Host-Specialist Lineages Dominate the Adaptive Radiation of Reef Coral Endosymbionts

ORIGINAL ARTICLE

doi:10.1111/evo.12270

HOST-SPECIALIST LINEAGES DOMINATE THE ADAPTIVE RADIATION OF REEF CORAL ENDOSYMBIONTS

Daniel J. Thornhill,1 Allison M. Lewis,2 Drew C. Wham,2 and Todd C. LaJeunesse2,3 1Department of Conservation Science and Policy, Defenders of Wildlife, 1130 17th Street NW, Washington, DC 20007 2Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802 3E-mail: [email protected]

Received April 8, 2013 Accepted September 4, 2013 Data Archived: Dryad doi: 10.5061/dryad.2247c

Bursts in species diversification are well documented among animals and plants, yet few studies have assessed recent adaptive radiations of eukaryotic microbes. Consequently, we examined the radiation of the most ecologically dominant group of endosymbiotic dinoflagellates found in reef-building corals, Symbiodinium Clade C, using nuclear ribosomal (ITS2), chloroplast (psbAncr), and multilocus microsatellite genotyping. Through a hierarchical analysis of high-resolution genetic data, we assessed whether ecologically distinct Symbiodinium, differentiated by seemingly equivocal rDNA sequence differences, are independent species lineages. We also considered the role of host specificity in Symbiodinium speciation and the correspondence between endosymbiont diversification and Caribbean paleo-history. According to phylogenetic, biological, and ecological species concepts, Symbiodinium Clade C comprises many distinct species. Although regional factors contributed to population-genetic structuring of these lineages, Symbiodinium diversification was mainly driven by host specialization. By combining patterns of the endosymbiont’s host specificity, water depth distribution, and phylogeography with paleo-historical signals of climate change, we inferred that present-day species diversity on Atlantic coral reefs stemmed mostly from a post-Miocene adaptive radiation. Host-generalist progenitors spread, specialized, and diversified during the ensuing epochs of prolonged global cooling and change in reef-faunal assemblages. Our evolutionary reconstruction thus suggests that Symbiodinium undergoes “boom and bust” phases in diversification and extinction during major climate shifts.

KEY WORDS: Climate change, coral symbiosis, dinoﬂagellate, ecological specialization, phylogeography, speciation, Symbio- dinium.

Adaptive radiations occur when lineages diversify in response to verswords (Baldwin et al. 1991). By comparison, relatively little is a variety of ecological opportunities (Gavrilets and Losos 2009; known about the adaptive radiations of microorganisms, including Glor 2010). These bursts of diversification occurred repeatedly at marine protists (see Falkowski et al. 2004). Their size, impover- different taxonomic, spatial, and temporal scales throughout the ished geologic record, and difficulty in acquiring uncontaminated history of life. Iconic examples of adaptive radiations include the specimens for genetic analysis have limited our understanding of Cambrian explosion (Valentine et al. 1999), the Cenozoic diversi- the tempo and mode of their diversification. fication of therian mammals (Janis 1993; Agadjanian 2003), and Indications of the nature of adaptive radiations in eukary- more recent radiations of Darwin’s finches of the Galapagos´ is- otic microbes can be derived from the major groups of mod- lands (Grant and Grant 2007), African cichlids (Seehausen 2006), ern eukaryotic phytoplankton—the dinoflagellates, diatoms, and Caribbean island Anolis lizards (Losos 2011), and Hawaiian sil- coccolithophorids—following the mass extinction of marine life

C 2013 The Author(s). Evolution C 2013 The Society for the Study of Evolution. 352 Evolution 68-2: 352–367 ADAPTIVE RADIATION OF SYMBIOTIC DINOFLAGELLATES

during the end-Permian (Falkowski et al. 2004). This diversifi- ological changes coincided with the onset of global cooling that cation was likely facilitated by the vacancy of many niches, the eventually led to periodic cycles of northern hemisphere glaciation endosymbiotic acquisition of red algal plastids by previously het- (2–3 Mya; Haq et al. 1987; Ruddiman and Raymo 1988; Beerling erotrophic lineages, and the competitive superiority of these phy- et al. 2002). This hypothesis suggested that hundreds of Symbio- toplankton in the low-nitrogen, low-trace-element oceans of the dinium lineages have evolved since the Miocene–Pliocene transi- Mesozoic (Falkowski et al. 2004). This example suggests that eco- tion. Phylogenetic patterns based on ITS2 nrDNA indicated that logical opportunities, including symbiotic interactions and major during this climatic upheaval, generalist Symbiodinium spp. ca- changes in climate and ocean chemistry, drove adaptive radiations pable of associating with many host taxa became widespread (and extinctions) in unicellular eukaryotic life. (LaJeunesse 2005). Over time these lineages putatively speciated, Symbiodinium spp. dinoflagellates, colloquially known as as numerous populations evolved habitat specificity for particu- zooxanthellae, are important microbes to coral–reef ecosys- lar host taxa (LaJeunesse 2005). This model of evolution implies tems because their photosynthesis enhances animal calcifica- that large numbers of Symbiodinium spp. are lost and gained dur- tion and supports animal metabolism through the supply of or- ing and after major climate shifts. Furthermore, speciation and ganic nutrients (Muscatine et al. 1981; Barnes and Chalker 1990; extinction events appear to be considerably more dynamic in en- Colombo-Pallotta et al. 2010). The genus Symbiodinium com- dosymbiont lineages compared to their hosts. prises many phylogenetically divergent Clades1 and numerous To further investigate these initial inferences about Sym- subcladal “types” (reviewed in Coffroth and Santos 2005). Anal- biodinium spp. diversification, we examined the phylogenetic yses of their diversity using nuclear ribosomal DNA, chloroplast relationships of Symbiodinium Clade C from the Atlantic. DNA, as well as microsatellites and their flanking sequences Although very low-density populations of “background,” cryptic, revealed complex phylogeographic patterns and differences in or ephemeral types can exist (Mieog et al. 2007; LaJeunesse host specificity among subclade lineages (e.g., LaJeunesse et al. et al. 2009), most colonies of symbiotic Cnidaria harbor a 2004, 2010; Finney et al. 2010; Silverstein et al. 2011; Thornhill dominant Symbiodinium whose in hospite population comprises et al. 2013). Many past investigations used the internal transcribed a single clonal genotype that may persist for years or more spacer regions (ITS1 and ITS2) to resolve putative Symbiodinium (Goulet and Coffroth 2003; Thornhill et al. 2006, 2009, 2013; species (e.g., LaJeunesse 2001; Sampayo et al. 2009; LaJeunesse Andras et al. 2011; Pettay et al. 2011). Therefore, these stable and et al. 2010). Equating species designations to ITS “types” has physiologically important symbionts are regarded as ecologically been met with skepticism (Correa and Baker 2009); however, evi- and evolutionarily significant to the host. To examine the diversity dence from additional genetic markers affirms that closely related of these Symbiodinium and their evolutionary origins, we com- ITS lineages are independent evolutionarily units (Sampayo et al. bined ITS2 genotyping, partial sequences of the psbA minicircle 2009; LaJeunesse et al. 2010; LaJeunesse and Thornhill 2011; noncoding region (psbAncr), and microsatellite population genetic Pochon et al. 2012). Following this logic, some species lineages markers (Moore et al. 2003; Babcock et al. 2006; LaJeunesse and were described formally with an emphasis on concordant data Thornhill 2011; Wham et al. 2013). Each of these approaches from multiple DNA loci, complemented by ecological, physio- differs in its resolution of evolutionary relationships among logical, and morphological information (LaJeunesse et al. 2012). Symbiodinium. Nuclear ribosomal DNA, for example, provides Genetic evidence suggests that present day diversity in sev- coarse phylogenetic resolution and enables comparisons across eral Symbiodinium Clades originates from adaptive radiations the entire genus (Rowan and Powers 1991; LaJeunesse 2001). during recent geological epochs (LaJeunesse 2005; Pochon et By contrast, the psbA minicircle noncoding region, which is al. 2006). One hypothesis posited that allopatry, major changes vertically inherited as one element of the unusual choloroplast in climate, and, most importantly, host specialization drove the genomes of peridinin-containing dinoflagellates, allows for tempo and mode of Symbiodinium diversification (LaJeunesse high-resolution comparisons within Clades and among closely 2005). To explain the dominance of Clade C, LaJeunesse (2005) related Symbiodinium lineages (Barbrook et al. 2006; LaJeunesse proposed that adaptive radiations occurred in this group as the and Thornhill 2011). Microsatellites similarly provide fine-scale Atlantic and Pacific Ocean basins separated (∼4–6 Mya) with the resolution of populations and strains within closely related Sym- shoaling and eventual closure of the Central American Seaway biodinium species, facilitating studies on dispersal, gene flow, (Chaisson and Ravelo 2000; Haug et al. 2001). These major ge- and allele exchange (e.g., Andras et al. 2011; Pettay et al. 2011; Thornhill et al. 2013). Our objectives were to examine whether (1)

1Note that throughout the text, the capitalized term “Clade” will be used when small albeit fixed sequence differences in rapidly evolving rDNA referring to one of the nine recognized subgroupings of Symbiodinium (i.e., (i.e., ITS2) approximate species lineages; (2) ecological special- Clades A–I), whereas the lower case term “clade” will be used in the typical ization to different kinds of host led to species evolution; and (3) context to refer to an ancestor and all of its descendants. high-resolution phylogeographic evidence supports the

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90˚W 80˚W 70˚W 500 km 25˚N

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Figure 1. Map of the Caribbean and western Atlantic showing collection locations and prevailing surface currents. hypothesis that major adaptive radiations occurred in Clade C tion, and depth of collected specimens (Table S1). Differences in response to post-Miocene climate cooling and loss in the in water depth distributions between phylogenetic groupings of connectivity between the Atlantic and Pacific. Symbiodinium were evaluated statistically using a homoscedas- tic two-sample T-test in StatPlus:mac LE2009 (AnalystSoft Inc., Vancouver, Canada). Methods SAMPLING LOCATIONS AND METHODS Symbiotic cnidarian samples analyzed in this study were collected MOLECULAR WORK from various reef habitats in regions throughout the Caribbean Skeletal fragments, clippings from oral discs, or concentrated and Atlantic, including Africa (Cape Verde Island), the Bahamas Symbiodinium pellets comprising approximately 5–10 mm2 of (Abaco and Lee Stocking Islands), Barbados, Belize (Carrie Bow preserved tissue were mechanically disrupted using a Biospec Cay), Brazil, Curac¸ao, Florida (Upper Florida Keys), the Gulf Beadbeater (Bartlesville, OK) for 1 min at maximum speed. Nu- of Mexico (Flower Garden Banks), Mexico (Puerto Morelos), cleic acids were then extracted using a modified Promega Wizard Panama (San Blas), and the U.S. Virgin Islands (St. Croix; Fig. 1). DNA extraction protocol (Madison, WI) following LaJeunesse In an effort to maximize the diversity of Clade C Symbiodinium et al. (2003, 2010). All samples were initially analyzed using de- genotypes recovered, samples were collected from a variety of naturing gradient gel electrophoresis (DGGE) fingerprinting (i.e., host taxa, reef habitats, and water depths with an emphasis on the genotyping) of the ribosomal internal transcribed spacer 2 (ITS2) scleractinian genera Agaricia, Orbicella (formerly the Montas- with 45–80% gradient gels on a C.B.S. ScientificTM DGGE-2001, traea annularis species complex), and Siderastrea. For compari- followed by excision and DNA sequencing of all discreet, promi- son, samples were also collected from two clonal cultures (rt-113 nent bands in the DGGE profile as described by LaJeunesse and rt-152) and select Indo-Pacific locations including Australia (2002). Profiles and sequences were assigned an alphanumeric (the Great Barrier Reef), Baja California, central Mexico (Ban- ITS2 type designation (sensu LaJeunesse 2001) representative of deras Bay), Hawaii, and Japan (Zamami Island). Collections were that fingerprint and symbiont type. This technique targets the nu- conducted using scuba equipment, with a several cm2 tissue biop- merically dominant sequence variants in the symbiont’s genome sies taken from each coral colony using hammer and chisel, punch (Thornhill et al. 2007) and is able to detect symbionts com- core, pliers, or scissors. A supplementary table is provided that prising approximately 10% or more of a symbiotic population details the host genus and species, sampling dates, source loca- (Thornhill et al. 2006). Because this study focused on the

