Population genetics of reef coral endosymbionts (Symbiodinium, Dinophyceae)

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1 Molecular Ecology (2017) 26, doi: /mec INVITED REVIEWS AND SYNTHESES Population genetics of reef coral endosymbionts (Symbiodinium, Dinophyceae) D. J. THORNHILL,* E. J. HOWELLS, D. C. WHAM, T. D. STEURY and S. R. SANTOS* *Department of Biological Sciences and Molette Biology Laboratory for Environmental and Climate Change Studies, Auburn University, 101 Rouse Life Sciences Building, Auburn, AL 36849, USA, Center for Genomics and Systems Biology, New York University Abu Dhabi, PO Box , Abu Dhabi, United Arab Emirates, Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA, School of Forestry and Wildlife Sciences, Auburn University, 3301 Forestry and Wildlife Building, Auburn, AL 36849, USA Abstract Symbiodinium is a diverse genus of unicellular dinoflagellate symbionts associating with various marine protists and invertebrates. Although the broadscale diversity and phylogenetics of the Symbiodinium complex is well established, there have been surprisingly few data on fine-scale population structure and biogeography of these dinoflagellates. Yet population-level processes contribute strongly to the biology of Symbiodinium, including how anthropogenic-driven global climate change impacts these symbionts and their host associations. Here, we present a synthesis of population-level characteristics for Symbiodinium, with an emphasis on how phylogenetic affinities, dynamics within and among host individuals, and a propensity towards clonality shape patterns on and across reefs. Major inferences include the following: (i) Symbiodinium populations within individual hosts are comprised mainly of cells belonging to a single or few genetic clones. (ii) Symbiont populations exhibit a mixed mode of reproduction, wherein at least one sexual recombination event occurs in the genealogy between most genotypes, but clonal propagation predominates overall. (iii) Mutualistic Symbiodinium do not perpetually persist outside their hosts, instead undergoing turnover and replacement via the continuous shedding of viable clonal cells from host individuals. (iv) Symbiont populations living in the same host, but on different reefs, are often genetically subdivided, suggesting low connectivity, adaptation to local conditions, or prolific asexual reproduction and low effective population sizes leading to disproportionate success within and among hosts. Overall, this synthesis forms a basis for future investigations of coral symbiosis ecology and evolution as well as delimitation of species boundaries in Symbiodinium and other eukaryotic microorganisms. Keywords: connectivity, coral reef, microsatellite, symbiosis, symbiotic dinoflagellate, zooxanthellae Received 26 October 2016; revision received 20 January 2017; accepted 23 January 2017 Introduction Populations are the fundamental units by which decent with modification occurs, a foundational principle in biology first proposed by Darwin (1859; Smocovitus 1996). Consequently, population-level processes such as Correspondence: Daniel J. Thornhill, Fax: +01 (703) ; thornhill.dan@gmail.com natural selection, genetic drift, mutation and migration offer valuable insights into both a species history and its future evolutionary trajectory. The continual integration of population genetics into various biological subfields (Hickerson et al. 2010), the rise of coalescent theory (Wakeley 2009) and the emergence of statistically rigorous, model-based inference (Beaumont et al. 2010) are providing remarkable new insights into the biology of populations and species, ranging from determining

2 SYMBIODINIUM POPULATION GENETICS 2641 levels and directionality of gene flow, identifying past refugia (Carnaval et al. 2009), to evaluating the origins and dispersal dynamics of introduced species (Lombaert et al. 2010; Ascunce et al. 2011). Furthermore, population genetics studies can discover signs of local adaptation (Kawecki & Eber 2004) and aid in the delimitation of species boundaries (Camargo et al. 2012). Relative to macroscopic organisms, investigating the population genetics of microbes presents unique challenges due to their size and an inherent difficulty in isolating and concentrating uncontaminated specimens for analyses such as multilocus DNA genotyping. However, such challenges must be overcome if we are to understand the critical roles microbes play in every ecosystem (Azam et al. 1983; Atlas & Bartha 1997; Prosser et al. 2007). Fortunately, microbes with an endosymbiotic lifestyle are conducive to population genetics as symbiotic hosts typically harbour concentrated numbers of relatively few