Diversity and Stability in Aquarium Corals1

J. Phycol. 45, 1030–1036 (2009) 2009 Phycological Society of America DOI: 10.1111/j.1529-8817.2009.00730.x

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS1

Robin T. Smith Department of Biology, Florida International University, Miami, Florida 33199, USA Jorge H. Pinzo´n and Todd C. LaJeunesse2 Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA

Indo-Pacific reef corals growing for years in existence of coral reef ecosystems worldwide (Hoegh- closed-system aquaria provide an alternate means to Guldberg et al. 2007). The sensitivity of these eco- investigate host–symbiont specificity and stability. systems is partially dependent on the resiliency The diversity of dinoflagellate endosymbionts (Sym- between corals and their dinoflagellate endo- biodinium spp.) from coral communities in private symbionts (Symbiodinium spp.). Hosts that do not and public aquaria was investigated using molecular- harbor the same symbiont respond differently to genetic analyses. Of the 29 symbiont types (i.e., spe- physiological stress (e.g., high temperature, irradi- cies) identified, 90%% belonged to the most prevalent ance; Rowan 2004, Warner et al. 2006, Berkelmans group of Symbiodinium harbored by Indo-Pacific reef and van Oppen 2006, LaJeunesse et al. 2007, corals, Clade C, while the rest belonged to Clade D. Sampayo et al. 2008); however, the extent to which Sixty-five percent of all types were known from field individual colonies adjust physiologically to environ- surveys conducted throughout the Pacific and mental stress through the replacement of one sym- Indian oceans. Because specific coral–dinoflagellate biont by another remains unclear (Baker 2001, partnerships appear to have defined geographic dis- Goulet 2006). Comparing the symbionts harbored tributions, correspondence of the same symbionts in by various corals, existing in different and ⁄ or unu- aquarium and field-collected specimens identifies sual environments from different geographic regions where particular colonies must have been regions, evaluates the range of what combinations collected in the wild. Symbiodinium spp. in clade D, can exist naturally. This understanding may improve believed to be ‘‘stress-tolerant’’ and ⁄ or ‘‘opportunis- the accuracy of predictions about how coral com- tic,’’ occurred in a limited number of individual col- munities respond to periods of major climate onies. The absence of a prevalent, or ‘‘weedy,’’ change over ecological and evolutionary timescales symbiont suggests that conditions under which (Buddemeier and Fautin 1993, Baker et al. 2004, aquarium corals are grown do not favor competitive LaJeunesse 2005). replacements of their native symbiont populations. Marine aquaria maintained around the world The finding of typical and diverse assemblages of offer a source of symbiotic cnidarians, including a Symbiodinium spp. among aquarium corals living high diversity of stony coral, with which to examine many years under variable chemical/physical condi- symbiont diversity and evaluate partner specificity tions, artificial and natural light, while undergoing and flexibility. In the United States, by the late fragmentation periodically, indicates that individual 1990s, there were an estimated 6 million pieces of colonies maintain stable, long-term symbiotic associ- live coral maintained in over 600,000 marine aqua- ations. ria, contributing to an industry worth $200–330 million annually (Green and Shirley 1999). Most Key index words: aquarium corals; coral trade; colonies sold in aquarium stores are collected from dinoflagellates; scleractinia; Symbiodinium;zoo- Indo-Pacific reefs (Green and Shirley 1999, Coˆte´ and xanthellae Reynolds 2006). The increased dissemination of Abbreviations: DGGE, denaturing gradient gel knowledge via the Internet as well as significant electrophoresis; DMSO, dimethyl sulfoxide; ITS, improvements in lighting and filtration over the last internal transcribed spacer decade, or two, have led to greater captive success for many scleractinians (Carlson 1999, Wabnitz et al. 2003). In addition, aquarium propagation through The combined impact of local anthropogenic dis- colony fragmentation is now common, especially turbances and global climate warming threatens the among advanced aquarists, and many thousands of asexually propagated pieces are traded between aquarists annually and offered for sale. This phenome- non has led to a dramatic increase in the diversity of 1 Received 20 October 2008. Accepted 25 February 2009. species in the hobby (Carlson 1999). 2Author for correspondence: e-mail [email protected].

