMNH 311616 Circe nummulina AMNH 311617 Petricola lapicida AMNH 305124 Petricolaria pholadiformis AMNH 311609 Tresus capax Rangia cuneata Macoma balthica Scissula similis 200.0 150.0 100.0 50.0 0.0

Figure 2.4. Biogeographical reconstruction of ancestral ranges in the Cardiidae. Colored circles and letters to the left of the species names indicate current biogeographical distributions and correspond to the distribution map. Pie charts at nodes represent the probabilities of the most likely ancestral ranges. Letters in parentheses correspond to the biogeographical distributions on the distribution map (Fig. 2.1). (CDI) Afrocardium richardi IM200730236 #!" #"

# #" (CD) Afrocardium exochumIM20099780 #! # #! (CDE) Lyrocardium lyratumIM200730001 #! #!" #" #!& 1 (C) Lyrocardium aurantiacumUF298808 # #!" (CD) Freneixicardia victorIM20099774

(B) Papillicardium turtoniNMSAW2602 '% (IJ) Papillicardium papillosumUF374115 "% #" (I) Acanthocardia paucicostataJJTP1396 B "% "% (I) Acanthocardia echinataBMNH20070236 #!" " "% "% (I) Acanthocardia aculeataJJTP1825 "

" (IJ) Acanthocardia tuberculataJJTP1827 " (I) Cerastoderma edule 01 "

"% " (I) Cerastoderma glaucumFMNH318977

(I) Monodacna colorataFMNH312499 " 2 (I) Parvicardium exiguumUF374117B " (I) Parvicardium scriptumUF374117A " " (I) Parvicardium vroomi 294

(I) Parvicardium minimum 194

(D) F122973 Hippopus hippopus ! (D) AF122974 Hippopus porcellanus

! (D) AF122977 Tridacna mbalavuana

(DE) Tridacna maximaNDH01 ! !& !& (D) AF122978 Tridacna squamosa ! ! D !& (DE) Tridacna croceaNDH02 ! ! #! (DE) Tridacna derasaRMNH40948 !& (D) AF122975 Tridacna gigas

(CD) Fragum suezienseIM200730189

(DE) Microfragum festivumIM200730232 !& #! #!& (C) Ctenocardia translataIM200730162 ! ! ! #! (D) Microfragum subfestivumIM200732254 ! #! X )! (CD) Ctenocardia fornicataIM20099775 !

#!" 3 !$ (D) Ctenocardia gustavi 311 (G) Americardia biangulatumUF351609 $ )$ (A) Ctenocardia mediaUF380487 )$ $( $ (G) Trigoniocardia granifera 333

! $ (GK) Apiocardia obovalisBMNH20080641

(CD) Fragum erugatum 133 C #! (CD) Fragum sueziense 31

(D) Fragum aff mundum 375 ! #! #! (D) Fragum carinatum 318

" (CD) Fragum whitleyiIM200730190 " 4 "# !" (CD) Fragum fragumIM200730139 !"# " (DE) Fragum unedo 131 " " (D) Corculum cardissaJJTP3978

" !" (CD) Lunulicardia retusaBMNH20060464

(D) Fragum mundum 78 " " " !" (CD) Fragum whitleyiIM200730199

" (D) Lunulicardia hemicardia 136

(D) Fragum loochooanum 121 !" " (CD) Fragum whitleyi 78

(K) Keenocardium buellowiBV33 & (K) Keenocardium blandumBV26 '%& Y #'& !" & (EFK) Serripes laperousiiCASIZ168221 *" "% '& #'& #' !"% #' '%& #'%& '%& #'& '( (FK) Ciliatocardium ciliatumFMNH326761 #'% #'& ' " 5 '% #'%& #'%& (% (EFIK) Serripes groenlandicusFMNH326762

(H) Clinocardium nuttalliiBV27

E "# (CD) Fulvia lineonotataUF305206 !"# " !"# "# "% !% !" (DE) Laevicardium lobulatumIM200730151 !"% (J) Cardium maxicostatumRMNH119660 ) %) (J) Cardium costatumRMNH119661

!" "% ) (J) Bucardium ringensRMNH119662 " % )

!"% (IJ) Cardium indicumRMNH119659 %) %) 6 (I) Europicardium capartiRMNH13182

(DE) Fulvia hungerfordiIM200730237 "% "# !% " # (DE) Fulvia hungerfordiIM200730142

!"% "# (DE) Fulvia hungerfordiBV18

! " (CD) Fulvia dulcisIM200730192

" (CD) Fulvia colorataIM20099791 "# !" !"# !" (CD) Fulvia australisIM200730159

*" " (D) Fulvia nsp1IM200743103 "% " !" W !" (CD) Fulvia australis 110 "$ "# !" " " !"# " " (D) Fulvia spIM200730235

!"# " (D) Fulvia apertaIM200730141 "# !" (D) Fulvia apertaRMNH41468 " " !"# (D) Fulvia boholensisUF304156

"# (CDE) Fulvia undatopicaBV32 "$ *" "# !" "% (E) Fulvia muticaBV19 " !"% (CD) Acrosterigma simplexUF425951 " Z (D) Acrosterigma punctolineatumIM200730197 " " (D) Acrosterigma transcendensIM20099805

!" (CD) Laevicardium attenuatumIM20099777

" (CD) Laevicardium biradiatumUF304138

!" (CD) Vasticardium angulatumIM20099835 " !" (D) Vasticardium flavumUF323694 " ! ! " " (CD) Vasticardium elongatumIM200630157

(C) Vasticardium lacunosumUF292823 !" ! " ! (D) Vasticardium vertebratumNTM43909 !" " !" !" " (CD) Vasticardium pectiniformeIM200740245

