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 endosym- biotic 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 lin- eages. 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 speci- ficity, 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