1030 SYMBIODINIUM SPP. IN AQUARIUM CORALS 1031

The conditions under which aquarium corals are a subset of these samples using primers designed by LaJeunesse cultivated may vary drastically and are different in et al. (2008). many ways from natural environments. Artificial DGGE analysis of PCR-amplified ITS rDNA mitigates prob- lems created by intragenomic variation by screening for lighting often differs from natural sunlight in spec- numerically common, evolutionarily persistent rDNA copies tral composition, photoperiod, and intensity (Carl- (LaJeunesse and Pinzoń 2007, Thornhill et al. 2007, Sampayo son 1999). The stress associated with collection, et al. 2009). Each new fingerprint is assigned an alpha-numeric shipment, and subsequent acclimation to the aquar- designator that refers to the sequence of the dominant band, ium environment can result in high mortality rates or multiple sequences in cases were the symbiont genome especially among species and ⁄ or individuals less suit- possesses codominant variants (see LaJeunesse 2002). Each able for captivity (C. Delbeek, personal communica- symbiont type (identified by its particular fingerprint or ‘‘bar- code’’) exhibits a specific geographic, depth, and host distri- tion). Similar to natural populations, aquarium bution (e.g., Rodriguez-Lanetty et al. 2004, Thornhill et al. specimens may experience bleaching and recovery, 2006, Barneah et al. 2007, Pochon et al. 2007, Sampayo et al. whereby a large proportion of the host’s symbiont 2007), and there is considerable evidence that they are community is lost and subsequently reestablished reproductively isolated (Santos et al. 2003, Pettay and LaJeu- (Carlson 1999). Therefore, the survival and coexis- nesse 2007, Sampayo et al. 2009). Collectively, these data satisfy tence of Indo-Pacific corals and soft corals in vari- requisites for the biological species, ecological species, and phylogenetic species concepts (Manhart and McCourt 1992). ous private and public aquaria presents an For each sample, both the profile and the sequences of the opportunity to evaluate how unusual (i.e., artificial) diagnostic band(s) were compared with established data sets of environments may affect the stability of coral– formerly characterized ITS2 fingerprints originating from dinoflagellate partnerships. Importantly, do these natural host populations collected from the Indo-Pacific. In environments favor the proliferation of certain each fingerprint, diagnostic bands were sequenced to confirm opportunistic symbionts? the identity of previously described profiles and to characterize novel profiles. New fingerprint profiles were given an alpha- Finally, analyses of the symbionts from captive numeric code. The capital letter designates the clade followed specimens may also expand the geographic coverage by a ‘‘species’’ number and lowercase letter(s) that corresponds in the assessment of Symbiodinium spp. diversity. to a codominant sequence variant when present. LaJeunesse Many of the corals in the aquarium trade originated (2002) provides additional explanation regarding the interpre- from the understudied reefs of Fiji, Tonga, Indone- tation and characterization of PCR–DGGE fingerprints. sia, and ⁄ or the Philippines (Green and Shirley A phylogenetic reconstruction based on maximum parsi- 1999), and it remains unknown whether hosts in mony was made using sequences generated in this study and from a selected subset of Clade C Symbiodinium identified in these regions contain previously characterized previous collections of Indo-Pacific reef cnidarians. Previous symbionts and ⁄ or possess new ‘‘species.’’ analyses have shown that maximum parsimony provides the best resolution among Symbiodinium spp. within each clade and MATERIALS AND METHODS is wholly consistent with maximum-likelihood and Bayesian protocols (LaJeunesse 2005). Small fragments of Indo-Pacific scleractinians were solicited The full ITS region (ITS 1, 5.8S, and ITS 2) of a colony openly from public aquaria and advanced private aquarists. identified as Acropora cervicornis from the Sea World Aquarium Environmental conditions of individual aquarium systems (Orlando, FL, USA) was PCR amplified using animal-specific differed substantially from one another in terms of species primers from DNA extractions of host and symbiont cells as composition, lighting configuration, and filtration mecha- described above. The