(CD) Vasticardium kenyanumIM20099785 8 !" "$ (D) Vasticardium philippinenseCASIZ114334 !" (C) Vasticardium insulareRMNH41367

(G) Papyridea aspersaUF371942 $ (G) Papyridea crockeriUF371362 $ $ (G) Mexicardia panamensisSBMNH149262 #$ #$ (A) Papyridea semisulcataUF286647

(A) Trachycardium egmontianumUF433840 #$ F #" $ "$ $ (G) Phlogocardium belcheriUF391879 # (GK) Dallocardia senticosumUF351612 #$ #$ (A) Acrosterigma magnumFMNH315294

(A) Laevicardium pristisJJTP2139 # (G) Laevicardium elenenseUF371589 #$ #$ # (AJ) Laevicardium pictumUF286690 # 7 #$ (A) Laevicardium serratumUF348860 #$ (AH) Dinocardium robustumBV22 #& (J) Laevicardium senegalenseRMNH119658

!" (D) Frigidocardium heliosIM200730161

" " (CD) Frigidocardium torresiIM200730234

(D) Frigidocardium kiranaUF294008

(G) Lophocardium cumingiBMNH20080645 "$ A (DE) Trifaricardium nomuraiIM200732271 "% (D) Microcardium aequiliratumIM200732265

"$ (G) Microcardium pazianumUF351595 "$ "$ "$ (D) Microcardium n spIM200732269 " (D) Microcardium tenuilamellosumIM200730196 " " "$ "$ (D) Microcardium velatumIM200732278 " "% (D) Microcardium trapezoidaleIM20099767 " (DE) Microcardium sakuraiiIM200732260 "

!" (C) Frigidocardium centumliratumIM20099803

(DE) Frigidocardium eosBV29 !"%

APPENDIX A

A.1. SAMPLING AND VOUCHER INFORMATION FOR THE CARDIIDAE USED IN THIS STUDY. ______Taxa Voucher Histone 3 16S 28S ______Acanthocardia echinata BMNH 20070236 S S S Acanthocardia aculeata JJTP 1825 S S — Acanthocardia paucicostata JJTP 1396 S S — Acanthocardia tuberculata JJTP 1827 S S — Acrosterigma magnum FMNH 315294 S S S Acrosterigma punctolineatum IM-2007-30197 S S S Acrosterigma simplex UF 425951 S S S Acrosterigma transcendens IM-2009-9805 S S S Acrosterigma transcendens IM-2009-9795 — S — Acrosterigma transcendens IM-2009-9779 S S S Afrocardium exochum IM-2009-9796 S S — Afrocardium exochum IM-2009-9789 S — — Afrocardium exochum IM-2009-9782 S — S Afrocardium exochum IM-2009-9780 S S S Afrocardium richardi IM-2007-30236 S S — Afrocardium richardi IM-2007-30229 S — — Apiocardia obovalis BMNH 20080641 S S — Bucardium ringens RMNH 119662 S — — Cardium costum RMNH 119661 S S — Cardium indicum RMNH 119659 S S — Cardium maxicostatum RMNH 119660 — S S Cerastoderma edule BMNH 20070234 S S S Cerastoderma edule BMNH 20030333 S S — Cerastoderma glaucum FMNH 318977 S S S Ciliatocardium ciliatum FMNH 326761 S S S

Table A.1- continued ______Taxa Voucher Histone 3 16S 28S ______Ciliatocardium ciliatum FMNH 278012 S — — Clinocardium nuttallii FMNH 170895 S — — Clinocardium nuttallii BV 27 (UBC 7) S S — Corculum cardissa RMNH 41473 S — — Corculum cardissa RMNH 41472 S S — Corculum cardissa JJTP 3978 S S S Corculum cardissa JJTP 3938 S S — Ctenocardia biangulata UF 351609 S S S Ctenocardia biangulata ANSP A15529 S S — Ctenocardia cf. fornicata IM-2007-30155 S S — Ctenocardia fornicata IM-2009-9775 S S S Ctenocardia gustavi IM-2007-30164 S S — Ctenocardia media UF 380487 S S — Ctenocardia translata IM-2007-30162 S S — Dallocardia senticostum UF 359633 S S — Dallocardia senticosum UF 351612 S S S Dallocardia senticosum UF 351587 S S S Dinocardium robustum BV22 S S S Europicardium caparti RMNH 13183 S S — Europicardium caparti RMNH 13182 S — — Fragum fragum IM-2007-30139 — S — Fragum fragum IM-2007-30138 — S — Fragum scruposum IM-2007-30199 S S — Fragum scruposum IM-2007-30190 S S — Fragum scruposum IM-2007-30158 S — S Fragum sueziense IM-2007-30189 S — — Fragum sueziense IM-2007-30167 S — —