microbial taxa (Goulet & Coffroth 2003a; Hilario et al. 2011; Pettay et al. 2011). Such endosymbiotic relationships have been critical drivers of evolutionary innovation throughout the history of life, from the inception of proto-eukaryotic cells to the many contemporary associations between microbes and their hosts, ranging from the mutualisms of chemosynthetic bacteria and deep-sea annelids to the parasitisms of various arthropods by Wolbachia (e.g. Sagan 1967; Wernegreen 2004; Dubilier et al. 2008; Hilario et al. 2011). An exemplary case of such symbioses is associations between invertebrates and dinoflagellate symbionts in the genus Symbiodinium, which have promoted the success of the coral reef ecosystem throughout shallow tropical oceans of the world (Fig. 1). Symbiodinium, colloquially known as zooxanthellae, provide photosynthates that support host metabolism, fecundity and growth as well as enhancing processes such as skeletal calcification in corals (Muscatine et al. 1981; Michalek- Wagner & Willis 2001; Yellowlees et al. 2008; Jones & Berkelmans 2011). The genetic diversity of Symbiodinium has been well studied (e.g. Coffroth & Santos 2005; Pochon & Gates 2010; Thornhill et al. 2014), with the genus comprising at least nine divergent clades (i.e. clades A through I). Most of these clades, in turn, can be divided into numerous nuclear ribosomal or chloroplast types 1 that approximate species-level diversity (Fig. 2), with many host species and individuals engaging in stable and specific associations with few species of Symbiodinium (Goulet & Coffroth 2003a; Thornhill et al. 2006a,b; Sampayo et al. 2008; Stat et al. 2009; Byler 1 Throughout the manuscript, we follow the alphanumeric-type nomenclature of LaJeunesse (2001) for symbiont types unless otherwise noted. Taxonomic names are used only for described species. et al. 2013). The diversity and distribution of Symbiodinium is of particular interest from the perspective of bleaching the stress-induced breakdown of the symbiosis most often linked to global climate change and resulting warming seawater temperatures (Hoegh-Guldberg 1999; Baker 2003; Weis 2008). In such cases, bleaching deprives host individuals of energy generated by their symbionts, often leading to mortality from starvation or increasing disease susceptibility (Thornhill et al. 2011), which has led to predictions of altered species abundances and community compositions of future coral reefs (Pandolfi et al. 2011). However, Symbiodinium species exhibit differential stress tolerance and it has been widely noted that the composition of Symbiodinium within hosts may shift following bleaching disturbance (Baker 2001; Thornhill et al. 2006a; Jones et al. 2008; Kemp et al. 2014; Silverstein et al. 2015). The genus Symbiodinium belongs to the Dinoflagellata, a phylum encompassing >2000 extant species from freshwater and marine environments and spanning a variety of lifestyles (Gomez 2012). Along with free-living and mutualistic forms, exo- and endoparasitic lineages are also common among dinoflagellates (Coats 1999) and the phylum itself is sister to the largely intracellular and near exclusively parasitic Apicomplexa within the superphylum Alveolata (Adl et al. 2007). This is notable as endosymbionts at either end of the continuum must first enter a host cell and then contend with the invertebrate innate immune response while in protracted and intimate contact with the host over the course of the association (Trench 1993; Weis et al. 2008; Voolstra et al. 2009; Hill & Hill 2012). While invertebrate Symbiodinium relationships are widely considered mutualistic, examples of parasitic states have been hypothesized or reported (Sachs & Wilcox 2006; Thornhill et al. 2008; Hill & Hill 2012; Banaszak et al. 2013; Dimond et al. 2013; Lesser et al. 2013). Thus, novel insights may be gleaned into the ecology and evolution of relationships involving Symbiodinium when examined via the same lens applied to parasites and pathogens. Numerous studies have examined Symbiodinium diversity at the levels of clades and types (e.g. Rowan & Powers 1991; LaJeunesse 2001; van Oppen et al. 2001; Sampayo et al. 2009; Pochon & Gates 2010; LaJeunesse & Thornhill 2011); however, far less work has occurred on the diversity and distributions of Symbiodinium at finer scales, such as populations and individual clones (Table 1). This is surprising because phenotypic, physiological and functional differences among individuals in populations are essential for evolution and will therefore shape the adaptive responses of these reef coral symbionts, and by extension their hosts, to events such as global climate change (Howells et al. 2012; Thornhill et al. 2014). Here, we review available knowledge about