ITS region was amplified with the ITSFor nisms, including one open system that utilized natural sunlight (GGG ATC CGT TTC CGT AGG TGA ACC TGC) and ITSRev (e.g., Waikiki Aquarium, Honolulu, Hawaii). Samples were (GGG ATC CAT ATG CTT AAG TTC AGC GGG T) primers mailed by aquarists or collected in person from six public and using the amplification protocol of 3 min at 94C followed by 40 six private aquaria across the continental U.S. and Hawaii cycles of 30 s at 92C, 30 s at 52C, and 30 s at 72C, and with a (Table S1 in the supplementary material). A total of 126 2 final extension of 10 min incubation at 72C. All sequencing specimens were fragmented (0.5 to 1.0 cm in area) and reactions were performed on purified PCR product using Big preserved in 20% dimethyl sulfoxide (DMSO) and sodium Dye Terminator (V1.1) and analyzed on a 3100 Genetic Analyzer chloride solution (Seutin et al. 1991; comprising 16 scleractin- (Applied Biosystems, Foster City, CA, USA). The sequence from ian, two alcyonarian, one corallimorpharian, and one zoanthid the putative A. cervicornis colony was blasted on the National genera). These specimens included taxa displaying an open Center for Biotechnology Information (NCBI) database to (horizontal) system of symbiont acquisition and those that check the similarity with archived Acropora ITS sequences. transfer their symbionts during egg production, referred to as a closed, or vertical, system of symbiont acquisition. The specimens analyzed in this study had been in captivity for RESULTS periods ranging from 2 months to 18 years, with most in cultivation for 7 or more years. Only a few colonies were Twenty-six Clade C Symbiodinium spp. as well as documented to have experienced repeated episodes of three Clade D Symbiodinium spp. were identified bleaching and recovery. from 126 specimens comprising 20 anthozoan gen- DNA extractions were conducted on small (<0.5 cm2) TM era (Table S1). Each specimen contained a single fragments using Promega’s DNA Wizard extraction protocol dominant symbiont population, and only one case (LaJeunesse et al. 2003). For each sample, the ribosomal internal transcribed spacer region 2 (ITS2) was analyzed using was a mixture of two symbionts detected in a col- denaturing gradient gel electrophoresis (DGGE) according to ony. A subset of samples analyzed using PCR–DGGE previously described methods (LaJeunesse 2002, LaJeunesse fingerprinting of ITS1 (data not shown) confirmed et al. 2003). The ITS1 region was also analyzed using DGGE in these findings. A high diversity of symbonts was 1032 ROBIN T. SMITH ET AL. found in aquaria where a high diversity of hosts was to the fainter bands existed between some of these sampled. ‘‘matching’’ fingerprints (Fig. 1a; LaJeunesse et al. More than half of the diversity detected matched 2004a). A new variant, C15h,inthePorites-Montipora with Symbiodinium sp. formally identified from sur- symbiont subclade, was identified in all 10 speci- veys of natural host populations, while 10 were mens of Montipora capricornis from six separate aqua- determined to be new types (i.e., species). The iden- ria, both private and public (Fig. 1b). The ITS2 tification of previously characterized Symbiodinium fingerprint of this symbiont is distinguished from spp. was verified by direct comparison of ITS–DGGE Symbiodinium C15 by the presence of a codominant fingerprints as well as through sequence compari- band designated ‘‘h.’’ While its sequence differs sons of the diagnostic bands from each profile from the C15 by one base change (Fig. 2), its (Fig. 1a). Differences between profiles with regard dominance in the genome of this host-specific Sym- biodinium indicates that it is evolutionarily diverged. Sequence comparisons verified that some symbionts in aquarium specimens matched those previously identified from field surveys, while others were clearly new types represented by distinct sequences that cor- responded to novel PCR–DGGE fingerprint profiles (Fig. 2). The phylogeny based on maximum parsimony shows that the diversity of Clade C Symbiodinium spp. occurring among aquarium corals is ‘‘scattered’’ throughout the entire phylogeny of this clade (Fig. 2; GenBank accession numbers for new Symbiodinium spp. found in aquaria are as follows: C8c, FJ646566; C15g, FJ646567; C15h, FJ646568; C15m, FJ646569; C31c, FJ646570, C88 and C88a, FJ646571 and FJ646572; C89, FJ646573; C96, FJ646574).