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Sample collection & and additional adjustments to the alignment made during visual in- DNA extraction spection of the output file. Sequences were deposited in GenBank (see Table S1 for Accession numbers) and the resulting alignments are available at http://datadryad.org (doi: 10.5061/dryad.2247c). ITS2 fingerprinting with PCR-DGGE & Ten polymorphic microsatellite loci developed for Clade band sequencing C Symbiodinium were used (loci Sgr 34, SgrSpl 13, SgrSpl 22, SgrSpl 24, SgrSpl 25, SgrSpl 26, SgrSpl 78, Spl 1, and Spl 16 [Wham et al. 2013] and locus C1.05 [Bay et al. 2009]) to produce Diagnosis of a multilocus genotypes for 67 samples of type C3 from Siderastrea Clade C type? siderea, as well as 83 samples of C7 and 43 samples of C7a/ Removal C12 from Orbicella spp. (Fig. 2 and Table S2). Each locus was No from Yes μ μ dataset amplified in 10 L reaction volumes containing 1 Lof10mM dNTPs, 0.2 U Taq DNA Polymerase (New England Biolabs), 1 μL psbAncr sequencing and phylogenetic analyses standard Taq Buffer (New England Biolabs, Ipswich, MA), and 1 μL25mMMgCl2,1μL of each forward and reverse primer Member of the C7, C7a/C12, at 10 μM, and 1 μL of approximately 50 ng DNA template. or C3Siderastrealineages? PCR amplification was preformed according to the specifications Not included in given by Wham et al. (2013). Following amplification, fragment No Yes microsatellite sizes were analyzed on an ABI 3730 Genetic Analyzer (Applied genotyping Biosystems) using a 500-bp standard (LIZ-labeled) at the Penn Multilocus microsatellite State University Nucleic Acid Facility. Fragments sizes were an- genotyping alyzed visually using GeneMarker version 1.91 (SoftGenetics, State College, PA). Although Symbiodinium, like most dinoflag-

Retrieval of a complete ellates, are haploid (Santos and Coffroth 2003), most Clade C multilocus genotype? members posses a duplicated genome in which many loci produce two alleles (Wham et al. 2013). Samples with background Not included < No genotypes ( 10%), defined by electropherograms that contained Yes in population genetic analysis additional allele peaks one-third or less than the intensity of the main peaks, were scored based on the dominant allele peaks. In Population genetic analyses cases where multiple genotypes were observed to codominate, the sample was removed from the dataset before further analysis Figure 2. Data collection pipeline for this study with decision (Fig. 2). This reduced the sample size of the data set, but enabled points highlighted in gray. us to confidently identify multilocus genotypes and conduct population genetic analyses with the remaining data. The multilocus evolution of Symbiodinium Clade C, results from other Clades allele scores and geographic data for these samples are available are not reported here (Fig. 2). at http://datadryad.org (doi: 10.5061/dryad.2247c). Each Clade C Symbiodinium sample was further analyzed by amplifying and sequencing the noncoding region of the psbA mini- DATA ANALYSIS circle (psbAncr) following the amplification settings and primers Sequences of the psbAncr fell into two distinct clusters— (7.4-Forw and 7.8-Rev) specified by Moore et al. (2003) and haplotypes from Symbiodinium with ITS2 sequences similar or LaJeunesse and Thornhill (2011). All successful reactions were identical to type C3 and haplotypes with ITS2 sequences similar sequenced using Big Dye 3.1 reagents (Applied Biosystems, Fos- or identical to type C1 (see Results). Hereafter these psbAncr lin- ter City, CA) at the DNA Core Facility (Penn State University) eages will be referred to as the C3 and C1 groups, respectively. using the forward primer (7.4-Forw). Only samples with a single Because of the high level of psbAncr variability among samples, unambiguous psbAncr sequence (in ∼90% of samples) were used, it was impossible to unambiguously align sequences from the C3 indicating the existence a single predominant Symbiodinium geno- group with those from the C1 group. Therefore, these two lineages type (LaJeunesse and Thornhill 2011). Chromatograms were vi- or groups of Symbiodinium Clade C were analyzed separately as sually inspected for accuracy in base calling (Sequence Navigator) follows. and the edited sequences aligned initially using the online appli- Phylogenetic reconstructions from the complete ITS2 and cation of ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) partial sequences of the psbAncr were conducted using Bayesian

EVOLUTION FEBRUARY 2014 355 DANIEL J. THORNHILL ET AL.

Inference using MrBayes version 3.2.1 (Huelsenbeck and (± 0.003) was calculated from the divergence between the psbAncr Ronquist 2001) implementing the Hasegawa–Kishino–Yano haplotypes associated with Eastern Pacific and Atlantic Porites (HKY; ITS2 data), general time reversible (GTR) + I + (C3 (assuming divergence occurred 3.1–4.6 Mya; LaJeunesse 2005). group psbAncr data), or symmetrical (SYM) + I + (C1 group Several calibration points for estimating timing of a most recent psbAncr data) models of substitution, as indicated by the Akaike In- common ancestor (tmrca) were determined from nodes in the formation Criterion by MrModeltest version 2.3 (Nylander 2004). phylogeny where Pacific and Atlantic lineages converged. Monte Each MCMC analysis was run for 1.0 × 106 generations and sam- Carlo Markov Chain length was set at 100 million. pled every 100 generations. Because of convergence of chains within 2.5 × 105 generations, the first 2500 trees were discarded as burn-in, and a 50% majority-rule consensus tree was calcu- Results lated from 7501 remaining trees. Symbiodinium types C3 and RESOLUTION AMONG SYMBIODINIUM CLADE C WITH C1 from Indo-Pacific hosts were used as outgroups. Posterior ITS rDNA probabilities (PP) were recorded to assess reliability of recovered ITS2 and partial 5.8S sequence data were compiled from 309 nodes. field-collected samples of Symbiodinium Clade C, comprising 38 Topologies based on Maximum Parsimony were also con- cnidarian genera from 12 western Atlantic and five Indo-Pacific structed under the default settings of PAUP∗ 4.0b10 (Swofford locations (Table S1). The aligned ITS2 sequence data set included 2000). For calculating phylogenies based on Maximum Parsi- 300 nucleotide positions, all of which (100%) could be unam- mony, and due to the high frequency of informative insertions and biguously aligned. Phylogenetic reconstruction from these ITS2– deletions (indels), sequence gaps were included as a fifth character 5.8S alignments resulted in a largely unresolved polytomy with state with the entire insertion or deletion weighted as one charac- member lineages unique to either the Pacific or the Atlantic. The ter. Outgroups were identical to those used in Bayesian Inference. limited divergence between these sequences produced few statis- A total of 1000 bootstrap replicates were performed to assess tically supported lineages. statistical significance of internal branching. For all Bayesian In- ference and Maximum Parsimony phylogenetic analyses, Symbio- dinium ITS2-DGGE genotype, host identity, and collection loca- HIGH-RESOLUTION PHYLOGENETIC ANALYSIS tion were mapped onto the resulting topologies as character states. OF THE C1-GROUP Uncorrected genetic distances (P) between haplotypes, in- In contrast to the ITS2 region, psbAncr sequences were highly vari- cluding all substitutions and with gaps considered via pairwise able across samples; in fact, samples belonging to the C1 group deletion, were calculated with MEGA version 5.2.2 (Tamura did not align to the psbAncr sequences of the C3 group. Therefore, et al. 2011). Sequences that differed by P < 0.030 were grouped these data were analyzed separately. The C1 group psbAncr align- and the mean genetic distances between groups were calculated ment of 69 sequences consisted of 863 nucleotide positions and in MEGA version 5.2.2. 63 unique haplotypes. Conserved regions in the alignment were Because recurring multilocus microsatellite genotypes (i.e., punctuated by areas with frequent nucleotide substitutions as well clones) bias allelic frequencies, clones were removed from the as numerous insertion–deletion polymorphisms (see also Moore data set (i.e., each genotype was represented only once; Fig. 2) et al. 2003). Phylogenetic analyses of these data characterized sev- with GenAlEx version 6.1 (Peakall and Smouse 2006). The re- eral well-supported, monophyletic psbAncr lineages (Fig. 3A) that sulting unique multilocus genotypes were analyzed using the are poorly resolved with ITS2 sequences (Fig. 3B). Some of the Bayesian clustering program STRUCTURE (Pritchard et al. monophyletic lineages corresponded to a particular ITS2 type des- 2000). The analysis in STRUCTURE was performed without a ignation, associated with a particular host taxon, or both (Fig. 3A). prior location, under an admixture and uncorrelated alleles model For instance, the host-specific C1 group lineages from the west- with a burn-in of 100,000 iterations and 1,000,000 iterations of ern Atlantic (Palythoa spp. [ITS2 type C1] and Porites spp. [types analysis at K values ranging from 1 to 10. The optimum K value C1aj and C80]) were distinct from host-specific Pacific lineages was determined by the Delta-K method (Evanno et al. 2005). (e.g., Porites panamensis [types C1 and C1v]). However, other The program BEAST version 1.7.4 (Drummond and lineages within the C1 group exhibited little host or geographic Rambaut 2007) was used to approximate divergence times among specificity and ITS2 type C1 occurred throughout the phylogeny. representatives of the C3 group estimated under a relaxed molec- For example, one of the largest sequence clusters, designated as ular clock using the HKY substitution model (base frequencies Symbiodinium goreaui (sensu stricto), contained psbAncr haplo- estimated). The tree prior was set at Yule Process (Gernhard 2008) types from both the Atlantic and Pacific (Fig. 3A). Symbiodinium and random starting tree for the tree model. The mean substi- goreaui was characterized by short internal branch lengths and lit- tution rate (ucld.mean) of 0.006 substitutions × site−1 × My−1 tle differentiation between psbAncr haplotypes associated with the

356 EVOLUTION FEBRUARY 2014 ADAPTIVE RADIATION OF SYMBIOTIC DINOFLAGELLATES

* Acropora

A culture rt-152 0.1 * culture rt-113 cultured isolate Indo-Pacific origin * Protopalythoa 0.58

* Siderastrea C1 (Symbiodinium goreaui)

Bartholomea Lebrunia Discosoma P. astreoides * Palythoa Tridacna * Corallimorpharia 0.88 C1 * 0.76 0.77 * Palythoa 0.85 * * C1aj Porites astreoides C80

B C1a-j C80 j a

Coscinarea 0.89 0.98 Siderastrea C1 Fungia Acropora f C1v * Galaxea Bartholomea C1b-f * Siderastrea 0.61 * Zoanthus C1 b 0.78 Siderastrea 1 base diff. Isaurus 0.56 Corallimorpharia Astreopora Discosoma 0.75 0.58 Galaxea 0.88 * C1 Porites panamensis 0.72 * (Eastern Pacific) * C1v * * 0.87 Psammocora Cyphastrea C1b-f Psammocora * 0.89 Fungia

Figure 3. High-resolution phylogeny of the C1 group of Symbiodinium based on a Bayesian Inference of chloroplast psbAncr (A). Nodal support indicated as posterior probabilities (PP) above 0.50 next to the relevant node or asterisks for a PP = 1.00. Colored symbols indicate the collection location of each sample corresponding to Figure 1. The corresponding Symbiodinium ITS2 types (based on DGGE ﬁngerprinting), symbiotic host afﬁliations, and cultured isolate designations are indicated to the right of each sample on the topology. By comparison, Maximum Parsimony analysis of nuclear ribosomal ITS2 (B) provides less genetic resolution among members of the C1 group. cnidarians Acropora, Bartholomea, Corallimorpharia, Dicosoma, HIGH-RESOLUTION PHYLOGENETIC ANALYSIS Lebrunia, Palythoa, Porites astreoides, Protopalythoa, Sideras- OF THE C3 GROUP trea, and the giant clam Tridacna. Although no psbAncr haplo- The psbAncr nucleotide alignment of the C3 group consisted of types were shared between locations, similar psbAncr haplotypes 242 sequences comprising 181 unique psbAncr haplotypes and of S. goreaui were identified throughout the western Atlantic in- 826 aligned nucleotide positions (indels included). The Bayesian cluding Belize, Brazil, the Florida Keys, the Gulf of Mexico, and Inference phylogenetic reconstruction of the C3 group produced Mexico, as well as from the Indo-Pacific. Thus, the C1 group con- numerous divergent lineages that grouped mostly by host origin tained both host-specific lineages and geographically widespread (Fig. 4A). By comparison, these lineages were poorly resolved host-generalists. with ITS2 sequences (Fig. 4B). Included in these analyses were

EVOLUTION FEBRUARY 2014 357 DANIEL J. THORNHILL ET AL.