3 2642 D. J. THORNHILL ET AL. Fig. 1 From left to right: isolated Symbiodinium cells (scale: 10 lm); coral polyps pigmented due to dense (10 6 cells per cm 2 ) endosymbiotic Symbiodinium populations (scale: 1 mm); shallow coral reef comprising various symbiotic invertebrates (scale: 10 cm); coral reef visible from outer space (scale: 100 km). Adapted from Howells (2011); photographs by E.J. Howells with the exception of satellite image by NASA. Symbiodinium at the population level and provide a synthesis of how factors such as their phylogenetic affinities, dynamics within and among host individuals and propensity towards clonality may interplay towards shaping population genetic patterns on and across reefs. Additionally, we utilize population genetic models to evaluate the relative importance of some of these factors as drivers of contemporary patterns as well as presenting testable hypotheses to help direct future investigations. Clonal proliferation vs. sex in Symbiodinium Symbiodinium frequently reproduces through asexual cellular division (Fitt & Trench 1983), a process that results in high levels of clonality that subsequently shapes patterns of their population genetic structure (Sections Free-living Symbiodinium, Symbiodinium diversity within individual hosts, and Symbiodinium population structure among reefs and regions ). A propensity towards asexual reproduction for the genus is not surprising given that clonal division is the dominant reproductive mode for other dinoflagellates (Pfiester & Anderson 1987) as well as relatives within the Apicomplexa (reviewed in Walker et al. 2013). More important, however, is the fact that this life history trait alone appears to impact multiple facets of these symbioses. Namely, when an individual Symbiodinium genotype can produce many clonal copies of itself, such a situation can be ecologically and evolutionarily advantageous, particularly when one or more of those clonal cells establish themselves in a new host, thus allowing the genotype to persist and spread as a result (Hume et al. 2016). We propose that this clonality significantly shapes the dynamics of symbiont infection and dispersal as well as the diversity and genetic structure within and between hosts, reefs and regions (Sections Free-living Symbiodinium, Symbiodinium diversity within individual hosts, and Symbiodinium population structure among reefs and regions ; see also Wulff 1985). Despite the prominence and importance of clonality, asexual division is unlikely to be the sole reproductive mode available to Symbiodinium. Many members of the Dinoflagellata possess a known sexual life stage (Walker 1984; Pfiester & Anderson 1987) and it would be rather remarkable for sexual recombination to be absent in Symbiodinium. Although karyogamy and meiosis have not yet been directly observed, numerous population genetic analyses utilizing either protein or DNA markers strongly suggest Symbiodinium undergoes sexual recombination at some frequency (e.g. Baillie et al. 1998; Baillie et al. 2000; LaJeunesse 2001; Santos et al. 2003; Pettay et al. 2011; Pettay & LaJeunesse 2013; Thornhill et al. 2014; Wilkinson et al. 2015) and this is supported by recent genomic analyses identifying the full complement of genes needed for meiosis and sexual recombination in Symbiodinium microadriaticum (type A1) and Symbiodinium minutum (type B1 Aiptasia ) (Chi et al. 2014; see also Levin et al. 2016). Finally, cells fitting the characteristics of gametes have also been observed in Symbiodinium cultures (Frommlet et al. 2012). In spite of multiple lines of evidence all pointing to a sexual stage in Symbiodinium, we currently do not know the frequency at which sexual recombination occurs in these dinoflagellates. However, reexamination of

4 SYMBIODINIUM POPULATION GENETICS 2643 A B C D E F G H Genetic diversity within Symbiodinium Clade Within clade Species Species or populations B224 B223 B211 B184 B183 B20 B11 B1i B1e B1 P5 P4 P3 P2 P1 Populations; clones or individuals C5 C4 C3 C2 C1 Fig. 2 Hierarchical breakdown of genetic diversity within Symbiodinium placed into a taxonomic context (upper portion of panel) as a function of utilized molecular marker (lower portion of panel). Using Symbiodinium Clade B as the example, lineages within a clade can be resolved from higher (i.e. within clade) to lower (i.e. species and/or populations) taxonomic levels via molecular markers spanning coarser (i.e. rdna) to finer (i.e. chloroplast [cp] hypervariable regions and/or flanking regions of microsatellite loci) resolutions, respectively. Ultimately, clones, and the populations they belong to, are defined with markers targeting