Fig. 1. (a) PCR–DGGE ITS 2 fingerprints of the Symbiodinium spp. from aquarium specimens (odd lanes) run adjacent to profiles from characterized wild populations (even lanes). ITS type designation is given above each set of paired lanes. The regions of origin for profiled wild populations are Hawaii (lanes 2 and 10) and Australia’s Great Barrier Reef (lanes 4, 6, 8, and 12). Fig. 2. An unrooted phylogeny of Clade C Symbiodinium spp. Slight differences in relative band intensities and ⁄ or the pres- characterized from aquarium corals using PCR–DGGE rDNA ence ⁄ absence of additional faint bands in a fingerprint correlate fingerprinting (LaJeunesse and Pinzoń 2007). This reconstruction with ecological differentiation (e.g., different host species) or is based on the maximum-parsimony analysis of ITS 2 sequence geographic isolation. For example, the C1b-f (C1f sensu LaJeu- data and shows the phylogenetic relationship of Symbiodinium nesse et al. 2004a) profile (lane 1) found in a colony of Pavona identified from aquarium corals with other type sequences sp. (Waikiki Aquarium) is slightly different than the C1b-f finger- previously described from various symbiotic cnidarians in the print occurring in Leptastrea purpurea colonies collected from the Indo-Pacific (designations not shown). The Symbiodinium spp. in reefs around Oahu (lane 2, LaJeunesse et al. 2004b). (b) The this study that matched previously identified ‘‘types’’ are indi- PCR–DGGE ITS 2 fingerprint of Symbiodinium C15h detected in a cated in brown; those that represent new diversity are indicated total of 10 Montipora capricornis colonies acquired separately from in blue. The dotted line separating C1 and C3 represents one four public and two private aquaria (het = heteroduplex compris- base change. The asterisk next to C1c and C3u indicates that the ing mismatched DNA strands of the ‘‘C15’’ band and the ‘‘h’’ fingerprint of this symbiont is characterized by the ‘‘c’’ or ‘‘u’’ band that form during the reannealing step of the PCR reaction). band only and lacks the ancestral ‘‘C1’’ or ‘‘C3’’ band, respec- DGGE, denaturing gradient gel electrophoresis; ITS, internal tively. DGGE, denaturing gradient gel electrophoresis; ITS, inter- transcribed spacer. nal transcribed spacer. SYMBIODINIUM SPP. IN AQUARIUM CORALS 1033

In one case, an unusual Symbiodinium sp. hinted therefore contain a wide diversity of Symbiodinium at the falsely perceived identity of the host. A speci- spp. provided that individual colonies maintain the men of what was believed to be A. cervicornis ana- same symbiont they possessed originally. lyzed from Sea World, Florida, contained a rare Biogeographic differences in partnerships arise species of Symbiodinium, C94a, common to several because of various factors that influence specificity Indo-Pacific Acropora spp. analyzed during this study and coevolution, including the differences in envi- (Fig. 2; Table S1). A. cervicornis is one of two species ronment, geographic isolation, and regional compo- of Acropora endemic to the Caribbean and typically sitions of host and symbiont taxa (Thompson 2005). contains Symbiodinium A3 in habitats where it is most While Symbiodinium diversity and distribution remain commonly found (Baker et al. 1997, LaJeunesse poorly described for most Indo-Pacific regions, geo- 2002, Thornhill et al. 2006). Some deep-dwelling graphic differences in host–symbiont combinations populations of this species do associate with a Clade may someday offer one option for determining the C Symbiodinium (Baker et al. 1997), but that symbi- region of origin for aquarium specimens (Fig. 3; c.f. ont is genetically different from the one identified LaJeunesse et al. 2005). For example, the colonies in this captive colony (C38a sensu LaJeunesse of Acropora harboring C3k probably originate from 2005). Because this colony was a color morph not the South Pacific collection sites in Fiji or Tonga. observed in natural populations of A. cervicornis,it Ocean currents connect these islands with the Great was suspected that it had been incorrectly identi- Barrier