C3 C31/ C31c C27 C3q * c C21a C3k Acropora * * * C17 C87

* C40 Indo-Pacific C21 a C3 0.96 * C3s 0.89 C7a (C12) C7 C3a C40 C7, C7a(C12), C7c C3b C16 * Orbicella 1 base diff. annularis C16a C3k B 0.81 complex 0.73 0.81 * * C27 Indo-Pacific * C3 Scolymia * C3 Mycetophyllia 0.90 0.88 C3 Orbicella franksi

0.52

0.68 C3, C3a, C3b, C3s Agaricia & Leptoseris

0.96 * C3 Mussa 0.98 0.67 C3 Scolymia Atlantic C3 Leptoseris * C3 Ricordia 0.67 0.98 C3 Siderastrea siderea * C3 Stephanocoenia 0.81 * C3 Erythropodium * C3 Leptoseris 0.72 0.84 * 0.60 C3h Indo-Pacific * 0.98 C3 Agaricia C3c Mycetophyllia reesi C3 Isophyllastrea

* C3 Siderastrea siderea

* 0.63 C3, C3b, C3q Agaricia & Leptoseris * C3 Agaricia * * C3

0.98 Atlantic * 0.83 C16a * C16 Stephanocoenia C87 * C3 Siderastrea C3 Diploria 0.57 * 0.63 C3 Mycetophyllia * 0.59 0.76 C31, C31c * 0.64 Montipora Indo-Pacific 0.1

Figure 4. High-resolution phylogeny of the C3 group in Symbiodinium based on Bayesian inference of chloroplast psbAncr (A). Nodal support indicated as posterior probabilities (PP) above 0.50 next to the relevant node or asterisks for a PP = 1.00. Dark grey arrowheads () denote ancestral branches in the radiation with low support values. Symbiodinium ITS2 types (based on DGGE ﬁngerprinting), symbiotic host afﬁliation, and major ocean basin are indicated to the right of each clade on the topology. Star symbols () indicate branch connections to subclades that are drawn in greater detail in Figures 5, S1, and S2. By comparison, Maximum Parsimony analysis of nuclear ribosomal ITS2 (B) provides less genetic resolution among members of the C3 group.

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several Indo-Pacific representatives (e.g., C3h, C27, and C31) A Symbiodinium Clade C associated with Orbicella sp. that shared a more recent common ancestry with western Atlantic representatives than with each other, indicating that the initial * diversification of these lineages predated the closure of the Central * American Seaway. The putatively ancestral C3 ITS2 genotype was * C7c found to represent distinct lineages throughout the phylogeny, whereas more derived ITS2 genotypes occurred as monophyletic (i.e., C3a, C3h, C3k, C3q, C7, C7c, C12, C16a, C27, C31/C31c, * C40), paraphyletic (i.e., C3c, C3s, C7a, C16, C87), or polyphyletic B C12 (i.e., type C3b) groups. The evolutionary relatedness inferred via Maximum Parsimony for many of these lineages was similar to * C7a that estimated by Bayesian Inference (data not shown). * Statistical support was generally low for most branching ncr * points, or ancestral nodes, that occurred deeper in the psbA C phylogeny of the C3 group (indicated with dark gray arrowheads in Fig. 3; PP = 0.52–0.72). PsbAncr lineages from the Atlantic all * C7 exhibited host-specific associations whereas many of the Indo- * * Pacific lineages used in our analysis associated with various host ncr taxa. Furthermore, two or more distinct psbA lineages associ- * ated with the Atlantic coral genera Agaricia, Leptoseris, Myce- D tophyllia, Orbicella, Scolymia, Siderastrea,andStephanocoenia (Fig. 4A). Finally, members of the C3 group occurred predomi- nantly in deeper environments (mean depth = 12.0 ± 0.5 m [SE]; Table S1; two-sample T-test, P < 0.0001) than the generally shal- * C3 5 changes lower habitats of the C1 group (mean depth = 6.5 ± 1.0 m [SE];

Table S1). Figure 5. (A) Maximum Parsimony topology based on the Sym- biodinium chloroplast psbAncr haplotypes associated with members of the bolder star coral, Orbicella annularis, species complex. GENETIC COHESION AMONG SYMBIODINIUM The basal node of this tree connects to the topology presented ASSOCIATED WITH ORBICELLA in Figure 4 (note symbol at the node labeled C7/C7a/C12 in ITS2 and psbAncr genotyping revealed a large group of Symbio- Fig. 4). Nodal support with bootstrap values greater that 95% are indicated by asterisks. Symbiodinium nuclear ribosomal ITS2- dinium (types C7, C7a, C7c, and C12) that associated specifi- DGGE genotypes are indicated to the right of each clade on cally with O. annularis, Orbicella faveolata,andOrbicella franksi the topology. Colored symbols indicate the collection location of throughout the Greater Caribbean. Phylogenetic analysis of the each sample corresponding to Figure 1. Panels B–D provide ex- ncr psbA haplotypes showed that these Symbiodinium comprised ample photographs of the O. annularis species complex, includ- a well-supported monophyletic group that splits into two distinct ing Orbicella faveolata (B), Orbicella annularis (C), and Orbicella subclades (Figs. 4, 5). These lineages are further divided into less franksi (D). resolved groupings that correlated with location and ITS2 type. For example, psbAncr haplotypes of type C7 Symbiodinium oc- were observed for Siderastrea siderea and the agaricid genera curred in Orbicella spp. from the western Caribbean, including Agaricia and Leptoseris (Figs. S1, S2 and Text S1, S2). Mexico and Belize. In contrast, psbAncr haplotypes of type C7a To investigate whether recent sexual recombination occurred occurred in Orbicella spp. from the eastern Caribbean province, within and between the two major groupings of Orbicella sym- including St. Croix and Barbados; closely related C12 psbAncr bionts, 10 microsatellite markers were examined in Symbiodinium haplotypes were obtained from the Bahamas, whereas type C7c C7, C7a/C12, and a third monophyletic lineage, the C3 type from was found only in Panama. Curac¸ao represented a well-sampled Siderastrea (i.e., C3Siderastrea). A total of 193 samples were geno- location where overlap between psbAncr haplotypes of C7 and C7a typed. Of these, 26 were removed because they contained clear occurred (Fig. 5). In all of these cases, psbAncr haplotypes were mixtures of more than one genotype and another 89 were removed distinct at a particular location and none were shared among local- because they constituted clone replicates. This left a total of 78 ities, further indicating that these Symbiodinium populations are distinct genotypes (C7, n = 31; C7a/C12, n = 14; C3Siderastrea, spatially structured. Similar fine-scale phylogeographic patterns n = 33) for analysis of genetic similarity. For summary statistics

EVOLUTION FEBRUARY 2014 359 DANIEL J. THORNHILL ET AL.

C7 C7a C3Siderastrea 36 100 Caribbean C1-group 34 Caribbean C3-group 50 32 K=3

0 100 8

50 6 K=5 4 0 Number of psbA Lineages 2

Belize Belize Mexico MexicoFlorida Curaçao St. Croix Curaçao St. Croix Bahamas Barbados Bahamas Barbados 1 2 3 4 5 6 7 8 9 10 11 Number of Host Associations Figure 6. STRUCTURE analysis partitioned unique multilocus microsatellite genotypes from Symbiodinium types C7 and C7a/C12 Figure 7. The specificity of Symbiodinium Clade C psbAncr lin- from Orbicella and C3 types from Siderastrea siderea into three eages for Caribbean host genera. Independent lineages consisted separate populations. Each colored vertical bar represents the mul- of haplotypes with a mean uncorrected genetic distance of less tilocus genotype of an individual and its statistical assignment to than 3%. This value was estimated based on findings of repro- a genetically cohesive group. Replicate clonal genotypes were re- ductive cohesion among haplotypes comprising Symbiodinium C7, moved from the analysis (n = 89 of 167 individuals) to avoid biases while being isolated reproductively from closely related C7a/C12 in the clustering due to clonality. Therefore, 78 distinct multilocus (see text for further details). Parsing the psbAncr data in this way genotypes were included in the STRUCTURE analysis. Population resulted in 40 sequence clusters that are presumed to represent clusters matched exactly with the symbionts’ nuclear ribosomal genetic variation among individuals of separately evolving lin- and chloroplast geneotypes, as well as the genus of host. The eages or species. Thirty-five Caribbean lineages of the C3 group likelihood of these partitions was maximized at K = 3andcor- were found to associate with only one and occasionally two host responded to reproductively isolated populations, or species. The genera. The C1 group, in addition to several host-specific lin- value at K = 5 further divides genetic diversity principally accord- eages, contained two lineages commonly associated with >9 host ingtosamplinglocation. genera. on these data, see Table S2. STRUCTURE analysis partitioned approximation of species richness in our sample set. Clustering the these genotypes into three separate populations: C7 and C7a/C12 sequences by genetic distance resulted in 40 distinct lineages with from Orbicella,aswellasC3Siderastrea (Fig. 6). Using the Evannov considerable host specificity (Fig. 7). The majority of lineages (35 method, the optimal number of clusters was 3 (K = 3; Fig. S3). of 40, 87.5%) associated with a single host genus (Fig. 7). The In Curaçao where the C7 and C7a lineages overlapped, genotypes remainder associated with two (three lineages), 10 (1 lineage), or of C7 clustered with C7 symbionts from the western Caribbean 11 (1 lineage) different host genera. Host-specialist lineages were rather than genotypes of C7a occurring several meters away. Like- concentrated in the C3 group (Fig. 7). wise, genotypes of C7a in Curaçao clustered more closely with Analysis of origination and divergence times among putative other C7a individuals from locations in the eastern Caribbean. species lineages of the C3 group using BEAST suggested that di- Recombinant genotypes occurred within populations of C7 and versification in this group began approximately 12–13 Mya (with C7a, but there was no evidence of genetic exchange between a calculated range of 7.9–20 Mya; Fig. 8A) as the planet began to these two Symbiodinium spp. Analyses run at higher K values cool following the mid-Miocene climactic optimum (Fig. 8B). maintained this partitioning and also suggested the existence of The radiation of the C3 group intensified approximately 4–6 genetic structure across populations of C7 and C3Siderastrea. Mya, but declined precipitously as cooling accelerated in the late Pliocene early Pleistocene (Fig. 8C). Error bars at branch nodes INFERENCE OF THE ORIGIN AND TIMING decreased considerably in the chronograph as divergence times OF AN ADAPTIVE RADIATION approached present day (Fig 8A). Scaling the patterns of allele exchange and genetic isolation from ncr types C7, C7a/C12, and C3Siderastrea across the psbA topology suggests that Clade C is speciose. To estimate the total number Discussion of Caribbean lineages, we clustered sequences according to their The genetic diversity of Symbiodinium is exceedingly large (e.g., genetic distance, using P ≥ 0.030 as the cut off between groups Coffroth and Santos 2005; Finney et al. 2010; LaJeunesse and (based on the mean distance between psbAncr haplotypes of C7 and Thornhill 2011), yet few hypotheses have been advanced to ex- C7a). Although the P ≥ 0.030 threshold is arbitrary, it provides an plain the ecological and evolutionary significance of this diversity

360 EVOLUTION FEBRUARY 2014 ADAPTIVE RADIATION OF SYMBIOTIC DINOFLAGELLATES

A 7.82 5.47 3.3

2.68 3.29

3.27 6.41 5.5 8.34 4.36

9.46 5.85

4.04 4.9 10.17 12.85 6.48 3.45

7.73 C7a 5.12 8.87 C7

11.13 7.48 5.96 5.28 4.21

6.92 4.64

9.0 2.84

11.79 8.07 4.91

2.24

7.25

C3Sid. 5.88 2.87 4.7

7.6 B 1.0 Mid-Miocene Climactic Optimum

δ18O

2.0

3.0

5 4.0 lineage origination 1

10MY 5MY 5.0 15 10 5 Miocene Pliocene Pleisto. Million Years Ago

Figure 8. Climate cooling and estimated tempo of lineage diversification among Symbiodinium. (A) Divergence times estimated among ecologically distinct psbAncr lineages of Caribbean and Pacific Symbiodinium in the C3 group and calibrated with the timing of major physical–chemical changes first measured between Atlantic and Pacific Oceans (4.6 Mya) as well as their physical separation (3.1 Mya). The numbers listed on the chronogram next to branch nodes (black dots) are estimated diversification times in millions of years before present. Gray bars are drawn to indicate the 95% confidence interval surrounding each node. Black arrowheads indicate calibration

EVOLUTION FEBRUARY 2014 361 DANIEL J. THORNHILL ET AL.