Table A.1- continued ______Taxa Voucher Histone 3 16S 28S ______Freneixicardia victor IM-2009-9774 — S S Frigidocardium torresi IM-2007-30234 — S — Frigidocardium torresi IM-2007-30191 — S S Frigidocardium cf. centumliratum IM-2009-9803 S S S Frigidocardium cf. centumliratum IM-2009-9786 S S S Frigidocardium cf. centumliratum IM-2009-9768 — S S Frigidocardium eos BV29 S S — Frigidocardium helios IM-2007-30161 — S — Frigidocardium kirana UF 294008 — S — Fulvia (Fulvia) aperta RMNH 41468 S S — Fulvia (Fulvia) aperta IM-2007-30141 — S — Fulvia (Fulvia) australis IM-2007-30159 S S — Fulvia (Fulvia) boholensis UF 304156 S S S Fulvia (Fulvia) colorata IM-2009-9793 — S S Fulvia (Fulvia) colorata IM-2009-9791 S S S Fulvia (Fulvia) colorata IM-2007-30156 S S S Fulvia (Fulvia) dulcis IM-2007-30225 S S — Fulvia (Fulvia) dulcis IM-2007-30192 S S — Fulvia (Fulvia) dulcis IM-2007-30168 S S — Fulvia (Fulvia) mutica BV19 S S S Fulvia (Fulvia) n.sp. 1 IM-2007-43104 S S — Fulvia (Fulvia) n.sp. 1 IM-2007-43103 S S S Fulvia (Fulvia) spec. IM-2007-30235 S S — Fulvia (Laevifulvia) hungerfordi BV18 S S S Fulvia (Laevifulvia) hungerfordi IM-2007-30237 S S — Fulvia (Laevifulvia) hungerfordi IM-2007-30231 S S — Fulvia (Laevifulvia) hungerfordi IM-2007-30195 S — —

Table A.1- continued ______Taxa Voucher Histone 3 16S 28S ______Fulvia (Laevifulvia) lineonotata UF 305206 S S S Fulvia (Laevifulvia) subquadrata IM-2007-30142 S S — Fulvia (Laevifulvia) undatopicta BV32 S S S Fulvia (Laevifulvia) undatopicta BV31 S S S Fulvia (Laevifulvia) undatopicta BV30 S S S Keenocardium blandum FMNH 12581 S — — Keenocardium blandum BV 26 (UBC 6) S S S Keenocardium blandum BV 25 (UBC 4) S — — Keenocardium buelowi BV33 S S — Laevicardium attenuatum IM-2009-9777 S S S Laevicardium biradiatum UF 285613 S S S Laevicardium biradiatum UF 304138 — S — Laevicardium elenense UF 371589 S S S Laevicardium elenense ANSP A15494 S — S Laevicardium laevigatum UF 348860 S S — Laevicardium laevigatum UF 322245 S S S Laevicardium lobulatum IM-2007-30151 S S — Laevicardium pictum UF 286690 S S — Laevicardium pristis JJTP 2139 — S — Laevicardium senegalense RMNH 119658 — S S Lophocardium cumingi BMNH 20080645 S S — Lunulicardia retusa BMNH 20060464 S S — Lyrocardium aurantiacum UF 298808 — S — Lyrocardium lyratum IM-2007-30001 S S — Maoricardium pseudolatum NTM 43910 S — — Microcardium aequiliratum IM-2007-32265 — S — Microcardium cf. pazianum UF 372357 S S S

Table A.1- continued ______Taxa Voucher Histone 3 16S 28S ______Microcardium n.sp. IM-2007-32269 S S S Microcardium pazianum UF 351595 — S S Microcardium sakuraii IM-2007-32260 S S S Microcardium sakuraii IM-2007-32245 — S — Microcardium tenuilamellosum IM-2007-30196 — S — Microcardium trapezoidale IM-2009-9767 — S — Microcardium trapezoidale IM-2009-9765 — S — Microcardium trapezoidale IM-2007-32259 — S — Microcardium velatum IM-2007-32278 S S S Microcardium velatum IM-2007-32277 S S — Microfragum festivum IM-2007-30232 S S — Microfragum subfestivum IM-2007-32254 S S — Monodacna colorata FMNH 312499 S — S Papillicardium papillosum UF 374115 S S S Papyridea aspersa UF 371942 S S S Papyridea crockeri UF 371362 S S — Papyridea semisulcata UF 286647 S S S Parvicardium exigum UF 374117B S — S Parvicardium scriptum UF 374117A S — S Parvicardium scriptum JJTP 271 S — — Serripes groenlandicus FMNH 326762 S S S Serripes laperousii CASIZ 168221 — — S Trachycardium belcheri UF 391879 S S S Trachycardium egmontianum UF 433948 S S — Trachycardium egmontianum UF 433840 S S S Tridacna crocea NDH02 — S — Tridacna derasa RMNH 40948 — S S

Table A.1- continued ______Taxa Voucher Histone 3 16S 28S ______Tridacna maxima NDH01 — S — Trifaricardium nomurai IM-2007-32271 S S S Vasticardium angulatum IM-2009-9835 S S S Vasticardium angulatum IM-2009-9834 S S S Vasticardium elongatum enode IM-2007-30157 S S S Vasticardium enode UF 299284 S S S Vasticardium flavum UF 323694 S S S Vasticardium flavum RMNH 41477 S — — Vasticardium insulare RMNH 41367 S S S Vasticardium kenyanum IM-2009-9785 — S S Vasticardium lacunosum UF 292823 S S — Vasticardium pectiniforme IM-2007-41443 — S S Vasticardium pectiniforme IM-2007-40245 — S S Vepricardium multispinosum NTM 43907 — S —