Reef where Symbiodinium C3k is known to be fied. A subsequent comparison of the coral’s ITS common among Acropora spp. (LaJeunesse et al. sequence (FJ550345) with sequences in the 2004a). Similarly, symbiont C3u is common among GenBank database showed only a two base differ- corals in the Indian Ocean, and so colonies with ence from Acropora formosa (GenBank U82732), this symbiont likely originate from western Indone- an Indo-Pacific species similar in morphology to sia (Fig. 3). Because so many distantly related coral A. cervicornis but distantly related (Veron 2000, van taxa have closely related symboints (Rowan and Oppen et al. 2001). Powers 1991), molecular genetic analyses targeting the symbiont minimize the need for having to develop population genetic markers for each coral DISCUSSION genus. Indeed, the population genetic analyses of Artificial communities of Indo-Pacific reef corals the symbionts may provide a surrogate for delineat- growing in closed-system aquaria provide unortho- ing geographic boundaries and levels of connectivity dox yet potentially useful systems for investigating among populations of various coral species and host–symbiont specificity and stability. Like most even entire reef communities (Santos et al. 2003). host communities throughout the Indo-Pacific Unlike the widespread Montipora-specific C26a and (Baker 2003, LaJeunesse et al. 2003, 2004a,b, 2008, C31, the presence of C15h, C31c, C88a, and C93 may Chen et al. 2005, Visram and Douglas 2006), Clade correspond more precisely to the geographic origin C Symbiodinium dominated aquarium coral micro- of a particular Montipora specimen. Symbiodinium cosms. A relatively high diversity of Symbiodinium was C15h was common among specimens of the plating identified among samples comprising only 20 host and vase-forming species M. capricornis and suggests genera. Aquarium assemblages are obviously artifi- that these colonies originated from regions around cial, so the high diversity of symbionts is probably Southeast Asia (Indonesia) because Montipora from explained by the nature of the coral trade where this region normally associates with members of a many colonies have separate origins of collection. A different Clade C subclade (Fig. 2, sensu LaJeunesse small number of host-generalist symbiont types usu- 2005, T. C. LaJeunesse unpublished data) and ally dominate most Indo-Pacific coral reef communi- because a substantial proportion of coral exports for ties, wherein a large number of host species and the aquarium trade originate from Indonesia (Fig. 3; colonies contain what appears to be the same Symbi- Wabnitz et al. 2003). The frequent exchange of odinium species in a given region (LaJeunesse et al. cloned coral fragments by aquarium enthusiasts may 2003, 2004a). High symbiont diversities relative to explain the high frequency of Symbiodinium C15h host diversities appear to occur naturally in Hawaii among M. capricornis specimens (Fig. 1b). However, (LaJeunesse et al. 2004b), the eastern Pacific C15h occurred in several different M. capricornis color (LaJeunesse et al. 2008), and Caribbean (LaJeunesse morphs representing different individuals. It there- et al. 2003). Similarly, no particular Symbiodinium fore indicates that these specimens originated from dominated the community of aquarium corals, and the same region (i.e., Southeast Asia). intercolony variability in symbiont type was observed Coral–algal combinations have distinct geo- among colonies from the same genus. Many coral graphic and ecological distributions that appear to taxa with open systems of symbiont acquisition relate to natural biogeographic provinces and exhibit geographic differences in their symbioses boundaries (LaJeunesse et al. 2004a). Continued with certain Symbiodinium (LaJeunesse et al. 2004a). surveys of natural Symbiodinium populations Assemblages of colonies brought together from especially in geographic locations near collec- different regions throughout the Indo-Pacific should tion ⁄ transshipping sites may identify particular types 1034 ROBIN T. SMITH ET AL.