(but see LaJeunesse 2005; Pochon et al. 2006; Hill and Hill 2012). likely responsible for the discrepancy between markers. Evolu- The recent availability of additional high-resolution phylogenetic tion of rDNA begins as a mutation in one copy, which spreads and population genetic (LaJeunesse and Thornhill 2011; Wham across copies via gene conversion or unequal crossing over, and et al. 2013) markers enables detailed examination of evolution- ultimately becomes numerically dominant within the genomes ary relationships among Symbiodinium. Here, we focused on the of individuals undergoing genetic exchange in a population— psbAncr, which exhibits levels of sequence evolution 10–20 times a process known as concerted evolution (i.e., Smith 1976; greater than that of ITS2 and unambiguously differentiates ge- Arnheim 1983; Dover 1986; Saito et al. 2002; Galluzzi et al. netically cohesive lineages comprising distinct haplotypes (LaJe- 2004). Concerted evolution exerts a stabilizing influence that can unesse and Thornhill 2011). When psbAncr data were compared prevent the spread of rare sequence variants, thereby maintaining with ITS2 and multilocus microsatellite genotyping, concordant the numerically dominant rDNA sequence and masking lineage patterns occurred across markers, indicative of species bound- divergence (Arnheim 1983; Dover 1986; Thornhill et al. 2007). aries. Below we examine these results in the context of different The shared C3 sequence that dominated the genomes of many species concepts and consider abiotic and biotic factors poten- Symbiodinim Clade C lineages is likely a shared ancestral state, a tially underlying the diversification of Symbiodinium Clade C. symplesiomorphy, which has not been replaced by a more derived sequence. DELIMITING SPECIES LINEAGES WITH Consequently, high-resolution genetic data indicate that HIGH-RESOLUTION GENETIC DATA ITS2-nrDNA only approximates species richness within Clade Although different species concepts emphasize different lines of C. In cases such as Symbiodinium type C3 or type B1 from the evidence, most share the idea that species are separately evolving Caribbean, fine-scale genetic markers demonstrate that apparent units (de Queiroz 2007; Hausdorf 2011). The phylogenetic species host-generalists actually consist of several specialized lineages concept, for example, identifies species using statistically signifi- (Santos et al. 2004; Finney et al. 2010). As a result, populations cant character differences resulting from evolutionary divergence of other provisional host-generalists, including ITS2 types A1, of populations over time (Donoghue 1985; de Queiroz 2007). Un- A3, C15, and D1a (D1–4), require reexamination with higher- der this concept, members of a species share more diagnostic ge- resolution genetic markers (see Pinzon´ et al. 2011; Wham et al. netic or morphological characters with each other—and are hence 2011). Future investigations may reveal that putative generalists monophyletic—than with individuals from other species. Phylo- instead comprise several independent and ecologically specialized genetic reconstructions based on the psbAncr revealed fine-scale species lineages, but others like S. goreaui may indeed associate genetic diversity in Symbiodinium Clade C, including numerous with many different host genera over a large geographic area, and monophyletic clades that were differentiated from each other by are thus true host-generalist symbionts (Fig. 3A). ncr many nucleotide substitutions and indels. These phylogenetic pat- The phylogenetic patterns of the psbA beg the questions: terns are consistent with species-level distinctions, in that sister do the discreet sequence clusters represent evolutionarily distinct lineages, as independently evolving metapopulations, appear to species? Or are they simply the accumulation of somatic muta- have lost shared ancestral haplotypes (Wakeley 2008). tions through clonal evolution? Given the clonality of many in The frequent phylogenetic correspondence, or reciprocal hospite Symbiodinium populations (e.g., Thornhill et al. 2013), monophyly, of the independently sorting ITS (nuclear) and psbAncr clonal evolution and periodic selective sweeps (Palys et al. 1997) (chloroplast) provides strong support for the hypothesis that Clade might explain the concordant phylogenetic patterns of the chloro- C comprises hundreds of species worldwide. Despite this, sev- plast and nuclear loci. We therefore tested for evidence of ge- eral genetically and ecologically distinct psbAncr lineages shared netic recombination using multilocus population genetic data. ncr dominant ITS2 sequences (e.g., type C3). In such instances, the Three sympatric ITS2/psbA lineages were examined, includ- unusual molecular evolution of the multicopy ribosomal array is ing C7 and C7a/C12 from the star corals, Orbicella spp., and

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 8. points for estimating timing of a most recent common ancestor (tmrca) between Paciﬁc and Atlantic lineages. The Atlantic Symbiodinium spp., examined by microsatellite analyses (Fig. 6) and associated with the reef corals Orbicella (C7 and C7a/C12) and

Siderastrea (C3Sid), are noted so as to delimit the ranges of interspecific versus interindividual sequence variation. Putative species lineages are shaded according to their geographic origin (black = Indo-Pacific, brown = Atlantic). Stippled lines represent branches with poor statistical support (Bayesian Inference). Red highlighting to the right of terminal branches indicates host-generalist lineages. (B) Climate curve over the past 17 My based on oxygen-isotope measurements from deep-sea foraminifera (from Zachos et al. 2008). (C) Basic estimation of the rate and tempo of lineage diversification in the chronogram (panel A). The gray shading in panels B and C encompasses the time horizon of major climate shifts corresponding to the Miocene–Pliocene boundary.

362 EVOLUTION FEBRUARY 2014 ADAPTIVE RADIATION OF SYMBIOTIC DINOFLAGELLATES

C3Siderastrea from the massive starlet coral, Siderastrea siderea, Helberg 2003; Baums et al. 2005), that relate to theoretically with 10 unlinked microsatellite loci. STRUCTURE analyses iden- expected patterns of allopatry based on major surface currents tified boundaries to sexual recombination between C7, C7a/C12, (Cowen et al. 2006) and long-standing environmental gradients and C3Siderastrea that corresponded to the symbionts’ phylogenetic (Chollett Ordaz 2011). Moreover, these phylogeographic patterns grouping and host affiliation (Fig. 6). Thus, host affiliation and are consistent with previous findings of genetic structure in Sym- phylogenetic groupings superseded location as the primary fac- biodinium populations at scales encompassing several hundred tors in determining clustering. Furthermore, individuals belong- kilometers, or less (e.g., Santos et al. 2003; Howells et al. 2009; ing to each ITS2/psbAncr lineage comprised recombinant (i.e., Thornhill et al. 2009; Andras et al. 2011). Limited connectivity scrambled) allele combinations, indicating sexual recombination. among Symbiodinium populations probably results from a combi- Members within each phylogenetic grouping, or lineage, appear nation of geographic barriers such as prevailing currents, limited capable of engaging in sexual recombination, but are reproduc- dispersal capacity of free-living cells, high local recruitment or tively isolated from the constituencies of other lineages. These retention, or density-dependent barriers preventing long-distance three lineages therefore operate as distinct species according to migrants from successfully penetrating habitats that are already the biological species concept. dominated by other Symbiodinium genotypes (reviewed in Thorn- A common driver of monophyly, reproductive isolation, and hill et al. 2009). Finally, different local and regional conditions speciation is the selection of individuals with gene combinations may also select for locally adapted genotypes (Chollett Ordaz adapted to a resource or habitat (Ackerly et al. 2006; Losos 2009; 2011; Howells et al. 2012). Glor 2010). Here, most psbAncr lineages contained individual hap- Taken together, these ecological, geographic, reproductive, lotypes recovered from only a single coral species or genus and population genetic, and phylogenetic patterns suggest that Sym- are thus functionally distinct species under the ecological species biodinium Clade C could consist of hundreds of species globally. concept. These clear ecological differences are consistent with the These findings also point to major biotic and abiotic factors—host adaption of many psbAncr clades to a particular host habitat, sug- specialization and geographic isolation—shaping Symbiodinium gesting the importance of distinct intracellular environments— evolution. The following section considers these observations in characteristic of different hosts—in the ecology and evolution of the context of the paleo-history of the Caribbean Basin in order these dinoflagellates. Indeed, host identity appears to be the pri- to make general inferences about their evolutionary history with mary ecological factor, or α-niche axis, of lineage diversification reef corals. in Symbiodinium (Figs. 3, 4, 6; LaJeunesse 2005; Ackerly et al. 2006; Finney et al. 2010; Glor 2010; LaJeunesse et al. 2010). A SYNTHESIS OF THE INTERPLAY BETWEEN In addition to the Symbiodinium associated with hosts that verti- PALEO-HISTORY, GEOGRAPHIC ISOLATION, AND cally transmit their symbionts, many host-specific lineages were HOST SPECIALIZATION found associated with Caribbean Cnidaria that must horizontally Phylogenetic comparisons between Cnidaria and Symbiodinium acquire symbionts from the environment at the start of each gen- indicate a lack of co-speciation over tens of millions of years eration. This indicates that vertical transmission is unnecessary (Rowan and Powers 1991; LaJeunesse 2005; Pochon et al. 2006; for the evolution of host-specialized Symbiodinium spp. Further- this study). A more dynamic process of partner recombination more, we found examples of hosts that harbored several phylo- and coevolution must be at work to explain the existence of nu- genetically independent and host-specific endosymbionts across merous closely related endosymbionts associated with distantly their range (e.g., Fig. S2), suggesting that endosymbionts evolved related hosts (LaJeunesse 2005). Evidence from our phylogeo- along similar ecological axes in different locations (i.e., paral- graphic and molecular clock analyses (Figs. 4, 8) indicate that lel evolution) and that speciation through ecological selection is an acceleration of lineage diversification occurred with the sep- a major recurrent theme throughout the evolutionary history of aration of the Atlantic from the Pacific during the late Miocene these dinoflagellates. and early Pliocene. Continental drift and tectonic uplift initiated Beyond host associations, many monophyletic and host- major changes in ocean circulation patterns that coincided with specific psbAncr clades within the C3-group contained geograph- the overall reduction in the planet’s temperature and atmospheric ically segregated haplotypes (e.g., in symbionts of Orbicella, CO2 concentration (Haug and Tiedemann 1998; Zachos et al. Siderastrea, Agaricia), suggesting that spatial isolation acts as 2001, 2008; Pearson and Palmer 2000). Terrestrial changes dur- a secondary or β-niche axis of divergence in Symbiodinium evo- ing this time included a drying of the climate, significant turnover lution (Ackerly et al. 2006; Glor 2010). This spatial partitioning in terrestrial mammals, and expansion of C4 grasses (Haq et al. mirrored the presence of distinct coral reef species assemblages 1987; Ruddiman and Raymo 1988; Janis 1993; Cerling et al. (Briggs 1974), as well as the genetic partitioning of several shal- 1997; Haug et al. 2001). In Caribbean marine environments, coral low water species in the Greater Caribbean (Fig. 1; Taylor and species diversity initially increased with the complete closure of

EVOLUTION FEBRUARY 2014 363 DANIEL J. THORNHILL ET AL.