APPENDIX B

B.1 CARDIID SEQUENCES INCORPORATED INTO THIS STUDY FROM GENBANK. ______Species Accession Source ______Cerastoderma edule AF122971 Nikula & Vainola, 2003 Cerastoderma glaucum AF122972 Nikula & Vainola, 2003 Hippopus hippopus AF122973 Schneider & Foighil, 1999 Hippopus porcellanus AF122974 Schneider & Foighil, 1999 Tridacna gigas AF122975 Schneider & Foighil, 1999 Tridacna derasa AF122976 Schneider & Foighil, 1999 Tridacna tevoroa AF122977 Schneider & Foighil, 1999 Tridacna squamosa AF122978 Schneider & Foighil, 1999 Tridacna maxima AF122979 Schneider & Foighil, 1999 Tridacna crocea AF122980 Schneider & Foighil, 1999 Parvicardium sp 299 EU733015 Kirkendale, 2009 Parvicardium vroomi isolate 294 EU733016 Kirkendale, 2009 Parvicardium vroomi isolate 296 EU733017 Kirkendale, 2009 Parvicardium scriptum isolate 283 EU733018 Kirkendale, 2009 Parvicardium papillosum isolate 279 EU733019 Kirkendale, 2009 Parvicardium papillosum isolate 278 EU733020 Kirkendale, 2009 Ctenocardia fornicata isolate 18 EU733021 Kirkendale, 2009 Ctenocardia victor isolate 3 EU733022 Kirkendale, 2009 Ctenocardia gustavi isolate 311 EU733023 Kirkendale, 2009 Trigoniocardia granifera isolate 334 EU733025 Kirkendale, 2009 Americardia media isolate 115 EU733026 Kirkendale, 2009 Americardia media isolate 387 EU733027 Kirkendale, 2009 Fragum sueziense isolate 31 EU733028 Kirkendale, 2009 Fragum sueziense isolate 56 EU733029 Kirkendale, 2009 Fragum carinatum isolate 318 EU733030 Kirkendale, 2009 Fragum scruposum isolate 316 EU733031 Kirkendale, 2009

Table B.1- continued ______Species Accession Source ______Fragum scruposum isolate 315 EU733032 Kirkendale, 2009 Fragum fragum isolate 60 EU733033 Kirkendale, 2009 Fragum unedo isolate 129 EU733034 Kirkendale, 2009 Fragum unedo isolate 131 EU733035 Kirkendale, 2009 Fragum mundum isolate 78 EU733036 Kirkendale, 2009 Fragum mundum isolate 379 EU733037 Kirkendale, 2009 Fragum mundum isolate 381 EU733038 Kirkendale, 2009 Corculum cardissa isolate 9 EU733039 Kirkendale, 2009 Corculum cardissa isolate 67 EU733040 Kirkendale, 2009 Cerastoderma edule isolate 301 EU733041 Kirkendale, 2009 Cerastoderma edule isolate 300 EU733042 Kirkendale, 2009 Nemocardium pazianum isolate 341 EU733043 Kirkendale, 2009 Fulvia australis isolate 110 EU733044 Kirkendale, 2009 Papyridea semisulcata isolate 80 EU733045 Kirkendale, 2009 Papyridea aspersa isolate 336 EU733046 Kirkendale, 2009 Lunulicardia hemicardia isolate 136 EU733047 Kirkendale, 2009 Parvicardium minimum isolate 194 EU733048 Kirkendale, 2009 Parvicardium minimum isolate 195 EU733049 Kirkendale, 2009 Parvicardium papillosum isolate 279 EU733051 Kirkendale, 2009 Parvicardium papillosum isolate 278 EU733052 Kirkendale, 2009 Ctenocardia fornicata isolate 18 EU733053 Kirkendale, 2009 Ctenocardia victor isolate 3 EU733054 Kirkendale, 2009 Ctenocardia gustavi isolate 311 EU733055 Kirkendale, 2009 Trigoniocardia granifera isolate 333 EU733056 Kirkendale, 2009 Trigoniocardia granifera isolate 334 EU733057 Kirkendale, 2009 Americardia media isolate 115 EU733058 Kirkendale, 2009 Americardia media isolate 387 EU733059 Kirkendale, 2009

Table B.1- continued ______Species Accession Source ______Fragum sueziense isolate 56 EU733060 Kirkendale, 2009 Fragum sueziense isolate 31 EU733061 Kirkendale, 2009 Fragum aff mundum isolate 375 EU733062 Kirkendale, 2009 Fragum aff mundum isolate 377 EU733063 Kirkendale, 2009 Fragum carinatum isolate 318 EU733064 Kirkendale, 2009 Fragum scruposum isolate 316 EU733065 Kirkendale, 2009 Fragum scruposum isolate 315 EU733066 Kirkendale, 2009 Fragum loochooanum isolate 385 EU733067 Kirkendale, 2009 Fragum loochooanum isolate 386 EU733068 Kirkendale, 2009 Fragum loochooanum isolate 382 EU733069 Kirkendale, 2009 Fragum loochooanum isolate 383 EU733070 Kirkendale, 2009 Fragum loochooanum isolate 121 EU733071 Kirkendale, 2009 Fragum fragum isolate 60 EU733072 Kirkendale, 2009 Fragum unedo isolate 129 EU733073 Kirkendale, 2009 Fragum unedo isolate 131 EU733074 Kirkendale, 2009 Fragum mundum isolate 78 EU733075 Kirkendale, 2009 Fragum mundum isolate 379 EU733076 Kirkendale, 2009 Fragum mundum isolate 381 EU733077 Kirkendale, 2009 Corculum cardissa isolate 9 EU733078 Kirkendale, 2009 Corculum cardissa isolate 67 EU733079 Kirkendale, 2009 Cerastoderma edule isolate 301 EU733080 Kirkendale, 2009 Cerastoderma edule isolate 300 EU733081 Kirkendale, 2009 Nemocardium pazianum isolate 341 EU733082 Kirkendale, 2009 Fulvia australis isolate 110 EU733083 Kirkendale, 2009 Papyridea semisulcata isolate 80 EU733084 Kirkendale, 2009 Papyridea aspersa isolate 336 EU733085 Kirkendale, 2009 Cerastoderma glaucum isolate 345 EU733086 Kirkendale, 2009