Fig. 3. The geographic distributions of Symbiodinium characterized from field studies that were also found in aquarium specimens. While some symbionts have wide geographic distributions, others appear to be diagnostic of certain geographic provinces and are color coordinated with countries that have supplied the live coral trade over the last few decades. ‘‘Endemic symbioses’’ could be used to identify the regional origin of imported specimens. of Symbiodinium specific to certain corals in the neous observations may wrongly influence interpre- region (i.e., endemics). Because closely related sym- tations of host–symbiont specificity. bionts associate with evolutionarily divergent host Symbiodinium in clade D are sometimes common taxa (especially among Indo-Pacific corals), molecu- among coral populations living in habitats or lar-genetic and biogeographic data of the symbiont regions that experience wide fluctuations in temper- diversity are far more advanced than what is pres- ature and ⁄ or irradiance (e.g., changes in turbidity; ently available for the animal. Symbiont identity Toller et al. 2001, Chen et al. 2003, Fabricius et al. may therefore provide a diagnostic tool to recognize 2004, Mostafavi et al. 2007, LaJeunesse et al. 2008). the general region of origin (e.g., Indonesia versus Studies in the Caribbean indicate that Symbiodinium Fiji) for an aquarium coral for purposes of owner D1a is an ecological opportunist occurring in a wide curiosity, conservation, and ⁄ or authenticating ship- range of host taxa, often coexisting with other Symbi- ments for enforcement purposes (Fig. 3; LaJeunesse odinium spp. and sometimes associating with et al. 2005). recently bleached and ⁄ or chronically stressed colo- Scleractinians associate with a range of Symbiodi- nies (Toller et al. 2001, Thornhill et al. 2006). nium spp. depending on the animal’s species, the Clade D Symbiodinium were detected in only a few depth of the colony, and biogeographic location of the specimens analyzed in this study. Among the (Baker 2003, LaJeunesse 2005). Over broad geo- three D types identified, each was found in hosts graphic areas and depth ranges, most species of coral that harbor Clade D naturally. For example, Symbi- associate with a small number of compatible symbiodinium D1 occurs in certain colonies of Acropora sp. ont partners relative to what is potentially available from the Great Barrier Reef (Stat et al. 2008); D1a-f (LaJeunesse 2002, Thornhill et al. 2006, Frade et al. (D1-4-6), found in several specimens of Pocillopora 2008, Goulet et al. 2008, LaJeunesse et al. 2008, damicornis was recently occurring in P. damicornis Sampayo et al. 2008). Host–symbiont specificity is colonies from the Andaman Sea, Thailand (T. C. probably influenced by numerous intrinsic and LaJeunesse unpublished data). Symbiont D1a extrinsic factors, but ultimately it depends on genetic (D1-4), known to associate with various host taxa in compatibility (Colley and Trench 1983, Fitt and some regions, was found in specimens of Euphyllia Trench 1983, LaJeunesse 2002, Rodriguez-Lanetty sp. and Hydnophora sp. Real-time PCR analyses may et al. 2004), the subcellular mechanisms of which are reveal clade D Symbiodinium existing at low unde- largely unknown (Trench 1993, Weis et al. 2008). tected background levels among the specimens sur- Symbiodinium C94a was initially collected in several veyed (LaJeunesse et al. 2007, Mioeg et al. 2007), species of Pacific Acropora and in a specimen labeled but the absence of a dominant ‘‘weedy’’ symbiont as A. cervicornis, from the Caribbean. This unusual indicates that symbioses in artificial systems are not finding prompted genetic analysis of the host, which stressed (Toller et al. 2001), and ⁄ or do not undergo subsequently determined that this ‘‘A. cervicornis’’ competitive replacements facilitated by the aquar- was instead a colony of Acropora formosa (= muricata) ium environment, and ⁄ or are incapable of changing from the Indo-Pacific. This example highlights the symbionts. Indeed, most coral colonies appear to importance of validating cases where new and associate stably with a particular symbiont over time- unexpected symbiotic combinations are identified scales ranging from years to decades, regardless of for the first time. Incorrect host identity, sample external environmental factors and ⁄ or bleaching switching during collection, and ⁄ or processing are history (Goulet and Coffroth 2003, Iglesias-Prieto among several factors that may account for et al. 2004, LaJeunesse et al. 2005, Thornhill et al. ‘‘strange’’ host–symbiont combinations. Such erro- 2006, Sampayo et al. 2008). SYMBIODINIUM SPP. IN AQUARIUM CORALS 1035