the Central American Seaway (Budd 2000). However, climate Why was the C3 group of Clade C Symbiodinium more evo- shifts continued during the Plio–Pleistocene transition, notably lutionarily successful in terms of the numbers of hosts and habitats including the onset of northern hemisphere glaciation and asso- occupied in the Atlantic (and Pacific)? Although answers to this ciated oscillations in temperature, pCO2, and eustasy, resulting question are speculative given the available data, some insight in continual ecological change (Zachos et al. 2001, 2008). These may be gained by comparing the ecologies of extant species with effects were more pronounced in the Greater Caribbean, which ex- the historic climatic conditions during the radiation of this group perienced significant losses in coral diversity relative to the Pacific over the past 5 My. It is noteworthy that members of the C3 group, (Budd 2000). Major changes in climate that induced reef faunal usually occurred in colonies from deeper environments (>10 m), turnover presumably affected Symbiodinium species assemblages where average temperatures and irradiance levels are lower and as well. less variable. By comparison, members of the C1 group occurred A once small number of species within Symbiodinium Clade in shallower habitats (∼5 m), where they would be subjected C, designated by ancestral ITS2 genotypes C3 and C1 that were to greater extremes in temperature and light conditions. Further- common to both ocean basins, served as the progenitors to the more, type C1 examined in Australia is substantially more heat radiation of the C1 and C3 groups. Presumably, these ancestors tolerant than type C3 (referred to as type C2 in Berkelmans and or their descendants displaced Symbiodinium spp. that existed van Oppen 2006; Abrego et al. 2008; Jones et al. 2008; but see before the changes in climate related to the Miocene–Pliocene Hume et al. 2013). This difference in diversity between the C1 transition. According to the psbA data, a major burst in clado- and C3 groups in the Greater Caribbean is significant because genesis began in the Atlantic during the late-Miocene (Fig. 8A). the Plio–Pliestocene was cold relative to the rest of the Ceno- By 3.1–4.6 Mya, genetic exchange between the Atlantic and Pa- zoic (Fig. 8B; Weyl 1968; Burchardt 1978; Wolfe 1978; Haq cific had ceased, resulting in independent evolutionary trajecto- et al. 1987; Ruddiman and Raymo 1988). It is plausible, there- ries among Symbiodinium populations located in each ocean basin fore, that a cool climate contributed to the diversification of the C3 (LaJeunesse 2005; LaJeunesse et al. 2010). The shifts in climate group. Alternatively, fewer shallow water niches may have been conditions and the taxonomic composition of hosts that occurred available to members of the C1 group in the Atlantic due to the during and following this event ostensibly favored the diversi- success of Symbiodinium Clade B (and to a lesser degree Clade fication and specialization of Symbiodinium according to host A) in animals from shallow Caribbean habitats (LaJeunesse 2002, identity and geographic location, resulting in the diverse assem- 2005; Finney et al. 2010). blage of endosymbiont species found on contemporary Caribbean In summary, climate change, favoring the success of a few reefs(Figs.4,5,S1,S2).PsbAncr and ITS2 sequence data sug- ancestors that subsequently diversified principally through host gest that Pacific lineages in the C3 group underwent a parallel specialization in geographic isolation, likely drove the evolution radiation during this time (LaJeunesse 2005); however, several of Symbiodinium species diversity over the past 12 My. Under this dominant host-generalist lineages remain in the Pacific C3 group scenario, divergent selection, acting on genetic variation within (Figs. 4, 8A). a population, favored adaptations that increased fitness in a par- There is precedent for the proliferation and shift in dom- ticular habitat (Dieckmann and Doebeli 1999; Schluter 2001), inance of other microbial groups during major environmental which is determined primarily by the intracellular host envi- change, perhaps as a result of adaptive innovation, reduced com- ronment. Disruptive selection likely opposed gene flow between petition, or extinctions that made available previously occupied sympatric subpopulations adapting to different hosts. Genotypes niches (Crouch et al. 2001; Falkowski et al. 2004). Surpris- capable of associating with multiple host species (i.e., multiple ingly, after the large number of extinctions associated the Plio- habitats) would be suboptimal because, under ecological the- Pleistocene transition, the Caribbean fauna of reef corals has re- ory, these phenotypes experienced reduced fitness relative to a mained stable for approximately 1.5 My despite frequent, evenly specialist within any particular niche (Dieckmann and Doebeli spaced oscillations in global temperatures (Jackson et al. 1996; 1999; Schluter 2001). Instead, reinforcement by disruptive se- Jackson and Johnson 2000; Pandolfi and Jackson 2001). During lection and assortative mating likely increased the prevalence of that period, reduced sea level and altered sea-surface currents genotypes that were competitively superior in symbioses with with each glacial cycle created oceanographic barriers to disper- a particular host taxon. This ongoing process would result in sal in the western Atlantic basin (Veron 1995) that hypothetically reproductively isolated Symbiodinium populations, and eventu- provided opportunities for sequence coalescence and the evolu- ally generate species assemblages partitioned mostly by host tion of numerous regionally localized psbAncr haplotypes (e.g., identity (i.e., ecological speciation; Dieckmann and Doebeli 1999; Figs. 5, S1, S2). Indeed, Pleistocene climates may have generated Schluter 2001, 2009). regionally localized species radiations involving Symbiodinium in Reef coral symbioses of the near future will encounter Clades B (LaJeunesse 2005) and D (LaJeunesse et al. 2010). a warmer climate and more acidic ocean than that of the

364 EVOLUTION FEBRUARY 2014 ADAPTIVE RADIATION OF SYMBIOTIC DINOFLAGELLATES

preceding 7 My (Burchardt 1978; Wolfe 1978; Ruddiman and Bay, L. K., E. J. Howells, and M. J. H. van Oppen. 2009. Isolation, char- Raymo 1988; Pachauri and Reisinger 2007). Many Symbiodinium acterisation and cross amplification of thirteen microsatellite loci for spp. face displacement and extinction with increased sea-surface coral endo-symbiotic dinoflagellates (Symbiodinium clade C). Conserv. Genet. Res. 1:199–203. temperatures and greater, or more erratic, seasonal fluctuations Beerling, D. J. 2002. Low atmospheric CO2 levels during the Permo- in temperature that favor opportunistic species (LaJeunesse et al. Carboniferous glaciation inferred from fossil lycopsids. Proc. Natl. 2009). Alternatively, some populations may acclimatize or adapt Acad. Sci. USA 99:12567–12571. to this environmental change or migrate with their host popula- Berkelmans, R., and M. J. H. van Oppen. 2006. The role of zooxanthellae in the thermal tolerance of corals: a ‘nugget of hope’ for coral reefs in an tions to more suitable locations. Although the specific outcomes era of climate change. Proc. R. Soc. Lond. B Biol. Sci. 273:2305–2312. of these processes are uncertain, our reconstruction of evolution- Briggs, J. C. 1974. Marine zoogeography. McGraw-Hill, New York. ary history suggests that Symbiodinium spp. respond to climate Budd, A. F. 2000. Diversity and extinction in the Cenozoic history of change through “boom and bust” phases in diversification and Caribbean reefs. Coral Reefs 19:25–35. Burchardt, B. 1978. Oxygen isotope paleotemperatures from the Tertiary pe- extinction. As with past changes in climate, the current warm- riod in the North Sea area. Nature 275:121–123. ing may facilitate the rise and spread of host-generalists that are Cerling, T. E., J. M. Harris, B. J. MacFadden, M. G. Leakey, J. Quade, competitively inferior under stable conditions but experience suc- V. 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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website:

Figure S1. Maximum Parsimony topology based on the psbAncr for haplotypes associated with Siderastrea siderea (ITS2 type C3). Figure S2. Maximum Parsimony topology based on the psbAncr for haplotypes associated with Agaricia and Leptoseris (ITS2 types C3, C3a, C3b, C3s, and C3q). Figure S3. Evanno’s Delta-K showing the change in likelihood between K = 3 and corresponds to populations designated by the two sister lineages (C7 and C7a/C12) associated with Orbicella and the Symbiodinium C3 Siderastrea with Siderastrea siderea. Table S1. Symbiodinium samples, their ITS2 designations along with host species, depth of collection, and geographic origin. Table S2. Summary statistics on allele number and evenness among unique multilocus genotypes representing Symbiodinium lineages associated with Orbicella and Siderastrea from the Greater Caribbean of the western Atlantic.

EVOLUTION FEBRUARY 2014 367 Supplemental for Siderastrea siderea associated Symbiodinium.−

PsbAncr haplotypes of Symbiodinium C3 associated with Siderastrea in the Greater Caribbean form a well-supported, monophyletic clade (Fig. S1). Phylogeographic patterns show that some populations are regionally localized. Within this clade there were various locally-endemic sub-clades and haplotypes, including lineages from the Bahamas, Barbados, Brazil and Curacao. A particular haplotype was only found in one sampling location.

Figure S1, Maximum Parsimony topology based on the psbAncr for haplotypes associated with Siderastrea siderea (ITS2 type C3). The basal node of this tree connects to the topology presented in Fig. 4 (note «symbol at the node labeled C3 Siderastrea siderea in Fig. 4). Nodal support indicated as posterior probabilities (PP) above 0.85 next to the relevant node or asterisks for a PP = 1.00 (recovered from separate analyses using Mr. Bayes software, which produced a concordant tree topology). Colored symbols indicate the collection location of each haplotype corresponding to Fig. 1. The branch relating Brazil haplotypes with those of the Greater Caribbean is interrupted by slash marks to indicate that this lineage may be polyphyletic.

Supplemental for Agaricidae associated Symbiodinium −

Multiple phylogenetically independent, or paraphyletic, PsbAncr clades were found associated with Caribbean agaricidae: Agaricia and Leptoseris spp. (Figs. 4, S2). Many of these correlated with geography and corresponded to diagnostic intragenomic ITS2 sequence variants common in the genome. The two larger clades are abbreviated in Fig. 4 with a star symbol. Samples designated as type C3b by ITS2 DGGE fingerprinting (data not shown) occurred at two positions in the phylogeny (designated by the superscripts 1 and 2) and represents the only case were ITS2 and psbA sequences did not correspond. This unusual finding was the only example of a polyphyletic ITS2 type in the psbAncr dataset and possibly resulted from homoplasy at the ITS2 level (or a highly unusual recombinant genotype). The C3b samples belonging to

1 separate groups could be differentiated according to minor differences in banding pattern upon close inspection of their ITS2-DGGE profiles (data not shown).

Figure S2 Legend, Maximum Parsimony topology based on based on the psbAncr for haplotypes associated with Agaricia and Leptoseris (ITS2 types C3, C3a, C3b, C3s, and C3q). Separate lineages insert at various locations in the phylogeny depicted in Figure 4 (note «symbol at the nodes in Fig. 4). Nodal support based on > 0.95 are indicated by asterisks. Colored symbols indicate the collection location of each haplotype corresponding to Fig. 1.

2 Supplemental Figure S3. Evanno’s Delta K showing the change in likelihood between between K=3 and corresponds to populations designated by the two sister lineages (C7 and C7a/C12) associated with Orbicella and the Symbiodinium C3Siderastrea with Siderastrea siderea.

Table S1. Symbiodinium samples, their ITS2 designations along with host species, depth of collection, and geographic origin. GenBank accession numbers for psbAncr sequences are provides and were used to reconstruct the phylogenies in Figures 3–5, S1-S2. Shallow, intermediate and deep refers to collection depths ranging from 1-5, 6-10, and > 10 meters, respectively.