Table B.1- continued ______Species Accession Source ______Cerastoderma glaucum isolate 346 EU733087 Kirkendale, 2009 Trigoniocardia obovalis EU733088 Kirkendale, 2009 Trigoniocardia obovalis EU733089 Kirkendale, 2009 Americardia biangulata isolate 331 EU733090 Kirkendale, 2009 Americardia biangulata isolate 332 EU733091 Kirkendale, 2009 Ctenocardia festivum isolate 201 EU733092 Kirkendale, 2009 Fragum fragum isolate 61 EU733093 Kirkendale, 2009 Fragum fragum isolate 48 EU733094 Kirkendale, 2009 Fragum fragum isolate 24 EU733095 Kirkendale, 2009 Lunulicardia retusa isolate 29 EU733096 Kirkendale, 2009 Lunulicardia retusa isolate 21 EU733097 Kirkendale, 2009 Lunulicardia retusa isolate 22 EU733098 Kirkendale, 2009 Lunulicardia hemicardia isolate 136 EU733099 Kirkendale, 2009 Fragum erugatum isolate 133 EU733100 Kirkendale, 2009 Fragum erugatum isolate 134 EU733101 Kirkendale, 2009 Fragum mundum isolate 116 EU733102 Kirkendale, 2009 Acrosterigma biradiatum isolate 79 EU733103 Kirkendale, 2009 Papyridea sp 335 EU733104 Kirkendale, 2009 Acanthocardia echinata isolate 204 EU733105 Kirkendale, 2009 Nemocardium tinctum isolate 138 EU733106 Kirkendale, 2009 Ctenocardia fornicata isolate 17 EU733107 Kirkendale, 2009

REFERENCES

Allen, M. B. and H. A. Armstrong. 2008. Arabia–Eurasia collision and the forcing of mid- Cenozoic global cooling. Palaeogeography, Palaeoclimatology, Palaeoecology 265:52- 58.

Barber, P. H., M. V. Erdmann, M. K. Moosa, and S. R. Palumbi. 2000. Biogeography: A marine Wallace's line? Nature 406:692+.

Barber, P. H., S. R. Palumbi, M. V. Erdmann, and M. K. Moosa. 2002. Sharp genetic breaks among populations of Haptosquilla pulchella (Stomatopoda) indicate limits to larval transport: patterns, causes, and consequences. Molecular Ecology 11:659-674.

Bellwood, D. R. and C. P. Meyer. 2009. Searching for heat in a marine biodiversity hotspot. Journal of Biogeography 36:569-576.

Bellwood, D. R. and P. W. Wainwright. 2002. The history and biogeography of fishes on coral reefs. Pages 5-32 in P. F. Sale, editor. Coral reef fishes: dynamics and diversity in a complex ecosystem. Academic Press, London.

Briggs, J. C. 2003. Marine centres of origin as evolutionary engines. Pages 1-18 Journal of Biogeography. Wiley-Blackwell.

Briggs, J. C. 2006. Proximate sources of marine biodiversity. Journal of Biogeography 33:1-10.

Brown, W. M., T. M. Collins, K. Frazer, A. R. Palmer, and G. J. Vermeij. 1996. Evolutionary history of northern hemisphere Nucella (gastropoda, muricidae): molecular, morphological, ecological, and paleontological evidence. Evolution 50:2287+.

Bush, A. B. G. 1997. Numerical simulation of the Cretaceous Tethys circumglobal current. Science 275:807.

Carter, J. G. and J. A. Schneider. 1997. Condensing lenses and shell microstructure in Corculum (Mollusca: Bivalvia). Journal of Paleontology 71:56-61.

Castresana, J. 2000. Selection of Conserved Blocks from Multiple Alignments for Their Use in Phylogenetic Analysis. Molecular Biology and Evolution 17:540-552.

Coscia, I., P. Robins, J. Porter, S. Malham, and J. Ironside. 2013. Modelled larval dispersal and measured gene flow: seascape genetics of the common cockle Cerastoderma edule in the southern Irish Sea. Conservation Genetics 14:451-466.

Cowman, P. F., D. R. Bellwood, and L. A. Rocha. 2013. The historical biogeography of coral reef fishes: global patterns of origination and dispersal. Journal of Biogeography 40:209- 224.

Cox, B. C. and P. D. Moore. 2010. Biogeography An Ecological and Evolutionary Approach. 8 edition. John Wiley & Sons, Inc., Hoboken, NJ.

Crame, J. A. 2000. Evolution of Taxonomic Diversity Gradients in the Marine Realm: Evidence From the Composition of Recent Bivalve Faunas. Paleobiology 26:188-214.

DeBoer, T. S., M. D. Subia, Ambariyanto, M. V. Erdmann, K. Kovitvongsa, and P. H. Barber. 2008. Phylogeography and Limited Genetic Connectivity in the Endangered Boring Giant Clam across the Coral Triangle. Conservation Biology 22:1255-1266.

Drummond, A. J., S. Y. W. Ho, M. J. Phillips, and A. Rambaut. 2006. Relaxed Phylogenetics and Dating with Confidence. PLoS Biol 4:e88.

Drummond, A. J. and A. Rambaut. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. v. 1.5.1. BMC Evolutionary Biology 7:214.

Drummond, A. J., M. A. Suchard, D. Xie, and A. Rambaut. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution.

Ekman, S. 1953. Zoogeography of the Sea. Sidgwick & Jackson, London.

Floeter, S. R., L. A. Rocha, D. R. Robertson, J. C. Joyeux, W. F. Smith-Vaniz, P. Wirtz, A. J. Edwards, J. P. Barreiros, C. E. L. Ferreira, J. L. Gasparini, A. Brito, J. M. Falcón, B. W. Bowen, and G. Bernardi. 2008. Atlantic reef fish biogeography and evolution. Journal of Biogeography 35:22-47.