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Repopulation of zooxanthellae in the Caribbean corals Montastraea annularis Please note: Wiley-Blackwell are not responsi- and M. faveolata following experimental and disease-associated ble for the content or functionality of any supple- bleaching. Biol. Bull. 201:360–73. mentary materials supplied by the authors. Any Trench, R. K. 1993. Microalgal-invertebrate symbioses: a review. queries (other than missing material) should be Endocytobiosis Cell Res. 9:135–75. Veron, J. E. N. 2000. Corals of the World. Australian Institute of directed to the corresponding author for the Marine Science, Townsville, Queensland Australia, vols. 1, 2, article. and 3. Table S1. List of Symbiodinium spp. “types” along with the corresponding host specimen in which each was identified, known years in culture, and the name of the private owner or public source aquarium. The geographic location of where the colony was originally collected is known for only a few specimens. Colonies observed to have bleached and recovered are identified by an asterisk next to the years in captivity. The asterisk next to C1c and C3u indicates that the fingerprint lacked the ancestral “C1” or “C3” band, respectively. GenBank accession numbers of diagnostic sequences are given in the text of the Results section. Source private/public Symbiont Host genus Host species and morphology Years aquaria C1 Pocillopora Pocillopora damicornis Pierce C1c* Pavona Pavona sp. D. Riddle C1d-t Pocillopora Pocillopora damicornis Sea World, FL C1d-t Pocillopora Pocillopora damicornis Sea World, FL C3 Acropora Acropora formosa (muricata) Sea World, FL C3 Acropora Acropora sp. Sea World, FL C3 Acropora Acropora sp. Sea World, FL C3 Acropora Acropora valida Sea World, FL C3 Acropora Acropora yongei 18 C. Delbeek; Solomon Islands C3 Acropora Acropora sp. >5 R. Smith C3 Discosoma Discosoma sp. >8 Penn State aquarium C3k Acropora Acropora valida >8 Penn State aquarium C3k Acropora Acropora sp. grey with white polyps >8 Penn State aquarium C3m Protopalythoa Protopalythoa sp. >8 Penn State aquarium C3u Acropora Acropora millepora "green" >8 Penn State aquarium C3u* Acropora Acropora sp. >8 Penn State aquarium C3u* Euphylia Euphylia ancora Sea World, FL C3u* Turbinaria Turbinaria Sea World, FL C3x Acropora Acropora yongei Omaha Zoo C3x Acropora Acropora sp. >5 R. Smith C15 Anacropora Anacropora spinosa Sea World, FL C15 Montipora Montipora capricornis >5 R. Smith C15 Montipora Montipora confusa >5 R. Smith C15 Montipora Montipora digitata Pierce C15 Montipora Montipora digitata Pierce C15 Montipora Montipora digitata R. Smith C15 Montipora Montipora digitata 1 Wilkes C15 Montipora Montipora digitata 1 Wilkes C15 Montipora Montipora digitata 1 Wilkes C15 Montipora Montipora digitata 9* Omaha Zoo C15 Montipora Montipora digitata (green) 9* Omaha Zoo C15 Montipora Montipora digitata (red) 8 Delbeek; Solomon Islands C15 Montipora Montipora digitata (green) 8 Delbeek; Solomon Islands C15 Montipora Montipora digitata 18 Delbeek; Palau C15 Montipora Montipora digitata 2 J. Adams C15 Montipora Montipora digitata 5 J. Adams; (Fiji) C15 Montipora Montipora digitata Sea World, FL C15 Montipora Montipora sp. Sea World, FL C15 Montipora Montipora sp. brown capricornis 8 C. Delbeek; Solomon Islands C15 Montipora Montipora sp. foliose form <1 Wilkes C15 Montipora Montipora sp. "green-plating" 8 Delbeek; Solomon Islands C15 Montipora Montipora spongodes 2 Adams C15 Montipora Montipora spongodes (green) >8 Penn State aquarium C15 Montipora Montipora stellata 4 J. Adams C15 Montipora Montipora stellata Sea World, FL C15 Pocillopora Pocillopora damicornis Pierce C15 Porites Porites cylindrica 1 Wilkes C15 Porites Porites