psbAncr Collection ITS-DGGE Genbank Host Genus Host Species Region Location Site Sample I.D type accession Depth number 27B_mexico_Montastraea_C7 C7 KF572268 Orbicella Orbicella faveolata Caribbean Mexico Puerto Morelos Shallow 30B_mexico_Montastraea_C7 C7 KF572248 Orbicella Orbicella faveolata Caribbean Mexico Puerto Morelos Shallow A02_10_Acropora_C3 C3 JQ043639 Acropora Acropora cerealis West Pacific GBR Heron Island Shallow A02_105_Platygyra_C3 C3 JQ043635 Platygrya Platygra daedalea West Pacific GBR Heron Island Shallow A02_12_Acropora_C3 C3 JQ043634 Acropora Acropora clathroata West Pacific GBR Heron Island Shallow A02_136_Coralimorph_C1 C1 KF572204 Coralimorpharia Coralimorpharia sp. West Pacific GBR Heron Island Intermediate A02_20_Acropora_C3 C3 JQ043640 Acropora Acropora gemmifera West Pacific GBR Heron Island Intermediate A02_27_Acropora_C3 C3 JQ043641 Acropora Acropora microclados West Pacific GBR Heron Island Shallow A02_30_Acropora_C3 C3 JQ043642 Acropora Acropora nasuta West Pacific GBR Heron Island Shallow A02_34_Acropora_C3 C3 JQ043643 Acropora Acropora palifera West Pacific GBR Heron Island Shallow A02_36_Acropora_C3 C3 KF572372 Acropora Acropora polystoma West Pacific GBR Heron Island Shallow A02_44_Acropora_C3 C3 JQ043644 Acropora Acropora tenuis West Pacific GBR Heron Island Shallow A02_47_Astreopora_C1 C1 KF572203 Astreopora Astreopora myriophthalana West Pacific GBR Heron Island Shallow A02_63_Favia_C3 C3 JQ043636 Favia Favia stellegera West Pacific GBR Heron Island Shallow A02_64_Favia_C3 C3 JQ043637 Favia Favia abdita West Pacific GBR Heron Island Shallow A02_70_Fungia_C1 C1 KF572205 Fungia Fungia sp. West Pacific GBR Heron Island Shallow A02_71_Galaxea_C1 C1 KF572195 Galaxea Galaxea fascicularis West Pacific GBR Heron Island Shallow A02_74_Goniastrea_C3 C3 JQ043638 Goniastrea Goniastrea aspera West Pacific GBR Heron Island Shallow A03_11A_Turbinaria_C3h C3h JQ043628 Turbinaria Turbinaria frondens West Pacific GBR Feather Reef North Deep A03_127_Symphyllia_C40 C40 KF572360 Symphyllia Symphyllia radians West Pacific GBR Feather Reef North Shallow A03_138_Coscinarea_C1 C1 KF572190 Coscinarea Coscinarea columna West Pacific GBR Feather Reef North Shallow A03_198_Tridacna_C1 C1 KF572172 Tridacna Tridacna maxima West Pacific GBR Rib reef south Shallow A03_2_Pachyseris_C3h C3h KF572373 Pachyseris Pachyseris speciosa West Pacific GBR Feather Reef North Deep A03_221_Podabacia_C3h C3h JQ043630 Podabacia Podabacia crustacea West Pacific GBR Rib Reef North Deep A03_238_Acropora_C3K C3K JQ043648 Acropora Acropora nobilis West Pacific GBR Rib Reef North Shallow A03_326_Turbinaria_C40 C40 KF572357 Turbinaria Turbinaria frondens West Pacific GBR Rib Reef South Intermediate A03_331_Turbinaria_C40 C40 KF572361 Turbinaria Turbinaria reniformis West Pacific GBR Rib Reef South Intermediate A03_333_Turbinaria_C40 C40 KF572358 Turbinaria Turbinaria frondens West Pacific GBR Rib Reef South Intermediate A03_337_Turbinaria_C40 C40 KF572359 Turbinaria Turbinaria reniformis West Pacific GBR Rib Reef South Intermediate A03_379_Echinopora C3 KF572363 Echinopora Echinopora lamellosa West Pacific GBR Curacao Island Intermediate A03_44_Pectinia_C3h C3h JQ043631 Pectinia Pectinia paeonia West Pacific GBR Feather Reef North Deep A03_50_Acropora_C1 C1 KF572189 Acropora Acropora tenuis West Pacific GBR Feather Reef North Deep A03_52_Leptoseris_C3h C3h JQ043627 Leptoseris Leptoseris yabei West Pacific GBR Feather Reef North Deep A03_56_Corallomorpharia_C1 C1 KF572173 Discosoma Discosoma rhodostoma West Pacific GBR Feather Reef North Deep

1 A03_6_Fungia_C3h C3h JQ043629 Fungia Fungia sp. West Pacific GBR Feather Reef North Deep A03_64_Fungia_C3h C3h KF572374 Fungia Fungia granulosa (?) West Pacific GBR Feather Reef North Deep A03_71_Pavona_C3h C3h JQ043630 Podabacia Podabacia crustacea West Pacific GBR Rib Reef North Deep A03_81_Acropora_C3K C3K JQ043645 Acropora Acropora humilis West Pacific GBR Feather Reef North Shallow A03_82_Acropora_C3K C3K JQ043646 Acropora Acropora nasuta West Pacific GBR Feather Reef North Shallow A03_83_Acropora_C3K C3K JQ043647 Acropora Acropora florida West Pacific GBR Feather Reef North Shallow A03_98_Galaxea C1 KF572194 Galaxea Galaxea fascicularis West Pacific GBR Feather Reef North Shallow ABA06_26_Siderastrea_C3 C3 KF572331 Siderastrea Siderastrea siderea Caribbean Bahamas Guana Cay, Abaco Shallow ABA06_28_Siderastrea_C3 C3 KF572332 Siderastrea Siderastrea siderea Caribbean Bahamas Guana Cay, Abaco Shallow B701_NNP1_Siderastrea_C3 C3 KF572319 Siderastrea Siderastrea siderea Caribbean Bahamas Lee Stocking Shallow B704_NNP4_Siderastrea_C3 C3 KF572318 Siderastrea Siderastrea siderea Caribbean Bahamas Lee Stocking Shallow B705_NNP5_Siderastrea_C3 C3 KF572317 Siderastrea Siderastrea siderea Caribbean Bahamas Lee Stocking Shallow Bah_428_M_faveolata_C12 C12 KF572239 Orbicella Orbicella faveolata Caribbean Bahamas Lee Stocking Deep Bah_441_M_franksi_C12 C12 KF572238 Orbicella Orbicella franksi Caribbean Bahamas Lee Stocking Deep Bah_442_M_franksi_C12 B1, C12 KF572240 Orbicella Orbicella franksi Caribbean Bahamas Lee Stocking Deep Bah_725_Montastraea_C12 C12 KF572237 Orbicella Orbicella franksi Caribbean Bahamas Lee Stocking Deep Bah01_10_Agaricia_C3 C3 KF572378 Agaricia Agaricia tenuifolia Caribbean Bahamas Lee Stocking Shallow Bah01_08_Agaricia_C3b C3b KF572385 Agaricia Agaricia agaricites Caribbean Bahamas Lee Stocking Shallow Bah02_10_C3_Leptoseris_C3 C3 KF572292 Leptoseris Leptoseries cuculata Caribbean Bahamas Lee Stocking Deep Bah02_43_C3_Leptoseris_C3 C3 KF572294 Leptoseris Leptoseries cuculata Caribbean Bahamas Lee Stocking Deep Bah02_44_C3_Leptoseris_C3 C3 KF572295 Leptoseris Leptoseries cuculata Caribbean Bahamas Lee Stocking Deep Bah02_62_Agaricia_C3 C3 KF572379 Agaricia Agaricia tenuifolia Caribbean Bahamas Lee Stocking Shallow Bah02_77_Siderastrea_C3 C3 KF572301 Siderastrea Siderastrea siderea Caribbean Bahamas Lee Stocking Shallow Baja04_124_Porites_panamensis C1 KF572208 Porites Porites panamensis Eastern Pacific Gulf of California La Paz Shallow Baja04_135_Psammocora_C1bf C1b-f KF572213 Psammocora Psammocora profundacella Eastern Pacific Gulf of California La Paz Intermediate Baja04_14_Porites_panamensis C1 KF572210 Porites Porites panamensis Eastern Pacific Gulf of California La Paz Shallow Baja04_143_Psammocora_C1bf C1b-f KF572214 Psammocora Psammocora superficialis Eastern Pacific Gulf of California La Paz Intermediate Baja04_30_Porites_panamensis C1 KF572209 Porites Porites panamensis Eastern Pacific Gulf of California La Paz Shallow Baja04_34_Porites_panamensis C1 KF572207 Porites Porites panamensis Eastern Pacific Gulf of California La Paz Shallow Bar05_100_Diploria_C3 C3 KF572356 Diploria Diploria strigosa Caribbean Barbados Atlantis Bank Reef Deep Bar05_102_Erythropodium_C3 C3 KF572288 Erythropodium Erythropodium caribaeorum Caribbean Barbados Atlantis Bank Reef Deep Bar05_104_Montastraea_C7a C7a KF572222 Orbicella Orbicella faveolata Caribbean Barbados Atlantis Bank Reef Deep Bar05_113_Agaricia_C3 C3 KF572413 Agaricia Agaricia lamarcki Caribbean Barbados Atlantis Bank Reef Deep Bar05_114_Leptoseris C3 KF572293 Leptoseris Leptoseris cucullata Caribbean Barbados Atlantis Bank Reef Deep Bar05_119_Agaricea_C3b C3b KF572337 Agaricia Agaricia agaricites Caribbean Barbados Atlantis Bank Reef Deep Bar05_133_Montastraea_C7a C7a KF572223 Orbicella Orbicella faveolata Caribbean Barbados Atlantis Bank Reef Deep Bar05_134_Montastraea_C7a C7a KF572224 Orbicella Orbicella faveolata Caribbean Barbados Atlantis Bank Reef Deep Bar05_135_Montastraea_C7a C7a KF572225 Orbicella Orbicella faveolata Caribbean Barbados Atlantis Bank Reef Deep Bar05_45_Palythoa_C1 C1 KF572179 Palythoa Palythoa caribaeorum Caribbean Barbados Bachelors Hall Shallow Bar05_75_Montastraea_C7a C7a KF572226 Orbicella Orbicella franksi Caribbean Barbados Atlantis Bank Reef Deep Bar05_80_Montastraea_C7a C7a KF572227 Orbicella Orbicella annularis Caribbean Barbados Atlantis Bank Reef Deep Bar05_84_Palythoa_C1 C1 KF572182 Palythoa Palythoa caribaeorum Caribbean Barbados Atlantis Bank Reef Deep

2 Bar05_89_Agaricea_C3b C3b KF572336 Agaricia Agaricia agaricites Caribbean Barbados Atlantis Bank Reef Deep Bar05_91_Stephanocoenia_C3 C3 KF572414 Stephanocoenia Stephanocoenia intersepta Caribbean Barbados Atlantis Bank Reef Deep Bar07_03_Siderastrea_C3 C3 KF572303 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Bar07_10_Siderastrea_C3 C3 KF572302 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Bar07_130_Agaricia_Cq Cq KF572338 Agaricia Agaricia humilis Caribbean Barbados Bachelors Hall Shallow Bar07_131_Agaricia_Cq Cq KF572339 Agaricia Agaricia humilis Caribbean Barbados Bachelors Hall Shallow Bar07_132_Agaricia_Cq Cq KF572340 Agaricia Agaricia humilis Caribbean Barbados Bachelors Hall Shallow Bar07_133_Agaricia_Cq Cq KF572341 Agaricia Agaricia humilis Caribbean Barbados Bachelors Hall Shallow BAR07_20_Siderastrea_C3 C3 KF572305 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Bar07_23_Siderastrea_C3 C3 KF572304 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Barbados_AB27_sideratrea_C3 C3 KF572307 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Barbados_AB28_Siderastrea_C3 C3 KF572306 Siderastrea Siderastrea siderea Caribbean Barbados Atlantis Bank Reef Deep Barbados_AT16_Porites_C80 C80 KF572188 Porites Porites astreoides Caribbean Barbados Atlantis Bank Reef Deep Barbados_BR16_Scolymia_C3 C3 KF572285 Scolymia Scolymia cubensis Caribbean Barbados Batt's Rock Intermediate Barbados_R14_Agaricea_C3b C3b KF572388 Agaricia Agaricia agaricites Caribbean Barbados Reef Balls Deep Barbados_RB31_Agaricea_C3b C3b KF572387 Agaricia Agaricia agaricites Caribbean Barbados Reef Balls Deep Barbados_RB9_Agaricea_C3b C3b KF572386 Agaricia Agaricia humilis Caribbean Barbados Reef Balls Deep BB07_190_C1bf C1bf KF572215 Psammocora Psammocora stelata Eastern Pacific Banderas Bay Punta Minta Shallow BB07_201_C1bf C1bf KF572216 Psammocora Psammocora stelata Eastern Pacific Banderas Bay Punta Minta Shallow BB07_240_Porites_panamensis C1v KF572212 Porites Porites panamensis Eastern Pacific Banderas Bay Pocitos Shallow BB07_250_Porites_panamensis C1v KF572211 Porites Porites panamensis Eastern Pacific Banderas Bay Pocitos Shallow Be02_110_Leptoseris_C3 C3 KF572405 Leptoseris Leptoseris cucullata Caribbean Belize Carrie Bow Deep Be02_111_Montastraea_C7 C7 KF572254 Orbicella Orbicella faveolata Caribbean Belize Carrie Bow Deep Be02_116_C3_Erythropodium C3 KF572289 Erythropodium Erythropodium caribaeorum Caribbean Belize Carrie Bow Deep Be02_118_Lebrunia C1 KF572164 Lebrunia Lebrunia danae Caribbean Belize Carrie Bow Deep BE02_135_Bartholomea_C1 C1 KF572193 Bartholomea Bartholomea annulata Caribbean Belize Carrie Bow Shallow Be02_139_Bartholomea C1 KF572163 Bartholomea Bartholomea annulata Caribbean Belize Carrie Bow Shallow Be02_170_Stephanocoenia_C3 C3 KF572344 Stephanocoenia Stephanocoenia intercepta Caribbean Belize Carrie Bow Deep Be02_171_Stephanocoenia_C3 C3 KF572345 Stephanocoenia Stephanocoenia intercepta Caribbean Belize Carrie Bow Deep Be02_178_Ricordia_C3 C3 KF572286 Ricordia Ricordia florida Caribbean Belize Carrie Bow Deep Be02_185_Protopalythoa C1 KF572155 Protopalythoa Protopalythoa grandis Caribbean Belize Carrie Bow Shallow Be02_187_C3_Erythropodium C3 KF572290 Erythropodium Erythropodium caribaeorum Caribbean Belize Carrie Bow Shallow Be02_192_Mreesi_C3c C3c KF572299 Mycetophyllia Mycetophyllia reesi Caribbean Belize Carrie Bow Deep Be02_193_Mreesi_C3c C3c KF572300 Mycetophyllia Mycetophyllia reesi Caribbean Belize Carrie Bow Deep Be02_25_Agaricia_C3a C3a KF572402 Agaricia Agaricia tenuifolia Caribbean Belize Carrie Bow Shallow Be02_26_Agaricia_C3a C3a KF572400 Agaricia Agaricia tenuifolia Caribbean Belize Carrie Bow Shallow Be02_29_Palythoa C1 KF572166 Palythoa Palythoa caribbeorum Caribbean Belize Carrie Bow Shallow Be02_30_Lebrunia C1 KF572165 Lebrunia Lebrunia danae Caribbean Belize Carrie Bow Shallow Be02_36_Stephanocoenia_C3 C3 KF572346 Stephanocoenia Stephanocoenia intercepta Caribbean Belize Carrie Bow Deep Be02_51_Leptoseris_C3 C3 KF572406 Leptoseris Leptoseris cucullata Caribbean Belize Carrie Bow Intermediate Be02_61_Agaricia_C3a C3a KF572401 Agaricia Agaricia agaricites Caribbean Belize Carrie Bow Intermediate Be02_74_Agaricia_C3a C3a KF572403 Agaricia Agaricia tenuifolia Caribbean Belize Carrie Bow Intermediate