Giribet, G. and W. Wheeler. 2002. On bivalve phylogeny: a high-level analysis of the Bivalvia (Mollusca) based on combined morphology and DNA sequence data. Invertebrate Biology 121:271-324.

Grigg, R. W. and R. Hey. 1992. Paleoceanography of the Tropical Eastern Pacific Ocean. Science 255:172-178.

Harzhauser, M., O. Mandic, W. E. Piller, M. Reuter, and A. Kroh. 2008. Tracing Back the Origin of the Indo-Pacific Mollusc Fauna: Basal Tridacninae from the Oligocene and Miocene of the Sultanate of Oman. Palaeontology 51:199-213.

Healy, J. M., P. M. Mikkelsen, and R. Bieler. 2008. Sperm ultrastructure in Hemidonax pictus (Hemidonacidae, Bivalvia, Mollusca): comparison with other heterodonts, especially Cardiidae, Donacidae and Crassatelloidea. Zoological Journal of the Linnean Society 153:325-347.

Huelsenbeck, J. P. and F. Ronquist. 2003. MrBayes: A program for the Bayesian inference of phylogeny. ver. 3.0.

Hughes, T. P., D. R. Bellwood, and S. R. Connolly. 2002. Biodiversity hotspots, centres of endemicity, and the conservation of coral reefs. Ecology Letters 5:775-784.

Iredale, T. and D. F. McMichael. 1962. A reference list of the marine Mollusca of New South Wales. The Australian Museum Memoir. 11:1-109.

Jones, M. T., S. R. Voss, M. B. Ptacek, D. W. Weisrock, and D. W. Tonkyn. 2006. River drainages and phylogeography: An evolutionary significant lineage of shovel-nosed salamander (Desmognathus marmoratus) in the southern Appalachians. Molecular Phylogenetics and Evolution 38:280-287.

Joyeux, J. C., S. R. Floeter, C. E. L. Ferreira, and J. L. Gasparini. 2001. Biogeography of tropical reef fishes: the South Atlantic puzzle. Journal of Biogeography 28:831-841.

Kafanov, A. I. 1974. Composition, taxonomy and evolution of the group Clinocardium (Mollusca: Cardiidae). Zoologicheskij Zhurnal (Journal of Zoology) 53:1466-1476.

Kafanov, A. I. 1980. Systematics of the Subfamily Clinocardiinae Kafanov, 1975 (BIVALVIA, CARDIIDAE). Malacologia 19:297-328.

Kafanov, A. I. 2001. Recent and fossil Clinocardiinae (Bivalvia, Cardiidae) of the World. V. Genus Ciliatocardium Kafanov, 1974 (part 1). Bulletin of the Mizunami Fossil Museum:139-171.

Kafanov, A. I. 2002. Recent and fossil Clinocardiinae (Bivalvia, Cardiidae) of the World. VI. Genus Ciliatocardium Kafanov, 1974 (part II). Bulletin of the Mizunami Fossil Museum:1-18.

Kafanov, A. I. and S. V. Popov. 1977. K sistemek ainozoiiskikhk ardiodey (Bivalvia). Paleontologicheskii Zhurnal 1977:55-64.

Kappner, I. and R. Bieler. 2006. Phylogeny of venus clams (Bivalvia: Venerinae) as inferred from nuclear and mitochondrial gene sequences. Molecular Phylogenetics and Evoloution 40:317-331.

Kaufman, E. G. 1973. Cretaceous Bivalvia. Pages 353-383 in A. Hallam, editor. Atlas of Palaeobiogeography. Elsevier Publishing Company, New York, New York.

Kawaguti, S. 1949. Observations on the Heart Shell, Corculum cardissa (L.), and its Associated Zooxanthellae. Pacific Science 4:43-49.

Kay, A. E. 1990. Cypraeidae of the Indo-Pacific: Cenozoic Fossil History and Biogeography. Bulletin of Marine Science 47:23-34.

Keen, A. M., editor. 1969. Superfamily Cardiacea Lamarck, 1809. Geological Society of America and University of Kansas, Lawrence, Kansas.

Keen, A. M. 1980. The pelecypod family Cardiidae: A taxonomic summary. Tulane Studies in Geology and Paleontology 16:1-40.

Kirkendale, L. 2009. Their Day in the Sun: molecular phylogenetics and origin of photosymbiosis in the 'other' group of photosymbiotic marine bivalves (Cardiidae: Fraginae). Biological Journal of the Linnean Society 97:448-465.

Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson, and D. G. Higgins. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948.

Lessios, H. A., B. D. Kessing, and D. R. Robertson. 1998. Massive Gene Flow across the World's Most Potent Marine Biogeographic Barrier. Proceedings: Biological Sciences 265:583-588.

Lydeard, C. and D. R. Lindberg. 2003. Molecular systematics and phylogeography of mollusks. Smithsonian Books, Washington D.C.

Maddison, D. R. a. M., W. P. 2003. MacClade 4: Analysis of phylogeny and character evolution. Version 4.06. Sinauer Associates, Sunderland, Massachusetts.

Maruyama, T., M. Ishikura, S. Yamazaki, and S. Kanai. 1998. Molecular phylogeny of zooxanthellate bivalves. Biological Bulletin 195:70-77.

Mikkelsen, P. M., R. Bieler, I. Kappner, and T. A. Rawlings. 2006. Phylogeny of Veneroidea (Mollusca: Bivalvia) based on morphology and molecules. Zoological Journal of the Linnean Society 148:439-521.