cylindrica 1 Wilkes C15 Porites Porites cylindrica 9 Omaha Zoo C15 Porites Porites cylindrica Sea World, FL C15 Porites Porites cylindrica Sea World, FL C15 Porites Porites rus 6 J. Adams C15 Porites Porites rus 6 J. Adams C15 Porites Porites sp. Cromer C15 Porites Porites sp. D. Riddle C15 Seriatopora Seriatopora hystrix brown with green polyps >8 Penn State aquarium C15g Montipora Montipora digitata Hurley C15h Montipora Montipora capricornis >5 R. Smith C15h Montipora Montipora capricornis 1 Wilkes C15h Montipora Montipora capricornis 6* Omaha Zoo C15h Montipora Montipora capricornis 5 Adams; Fiji C15h Montipora Montipora capricornis "purple-plating" >8 Penn State aquarium C15h Montipora Montipora capricornis "purple-brown" >8 Penn State aquarium C15h Montipora "orange" capricornis 4 Birch Aquarium, San Diego C15h Montipora Montipora sp. 4 J. Adams C15h Montipora "folios" Montipora sp. <1 Wilkes C15h Montipora green-, brown-plating >8 Penn State aquarium C15h Montipora Montipora sp. "Superman" >8 Penn State aquarium C15m Montipora Montipora digitata bright orange >8 Penn State aquarium C1b-f Pavona Pavona cactus Sea World, FL C1b-f Pavona Pavona decussata 4 J. Adams C1b-f Pavona Pavona decussata Sea World, FL C21/C94a Acropora Acropora granulosa purple and green >8 Penn State aquarium C21 Acropora Acropora microphthalma Sea World, FL C21 Acropora Acropora millepora >8 Penn State aquarium C21 Acropora Acropora sp. Sea World, FL C21 Acropora Acropora sp. Sea World, FL C21 Acropora Acropora sp. Pierce C21 Acropora Acropora tenuis Sea World, FL C21 Acropora Acropora sp. aquamarine blue >8 Penn State aquarium C21 Acropora Acropora sp. green morph >8 Penn State aquarium C21 Montipora Montipora capricornis Pierce C21 Pachyseris Pachyseris gemmae Sea World, FL C26a Montipora Montipora aequituberculata Penn State aquarium C26a Montipora Montipora capricornis Hurley C26a Montipora Montipora capricornis >5 Smith C26a Montipora Montipora sp. Sea World, FL C27 Euphyllia Euphyllia divisa Sea World, FL C27 Fungia Fungia sp. Sea World, FL C31 Montipora Montipora sp. <1 J. Adams C31c Montipora Montipora capitata Sea World, FL C40 Acropora Acropora sp. >5 R. Smith C40 Pectinia Pectinia sp. Sea World, FL C65 Lobophytum Lobophytum sp. >8 Penn State aquarium C65 Sinularia Sinularia sp. green branch tips >8 Penn State aquarium C88a Montipora Montipora capricornis green morph 9* Omaha Zoo C88a Montipora Montipora sp. Sea World, FL C8c Stylophora Stylophora pistillata green w/ brown polyps >8 Penn State aquarium C8c Stylophora Stylophora pistillata pink morph >8 Penn State aquarium C89 Echinopora Echinopora sp. >8 Penn State aquarium C89 Polyphyllia Polyphyllia sp. >8 Penn State aquarium C94a Acropora Acropora cervicornis Sea World, FL C94a Acropora Acropora humilis >8 Penn State aquarium C94a Acropora Acropora millepora pink morph >8 Penn State aquarium C94a Acropora Acropora valida purple branch tip morph >8 Penn State aquarium C94a Acropora Acropora sp. blue morph >8 Penn State aquarium C94a Acropora Acropora sp. blue branch tip morph >8 Penn State aquarium C94a Acropora Acropora sp. green table-top >8 Penn State aquarium C94a Acropora Acropora sp. >8 Penn State aquarium C94a Acropora Acropora sp. >8 Penn State aquarium C94a Acropora Acropora sp. >8 S. Joshi C94a platyhelminth flat worm from Acropora listed above >8 S. Joshi C96 Montipora Montipora sp. Sea World, FL C101 Acropora Acropora formosa blue morph >8 Penn State aquarium C101 Acropora Acropora valida purple morph >8 Penn State aquarium C101 Acropora Acropora sp. "Miami Orchid" >8 Penn State aquarium C101 Acropora Acropora sp. bright yellow morph >8 Penn State aquarium D1 Acropora Acropora sp. orange-morph >8 Penn State aquarium D1a (D1-4) Euphyllia Euphyllia sp. >8 Penn State aquarium D1a (D1-4) Hydnophora Hydnophora sp. >8 Penn State aquarium D1a-f Pocillopora Pocillopora damicornis Sea World, FL D1a-f Pocillopora Pocillopora damicornis Sea World, FL D1a-f Pocillopora Pocillopora damicornis Sea World, FL D1a-f Pocillopora Pocillopora damicornis >8 Penn State aquarium D1a-f Seriatopora Seriatopora hystrix Sea World, FL D1a-f Seriatopora Seriatopora hystrix >8 Penn State aquarium D1a = D1-4 = Symbiodinium trenchi