3 Be02_81_Agaricia_C3 C3 KF572404 Agaricia Agaricia agaricites Caribbean Belize Carrie Bow Intermediate Be02_90_Palythoa_C1 C1 KF572180 Palythoa Palythoa caribbeorum Caribbean Belize Carrie Bow Intermediate Be02_91_Agaricia_C3 C3 KF572342 Agaricia Agaricia agaricites Caribbean Belize Carrie Bow Deep Be02_97_Agaricia_C3 C3 KF572343 Agaricia Agaricia agaricites Caribbean Belize Carrie Bow Deep Be02_98_Porites_C1a_j C1aj KF572187 Porites Porites astreoides Caribbean Belize Carrie Bow Deep Be02_99_Stephanocoenia_C3 C3 KF572347 Stephanocoenia Stephanocoenia intersepta Caribbean Belize Carrie Bow Deep Be03_21_Stephanocoenia_C16 C16 KF572350 Stephanocoenia Stephanocoenia intersepta Caribbean Belize Carrie Bow Intermediate Be03_22_Stephanocoenia_C16 C16 KF572351 Stephanocoenia Stephanocoenia intersepta Caribbean Belize Carrie Bow Intermediate Be03_25_Stephanocoenia_C16 C16 KF572352 Stephanocoenia Stephanocoenia intersepta Caribbean Belize Carrie Bow Intermediate Belize2_483_M_faveolata_C7 C7 KF572270 Orbicella Orbicella faveolata Caribbean Belize Carrie Bow Deep Belize2_487M_faveolata_C7 C7 KF572271 Orbicella Orbicella faveolata Caribbean Belize Carrie Bow Deep Belize3_224_M_faveolata_C7 C7 KF572269 Orbicella Orbicella faveolata Caribbean Belize Carrie Bow Deep Belize3_303M_faveolata_C7 C7 KF572247 Orbicella Orbicella faveolata Caribbean Belize Carrie Bow Deep CB13_02_Belize_Siderastrea_C3 C3 KF572316 Siderastrea Siderastrea siderea Caribbean Belize Carrie Bow Intermediate CB14_02_Belize_Siderastrea_C3 C3 KF572313 Siderastrea Siderastrea siderea Caribbean Belize Carrie Bow Intermediate CB32_02_Belize_Siderastrea_C3 C3 KF572314 Siderastrea Siderastrea siderea Caribbean Belize Carrie Bow Intermediate CB33_02_Belize_Siderastrea_C3 C3 KF572315 Siderastrea Siderastrea siderea Caribbean Belize Carrie Bow Intermediate C40 KF572370 Isopora Isopora palifera Indian Ocean Chagos Archipelago Shallow Chagos_Isopora21_C40 Culture_113 C1 KF572161 N.A. N.A. N.A. N.A. N.A. N.A. Culture_152 C1 KF572162 N.A. N.A. N.A. N.A. N.A. N.A. Cur09_106_Agaricea_C C3 KF572397 Agaricia Agaricia tenuifolia Caribbean Curacao Slangenbaai Intermediate Cur09_107A_Siderastrea_C3 C3 KF572320 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_109_Agaricea_C C3 KF572395 Agaricia Agaricia tenuifolia Caribbean Curacao Slangenbaai Intermediate Cur09_111_Agaricea_C C3 KF572396 Agaricia Agaricia tenuifolia Caribbean Curacao Slangenbaai Intermediate Cur09_114_Siderastrea_C3 C3 KF572322 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_23A_Mycetophyllia C3 KF572423 Mycetophyllia Mycetophyllia sp. Caribbean Curacao Slangenbaai Deep Cur09_25_Scolymia C3 KF572298 Scolymia Scolymia cubensis Caribbean Curacao Slangenbaai Deep Cur09_26_Agaricia C3 KF572409 Agaricia Agaricia graheami Caribbean Curacao Slangenbaai Deep Cur09_27A_Siderastrea_C3 C3 KF572323 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_27C_Siderastrea_C3 C3 KF572324 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_28_Agaricia C3 KF572408 Agaricia Agaricia graheami Caribbean Curacao Slangenbaai Deep Cur09_31A_Siderastrea_C3 C3 KF572325 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_31B_Siderastrea_C3 C3 KF572326 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_31C_Siderastrea_C3 C3 KF572327 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_32_Mycetophyllia C3 KF572424 Mycetophyllia Mycetophyllia sp. Caribbean Curacao Slangenbaai Deep Cur09_34A_Mycetophyllia_C3 C3 KF572354 Mycetophyllia Mycetophyllia sp. Caribbean Curacao Slangenbaai Deep Cur09_40C_Mycetophyllia_C3 C3 KF572355 Mycetophyllia Mycetophyllia sp. Caribbean Curacao Slangenbaai Deep Cur09_45A_Siderastrea_C3 C3 KF572328 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_45B_Siderastrea_C3 C3 KF572329 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_45C_Siderastrea_C3 C3 KF572330 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_47B_Siderastrea_C3 C3 KF572321 Siderastrea Siderastrea siderea Caribbean Curacao Slangenbaai Intermediate Cur09_93_Agaricia_C3 C3 KF572398 Agaricia Agaricia tenuifolia Caribbean Curacao Slangenbaai Intermediate

4 C7a KF572242 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_20_Montastraea C7a KF572243 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_22_Montastraea C7a KF572246 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_23_Montastraea C7a KF572241 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_25_Montastraea C7a KF572244 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_27a_Montastraea C7a KF572245 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_27b_Montastraea C7a KF572233 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_44_Montastraea C7a KF572234 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_46_Montastraea C7a KF572232 Orbicella Orbicella faveaolata Caribbean Curacao Intermediate Cur11_47_Montastraea C1 KF572184 Palythoa Palythoa caribaeorum East Atlantic Cape Verde Intermediate CVX09_01_Palythoa_C1 F1362_AG_Mfranksi2_C3 C3 KF572274 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep F1363_AG_M_franksi_C3 B1, C3 KF572276 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep F1377_AG_Mfranksi2_C3 C3 KF572275 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep F1378_AG_Mfranksi3_C3 C3 KF572272 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep F1379_AG_Mfranksi4_C3 C3 KF572273 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep F1380_AG_M_franksi_C3 C3 KF572277 Orbicella Orbicella franksi Caribbean Florida Keys Alligator Reef Deep FGB_70_Siderastrea C1 KF572160 Siderastrea Siderastrea siderea Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_17_Mussa_C3 C3 KF572281 Mussa Mussa angulosa Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_18_Mussa_C3 C3 KF572280 Mussa Mussa angulosa Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_30_C3_Leptoseris_C3 C3 KF572335 Leptoseris Leptoseris cucullata Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_31_C3_Leptoseris_C3 C3 KF572334 Leptoseris Leptoseris cucullata Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_32_C3_Leptoseris_C3 C3 KF572333 Leptoseris Leptoseris cucullata Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_34_Siderastrea C1 KF572157 Siderastrea Siderastrea siderea Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_35_Agaricia_C3s C3s KF572384 Agaricia Agaricia agaricites Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_39_Stephanocoenia_C87 C87 KF572416 Stephanocoenia Stephanocoenia intersepta Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_40_Stephanocoenia_C87 C87 KF572417 Stephanocoenia Stephanocoenia intersepta Caribbean Flower Garden Banks West Bank-Bouy Deep FGB05_53_Agaricia_C3s C3s KF572383 Agaricia Agaricia agaricites Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_54_Agaricia_C3s C3s KF572382 Agaricia Agaricia agaricites Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_55_Agaricia_C3s C3s KF572381 Agaricia Agaricia agaricites Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_64a_Mussa_C3 C3 KF572282 Mussa Mussa angulosa Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_65_Mussa_C3 C3 KF572279 Mussa Mussa angulosa Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_66_Mussa_C3 C3 KF572278 Mussa Mussa angulosa Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_67_Scolemia_C3 C3 KF572297 Scolemia Scolemia cubensis Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_69_Siderastrea C1 KF572158 Siderastrea Sideratrea siderea Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_76_Scolemia_C3 C3 KF572296 Scolemia Scolemia cubensis Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_78_Stephanocoenia_C87 C87 KF572418 Stephanocoenia Stephanocoenia intersepta Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_81_Agaricia_C3s C3s KF572380 Agaricia Agaricia agaricites Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_77_Stephanocoenia_C87 C87 KF572415 Stephanocoenia Stephanocoenia intersepta Caribbean Flower Garden Banks East Bank-Bouy Deep FGB05_89_Siderastrea C1 KF572156 Siderastrea Siderastrea siderea Caribbean Flower Garden Banks East Bank-Bouy Deep C1 KF572159 Acropora Acropora palmata Caribbean Flower Garden Banks Deep FGB05_90_Acropora FL01_31_C3_Erythropodium_C3 C3 KF572291 Erythropodium Erythropodium caribaeorum Caribbean Florida Keys Key Largo Intermediate FL01_35_Palythoa C1 KF572178 Palythoa Palythoa caribaeorum Caribbean Florida Keys Key Largo Shallow