Nevesskaja, L. A., N. P. Paramonova, and S. V. Popov. 2001. History of Lymnocardiinae (Bivalvia, Cardiidae). Palaeontological Journal 35:S147-217.

Nikula, R. and R. Vainola. 2003. Phylogeography of Cerastoderma glaucum (Bivalvia : Cardiidae) across Europe: a major break in the Eastern Mediterranean. Marine Biology 143:339-350.

Ponder, W. F., P. H. Colman, C. M. Yonge, and M. H. Colman. 1981. The taxonomic position of Hemidonax Mörch, 1871 with a review of the genus (Bivalvia: Cardiacea). Journal of the Malacological Society of Australia 5 (1-2):41-64.

Ponder, W. F. and D. R. Lindberg. 2008. Phylogeny and evolution of the mollusca. University of California Press, Berkeley and Los Angeles

Posada, D. 2008. jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution 25:1253-1256.

Ree, R. H., B. R. Moore, C. O. Webb, and M. J. Donoghue. 2005. A Likelihood Framework for Inferring the Evolution of Geographic Range on Phylogenetic Trees. Evolution 59:2299- 2311.

Ree, R. H. and I. Sanmartín. 2009. Prospects and challenges for parametric models in historical biogeographical inference. Journal of Biogeography 36:1211-1220.

Ree, R. H. and S. A. Smith. 2008. Maximum Likelihood Inference of Geographic Range Evolution by Dispersal, Local Extinction, and Cladogenesis. Systematic Biology 57:4-14.

Reece, J., B. Bowen, D. Smith, and A. Larson. 2011. Comparative phylogeography of four Indo- Pacific moray eel species (Muraenidae) reveals comparable ocean-wide genetic connectivity despite five-fold differences in available adult habitat. Marine Ecology Progress Series 437:269-277.

Rocha, L. A., D. R. Robertson, J. Roman, and B. W. Bowen. 2005. Ecological Speciation in Tropical Reef Fishes. Proceedings: Biological Sciences 272:573-579.

Roy, K., G. Hunt, D. Jablonski, A. Z. Krug, and J. W. Valentine. 2009. A macroevolutionary perspective on species range limits. Proceedings of the Royal Society B: Biological Sciences 276:1485-1493.

Roy, K. and J. D. Witman. 2009. Spatial patterns of species diversity in the shallow marine invertebrates: Patterns, processes, and prospects. Pages 101-121 in J. D. Witman and K. Roy, editors. Marine Macroecology. The University of Chicago Press, Chicago, Il.

Sanmartín, I., P. van der Mark, and F. Ronquist. 2008. Inferring dispersal: a Bayesian approach to phylogeny-based island biogeography, with special reference to the Canary Islands. Journal of Biogeography 35:428-449.

Santini, F. and J. C. Tyler. 2003. A phylogeny of the families of fossil and extant tetraodontiform fishes (Acanthomorpha, Tetraodontiformes), Upper Cretaceous to Recent. Zoological Journal of the Linnean Society 139:565-617.

Santini, F. and R. Winterbottom. 2002. Historical biogeography of Indo-western Pacific coral reef biota: is the Indonesian region a centre of origin? Journal of Biogeography 29:189- 205.

Savazzi, E. and J. Salgeback. 2004. A comparison of morphological adaptations in the cardiid bivalves Cardium and Budmania. Paleontological Research 8:221-239.

Schneider, J. A. 1992. Preliminary Cladistic-Analysis ofthe Bivalve Family Cardiidae American Malacological Bulletin 9:145-155.

Schneider, J. A. 1995. Phylogeny of the Cardiidae (Mollusca, Bivalvia): Protocardiinae, Laevicardiinae, Lahilliinae, Tulongocardiinae subfam n and Pleuriocardiinae subfam n. Zoologica Scripta 24:321-346.

Schneider, J. A. 1998a. Phylogeny of stem-group eucardiids (Bivalvia : Cardiidae) and the significance of the transitional fossil Perucardia. Malacologia 40:37-62.

Schneider, J. A. 1998b. Phylogeny of the Cardiidae (Bivalvia): Phylogenetic relationships and morphological evolution within the subfamilies Clinocardiinae, Lymnocardiinae, Fraginae and Tridacninae. Malacologia 40:321-373.

Schneider, J. A. 2002. Phylogeny of cardiid bivalves (cockles and giant clams): revision of the Cardiinae and the importance of fossils in explaining disjunct biogeographical distributions. Zoological Journal of the Linnean Society 136:321-369.

Schneider, J. A. and J. G. Carter. 2001. Evolution and phylogenetic significance of cardioidean shell microstructure (Mollusca, Bivalvia). Journal of Paleontology 75:607-643.

Schneider, J. A. and D. O. Foighil. 1999. Phylogeny of giant clams (Cardiidae : Tridacninae) based on partial mitochondrial 16S rDNA gene sequences. Molecular Phylogenetics and Evolution 13:59-66.

Spalding, M. D., G. R. Allen, N. Davidson, Z. A. Ferdana, M. Finlayson, H. E. Fox, B. S. Halpern, M. A. Jorge, A. Lombana, S. A. Lourie, K. D. Martin, E. McManus, J. Molnar, C. A. Recchia, and J. Robertson. 2007. Marine ecoregions of the world: a bioregionalization of coastal and shelf areas. BioScience 57:573-583.

Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690.

Stewart, R. B. 1930. Gabb's California Cretaceous and Tertiary type Lamellibranchs. Academy of Natural Sciences Philadelphia Special Publication 3:1-314.

Stille, P., M. Steinmann, and S. R. Riggs. 1996. Nd isotope evidence for the evolution of the paleocurrents in the Atlantic and Tethys Oceans during the past 180 Ma. Earth and Planetary Science Letters 144:9-19.

Streelman, J. T., M. Alfaro, M. W. Westneat, D. R. Bellwood, and S. A. Karl. 2002. Evolutionary history of the Parrotfishes: Biogeography, ecomorphology, and comparative diversity. Evolution 56:961-971. ter Poorten, J. J. 2009. The Cardiidae of the Panglao Marine Biodiversity Project 2004 and the Panglao 2005 Deep-Sea Cruise with descriptions of four new species (Bivalvia). Vita Malacologia 8:9-96.

Thomas, D. J., T. J. Bralower, and C. E. Jones. 2003. Neodymium isotopic reconstruction of late Paleocene–early Eocene thermohaline circulation. Earth and Planetary Science Letters 209:309-322.

Vermeij, G. J. 1980. Biogeography and Adaptation: Patterns of Marine Life. 2nd edition. Harvard University Press, Cambridge, Ma.

Vidal, J. 1999. Taxonomic review of the elongated cockles: genera Trachycardium, Vasticardium, and Acrosterigma (Mollusca: Cardiidae). Zoosystema 21:259-335.

Vidal, J. 2000. Genus Veprocardium Iredale 1929 (Bivalvia, Cardiidae) with description of a new species from Thailand, Vepricardium albohamatum Hylleberg & Vidal. Phuket Marine Biological Center Special Publication 21:447-464. von der Heydt, A. and H. A. Dijkstra. 2006. Effect of ocean gateways on the global ocean circulation in the late Oligocene and early Miocene. Paleoceanography 21:PA1011.

Voskuil, R. P. A. and W. J. H. Onverwagt. 1991a. The recent species of Maoricardium Marwick, 1944 (Mollusca: Bivalvia) with description of a new species. Basteria 55:25-33.

Voskuil, R. P. A. and W. J. H. Onverwagt. 1991b. The taxonomy of the genus Trachycardium (Part 1) with descriptions of three new species (Mollusca: Bivalvia). Vita Marina 41:56- 72.

Wiens, J. J. and M. J. Donoghue. 2004. Historical biogeography, ecology and species richness. Trends in Ecology & Evolution 19:639-644.

Willig, M. R., D. M. Kaufman, and R. D. Stevens. 2003. Latitudinal gradients of biodiversity: Pattern, process scale, and synthesis. Annual Review in Ecology and Evolution 34:273-309.

Wilson, B. R. and S. E. Stevenson. 1977. Cardiidae (Mollusca, Bivalvia) of Western Australia. Special Publication Western Australian Museum 9:1-114.

BIOGRAPHICAL SKETCH

Nathanael D. F. Herrera

Department of Biological Sciences Florida State University Tallahassee, Florida 32306

CIRRICULUM VITAE June 2013

PERSONAL Born 21 July, 1983; Asheville, North Carolina United States citizen

EDUCATION

M. S., Biological Sciences (anticipated Summer 2013) – Florida State University, Tallahassee, Florida. Interests: Systematics, Historical biogeography, and evolutionary biology Research Topic: Molecular phylogenetics and historical biogeography of the Cockles and Giant Clams (Bivalvia: Cardiidae) Major Advisor: Professor Scott J. Steppan

B. S., Biological Sciences (2009) Florida State University, Tallahassee, Florida.

TEACHING EXPERIENCE

Biology Teaching/Learning Workshop, Florida State University, Department of Biological Sciences. 25 August, 2009. Workshop Director: Dr. Ann S. Lumsden.

Graduate Teaching Assistantship, Anatomy and Physiology I (BSC 2085L), Instructor: Yung K. Su. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2009)

Graduate Teaching Assistantship. Comparative Vertebrate Anatomy (ZOO3713C), Instructor: Gregory M. Erickson. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Spring 2010)

Graduate Teaching Assistantship, Animal Diversity (BSC 2011L), Instructor: Patricia A. Spears. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Summer 2010) Graduate Teaching Assistantship. Lower Vertebrate: Herpetology (ZOO4343C), Instructor: Gregory M. Erickson. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2010)

Graduate Teaching Assistantship, Anatomy and Physiology II (BSC 2086L), Instructor: Yung K. Su. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Spring 2011)

Graduate Teaching Assistantship, Animal Diversity (BSC 2011L), Instructor: Patricia A. Spears. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Summer 2011)

Graduate Teaching Assistantship, Evolution (PCB 4674), Instructor: Scott J. Steppan. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2011)

Graduate Teaching Assistantship, Biological Sciences II (BSC 2011), Instructor: Patricia A. Spears. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Summer 2012)

Graduate Teaching Assistantship, Biogeography (BSC 4933), Instructor: Scott J. Steppan. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2012)

Graduate Teaching Assistantship, Animal Diversity (BSC 2011L), Instructor: Patricia A. Spears. Department of Biological Sciences, Florida State University, Tallahassee, FL. (Summer 2013)

RESEARCH EXERIENCE

Graduate Student, Department of Biological Sciences, Florida State University, Tallahassee, FL. August 2009 – Present.

Undergraduate Research Volunteer. Gregory M. Erickson, Department of Biological Sciences, Florida State University, Tallahassee, FL. (August 2008 – May 2009)

MEMBERSHIP in PROFESSIONAL SOCIETIES Society of Systematic Biologists Society for the Study of Evolution American Malacological Society

PUBLICATIONS Pfaller, J. B., Herrera, N. D., Gignac, P. M., and Erickson, G. M. (accepted September 2009). Ontogenetic scaling of cranial morphology and bite-force generation in the loggerhead musk turtle. J. Zool. (Lond.) 280, 280-289