5 FL01_6_Agaricia_C3a C3a KF572399 Agaricia Agaricia agaricites Caribbean Florida Keys Key Largo Shallow FL01_8_Agaricia_C3b C3b KF572389 Agaricia Agaricia humilis Caribbean Florida Keys Key Largo Shallow FL02_63_Palythoa_C1 C1 KF572181 Palythoa Palythoa caribaeorum Caribbean Florida Keys Alligator Reef Deep FL02_81_Lebrunia C1 KF572168 Lebrunia Lebrunia danae Caribbean Florida Keys Admiral Reef Shallow C1 KF572153 Acropora Acropora cervicornis Caribbean Florida Keys Shallow FLKeys_6298_Acorpora1 C1 KF572154 Acropora Acropora cervicornis Caribbean Florida Keys Shallow FLKeys_6302_Acropora2 Haw02_113_C27 C27 JQ043668 Pavona Pavona duerdeni Central Pacific Oahu Kaneohe Bay Deep Haw02_18b_Montipora_31 C31 JQ043612 Montipora Montipora capitata Central Pacific Oahu Kaneohe Bay Shallow Haw02_23_C27 C27 JQ043667 Pavona Pavona varians Central Pacific Oahu North shore Deep Haw02_41_C27 C27 JQ043673 Pavona Pavona duerdeni Central Pacific Oahu North shore Deep Haw02_43_C27 C27 JQ043672 Pavona Pavona duerdeni Central Pacific Oahu North shore Deep Haw02_45b_Montipora_31 C31 KF572371 Montipora Montipora capitata Central Pacific Oahu North shore Deep Haw02_51_Fungia_C1bf C1bf KF572218 Fungia Fungia scutaria Central Pacific Oahu North shore Deep Haw02_5b_Montipora_C31 C31 JQ043613 Montipora Montipora capitata Central Pacific Oahu Kaneohe Bay Shallow Haw02_6_Fungia_C1bf C1bf KF572219 Fungia Fungia scutaria Central Pacific Oahu Kaneohe Bay, Shallow Haw02_63_Psammocora_C1bf C1bf KF572220 Psammocora Psammocora haimeana Central Pacific Oahu Kaneohe Bay Shallow Haw02_7_Fungia_C1bf C1bf KF572221 Fungia Fungia scutaria Central Pacific Oahu Kaneohe Bay, Shallow Haw02_73_C27 C27 JQ043671 Pavona Pavona varians Central Pacific Oahu Kaneohe Bay, Shallow Haw02_86_Cyphastrea_C1bf C1bf KF572217 Cyphastrea Cyphastrea ocellina Central Pacific Oahu Kaneohe Bay, Shallow Haw02_95_C27 C27 JQ043674 Pavona Pavona varians Central Pacific Oahu Kaneohe Bay, Shallow JPB08_08_Agaricea_C3b C3b KF572394 Agaricia Agaricia agaricites Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_17_Siderastrea_C3 C3 KF572376 Siderastrea Siderastrea stellata Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_19_Agaricea_C3b C3b KF572390 Agaricia Agaricia agaricites Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_24_Siderastrea_C3 C3 KF572375 Siderastrea Siderastrea stellata Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_31_Agaricea_C3b C3b KF572391 Agaricia Agaricia agaricites Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_34_Protopalythoa C1 KF572170 Protopalythoa Protopalythoa variabilis Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_35_Agaricea_C3b C3b KF572393 Agaricia Agaricia agaricites Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow JPB08_39_Protopalythoa_C1 C1 KF572177 Protopalythoa Protopalythoa variabilis Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_42_Palythoa C1 KF572171 Palythoa Palythoa caribeaorum Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_47_Protopalythoa_C1 C1 KF572176 Protopalythoa Protopalythoa variabilis Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_48_Protopalythoa_C1 C1 KF572175 Protopalythoa Protopalythoa variabilis Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_52_Agaricea_C3b C3b KF572392 Agaricia Agaricia agaricites Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_55_Porites_astreoides C1 KF572169 Porites Porites astreoides Northeastern Brazil Paraíba, João Pessoa Cabo Branco Shallow JPB08_6_Siderastrea_C3 C3 KF572377 Siderastrea Siderastrea stellata Northeastern Brazil Paraíba, João Pessoa Picãozinho Shallow C1 KF572192 Siderastrea Siderastrea stellata Northeastern Brazil Baia da Traicao Shallow JPB08_61_Siderastrea_C1 C1 KF572174 Protopalythoa Protopalythoa variabilis Northeastern Brazil Baia da Traicao Shallow JPB08_63_Protopalythoa_C1 C1 KF572198 Isaurus Isaurus sp. Southwest Brazil Praia do Forte Shallow JPB08_75_Isaurus_C1 C1 KF572200 Siderastrea Siderastrea stellata Southwest Brazil Praia do Forte Shallow JPB08_76_Siderastrea_C1 C1 KF572201 Siderastrea Siderastrea stellata Southwest Brazil Praia do Forte Shallow JPB08_77_Siderastrea_C1 C1 KF572202 Siderastrea Siderastrea stellata Southwest Brazil Praia do Forte Shallow JPB08_79_Siderastrea_C1 C1 KF572196 Zoanthus Zoanthus sociatus Southwest Brazil Praia do Forte Shallow JPB08_82_Zoanthus_C1 C1 KF572191 Zoanthus Zoanthus sociatus Southwest Brazil Praia do Forte Shallow JPB08_84_Zoanthus_C1

6 C1 KF572197 Siderastrea Siderastrea stellata Southwest Brazil Lauro de Freitas Shallow JPB08_92_Siderastrea_C1 LB5_mexico_Montastraea_C7 C7 KF572249 Orbicella Orbicella faveolata Caribbean Mexico Puerto Morelos Shallow Montastraea_Cur11_31 C7 KF572261 Orbicella Orbicella faveolata Caribbean Curacao Playa Kalki Intermediate Montastraea_Cur11_66 C7 KF572250 Orbicella Orbicella annularis Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_67 C7 KF572251 Orbicella Orbicella franksi Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_68 C7 KF572252 Orbicella Orbicella franksi Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_72 C7 KF572253 Orbicella Orbicella franksi Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_73 C7 KF572262 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_74 C7 KF572263 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_75 C7 KF572264 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_80 C7 KF572265 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_81 C7 KF572266 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate Montastraea_Cur11_84 C7 KF572267 Orbicella Orbicella faveolata Caribbean Curacao Playa Largu Intermediate PM99_20_Isophyllastrea_C3 C3 KF572284 Isophyllastrea Isophyllastrea rigida Caribbean Puerto Morelos Shallow PM99_22_Leptoseris_C3 C3 KF572407 Leptoseris Leptoseris cucullata Caribbean Puerto Morelos Petem Deep PM99_29_Mycetophyllia_C3 C3 KF572419 Mycetophyllia Mycetophyllia lamarckiana Caribbean Puerto Morelos Petem Deep PM99_30_Mycetophyllia_C3 C3 KF572420 Mycetophyllia Mycetophyllia lamarckiana Caribbean Puerto Morelos Petem Deep PM99_31_Mycetophyllia_C3 C3 KF572421 Mycetophyllia Mycetophyllia sp. Caribbean Puerto Morelos Petem Deep PM99_32_Mycetophyllia_C3 C3 KF572353 Mycetophyllia Mycetophyllia sp. Caribbean Puerto Morelos Petem Deep PM99_33_Mycetophyllia_C3 C3 KF572422 Mycetophyllia Mycetophyllia danana Caribbean Puerto Morelos Petem Deep PM99_35_Porites_C1a_j C1aj KF572185 Porites Porites astreoides Caribbean Puerto Morelos Petem Deep PM99_36_Porites_C1a_j C1aj KF572186 Porites Porites astreoides Caribbean Puerto Morelos Petem Deep PM99_72_Discosoma_C1 C1 KF572199 Discosoma Discosoma carlgreni Caribbean Puerto Morelos Bocana Shallow PM99_73_Discosoma C1 KF572167 Discosoma Discosoma sanctithomae Caribbean Puerto Morelos Bocana Shallow PM99_74_C3_Ricordia_C3 C3 KF572287 Ricordea Ricordea florida Caribbean Puerto Morelos Petem Deep RB38_Palythoa_C1 C1 KF572183 Palythoa Palythoa caribaeorum Caribbean Barbados Reef Balls Deep SPA45_Montipora_C31 C31 JQ043602 Montipora Montipora sp. Indo-Pacific Aquarium trade N.A. N.A. SPA59_SW2_Montipora_C31c C31c JQ043601 Montipora Montipora capitata Indo-Pacific Aquarium trade N.A. N.A. STX04_15_Siderastrea_C3 C3 KF572308 Siderastrea Siderastrea siderea Caribbean St. Croix leeward side Shallow STX04_31_Montastraea_C7a C7a KF572228 Orbicella Orbicella annularis Caribbean St. Croix leeward side Deep STX04_32_Montastraea_C7a C7a KF572229 Orbicella Orbicella franksi Caribbean St. Croix leeward side Deep STX04_34_Montastraea_C7a C7a KF572230 Orbicella Orbicella faveolata Caribbean St. Croix leeward side Deep STX04_35_Montastraea_C7a C7a KF572231 Orbicella Orbicella faveolata Caribbean St. Croix leeward side Deep STX04_36_Stephanocoenia C16a KF572348 Stephanocoenia Stephanocoenia intersepta Caribbean St. Croix leeward side Deep STX04_37_Stephanocoenia C16a KF572349 Stephanocoenia Stephanocoenia intersepta Caribbean St. Croix leeward side Deep STX04_54a_Siderastrea_C3 C3 KF572309 Siderastrea Siderastrea siderea Caribbean St. Croix leeward side Deep STX04_55a_Siderastrea_C3 C3 KF572310 Siderastrea Siderastrea siderea Caribbean St. Croix leeward side Deep STX04_58_Agaricia_C3 C3 KF572411 Agaricia Agaricia agaricities Caribbean St. Croix leeward side Deep STX04_59_Agaricia_C3 C3 KF572412 Agaricia Agaricia agaricities Caribbean St. Croix leeward side Deep STX04_61_Agaricia_C3 C3 KF572410 Agaricia Agaricia agaricities Caribbean St. Croix leeward side Deep SW37_Pectinia_C40 C40 KF572369 Pectinia Pectinia sp. Indo-Pacific Aquarium trade N.A. N.A. C40 KF572364 Cyphastrea Cyphastrea chalcidicum Western Australia Ningaloo Reef Intermediate WA10_Cyphastrea_C40

7 C40 KF572366 Hydnophora Hydnophora rigida Western Australia Ningaloo Reef Intermediate WA112_Hydnophora_C40 C40 KF572367 Echinopora Echinopora lamellosa Western Australia Ningaloo Reef Intermediate WA29_Echinophora_C40 C40 KF572362 Acropora Acropora latistella Western Australia Dampier Intermediate WA908_Acropora_C40 C40 KF572368 Hydnophora Hydnophora rigida Western Australia Ningaloo Reef Intermediate WA93_Hydnophora_C40 C40 KF572365 Cyphastrea Cyphastrea chalcidicum Western Australia Ningaloo Reef Intermediate WA99_Cyphastrea_C40 YBD3_Montastraea_C7 C7 KF572259 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate YBD4_Montastraea_C7 C7 KF572260 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate YBD59_Montastraea_C7 C7 KF572258 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate 98_78_Montastraea_C7 C7 KF572255 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate 98_79_Montastraea_C7 C7 KF572256 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate 98_80_Montastraea_C7 C7 KF572257 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate 98_83_Montastraea_C7c C7c KF572236 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate 98_84_Montastraea_C7c C7c KF572235 Orbicella Orbicella faveolata Caribbean Panama San Blas Intermediate Zam03_10M_28_Alveopora_C27 C27 JQ043670 Pachyseris Pachyseris speciosa West Pacific Ryukyus Isls. Zamami Island Intermediate Zam03_10M_33_Alveopora_C27 C27 JQ043669 Alveopora Alveopora sp. West Pacific Ryukyus Isls. Zamami Island Intermediate Zam03_10m_68_Montipora_C31 C31 JQ043599 Montipora Montipora sp. West Pacific Ryukyus Isls. Zamami Island Intermediate Zam03_10m_87_Montipora_C31 C31 JQ043604 Montipora Montipora venosa West Pacific Ryukyus Isls. Zamami Island Intermediate Zam03_3m_87_Montipora_C31 C31 JQ043603 Montipora Montipora sp. West Pacific Ryukyus Isls. Zamami Island Shallow Zam03_3m_92_Montipora_C30 C30 JQ043605 Montipora Montipora efflorescens West Pacific Ryukyus Isls. Zamami Island Shallow Zam03_3m_95_Montipora_C31 C31 JQ043600 Montipora Montipora capitata West Pacific Ryukyus Isls. Zamami Island Shallow Zam03_3m_25_Astreopora_C1 C1 KF572206 Astreopora Astreopora myriophthalma West Pacific Ryukyus Isls. Zamami Island Shallow Zam03_41_Fungia_C27 C27 JQ043676 Fungia Fungia sp. West Pacific Ryukyus Isls. Zamami Island Shallow

8 Table S2. Summary statistics on allele number and evenness among unique multilocus genotypes representing Symbiodinium lineages associated with Orbicella and Siderastrea from the Greater Caribbean of the western Atlantic. Standard error (numbers in smaller font) is given below each mean.

C7 C7a/C12 C3Siderastrea Nunique 31 14 33 Na 3.200 3.400 7.100 0.573 0.600 0.767 Ae 2.055 2.356 3.697 0.347 0.412 0.442 Ho 0.000 0.000 0.599 0.000 0.000 0.077 He 0.377 0.445 0.673 0.094 0.090 0.057

Na = Average no. of alleles per locus Ae = No. of effective alleles per locus [1 / (Sum pi^2)] Ho = Observed heterozygosity [No. of Hets / N] He = Expected heterozygosity [1 - Sum pi^2]