polymorphic loci at specific regions (i.e. microsatellites) or across (i.e. DNA fingerprinting, randomly amplified polymorphic DNA [RAPDs]) the Symbiodinium genome. While diversity within lineages of Symbiodinium Clades A E has been defined using such a suite of approaches (designed in black), lineages from Clades F I have been less well characterized to the level of clones (signified in grey). Figure following Coffroth & Santos (2005). I 18S/28S rdna cp23s-rdna; ITS1-rDNA; ITS2-rDNA; mtdna cytb ITS2-rDNA cp hypervariable region; microsatellite flanking regions Utilized genetic marker Microsatellite alleles; DNA fingerprints; RAPDs available population genetic data may provide clues into this phenomenon. For example, the reassortment of alleles in a population undergoing sexual reproduction is expected to produce a low variance in allelic diversity when genotypes are compared in a pairwise fashion (Fig. 3a). In contrast, allelic diversity typically exhibits linkage disequilibrium across multiple loci in an asexual population, resulting in greater variance of pairwise differences among genotypes (Fig. 3a). When a distribution of pairwise differences among genotypes of Symbiodinium trenchii (type D1a or D1-4) from Thailand (LaJeunesse et al. 2014) is compared to these simulated patterns, it appears similar to that of a sexual population (Fig. 3b), with an average of >200 recombination events between any two genotypes and their most recent common ancestor (Appendix S1). However, an analysis of index of association (Maynard Smith et al. 1993) leads to a rejection of panmixia (P < ), implying the presence of significant linkage disequilibrium in this S. trenchii population, in spite of recombination. Taken together, these contrasting patterns are indicative of an epidemic, or mixed mode, of reproduction for Symbiodinium (Maynard Smith et al. 1993; Santos et al. 2003), where at least one sexual recombination event occurs between most genotypes in a population but clonal propagation dominants overall. Given that rates of sex are measureable in Symbiodinium, population genetic analyses can be applied to delimit Symbiodinium species using the biological species concept (Mayr 1942). For instance, if sympatric Symbiodinium have opportunities for allele exchange among genotypes through sexual recombination, then those individuals and populations exchanging alleles are members of the same biological species whereas those failing to do so operate as distinct species. Such a situation is consistent with the partitioning and

5 2644 D. J. THORNHILL ET AL. Table 1 Summary of results from population genetic studies on Symbiodinium. Data categories assembled below include Symbiodinium types/species investigated; their associated hosts, as well as mechanism of symbiont transmission; frequency of the detection of multiple Symbiodinium genotypes within a host individual; percentage of host individuals within the study that hosted symbiont genotypes that were not detected in other host individuals; primary scale of spatial or temporal genetic structure found in the study; and source of the data, including molecular method utilized and citation Symbiodinium type and region Host species and mode of symbiont transmission* Within-host occurrence of multiple genotypes Occurrence of unique genotypes among hosts Spatial and temporal genetic structure Data type ; reference A3 Caribbean B1 Caribbean B1Aiptasia S. minutum Global C1d South Pacific C1.3a Great Barrier Reef C3 Great Barrier Reef Acropora palmata (h/v) 16% (n = 759) 0 19% Structure at 100s of km. Clonal propagation of host and symbiont at 10s of m scales Acropora palmata (h) 6% (n = 25) 53% Structure at 100s of km. Differentiation among sympatric host species Acropora cervicornis (h) 0% (n = 43) 63% Structure absent up to 850 km. Differentiation among sympatric host species Orbicella annularis (h) 0% (n = 6) 100% Spatial structure not investigated. Differentiation among sympatric host species Stephanocoenia intersepta (h) 17% (n = 36) 80% Structure at 100s of km. Differentiation among sympatric host species 10 microsatellites (m); Baums et al. (2014) 10 microsatellites (m); Pinzon (2011) Eunicea flexuosa (h) 47% (n = 610) <2% Structure at 10s of km 5 microsatellites (m); Wirshing et al. (2013) Gorgonia ventalina (h) 28% (n = 747) 84% Structure at 10s of km and less. Divergence of 6 microsatellites (m); Kirk et al. (2009) shallow (<7 m) and deep (>12 m) sites 18% (n = 1624) 91% Structure at 10s of km and less. Divergence 10 microsatellites (m); Andras et al. (2011) between hosts of different ages Orbicella annularis (h) 1% (n = 24) 4% Structure at 10s of km and less. Temporal 33% (n = 90) genotypic stability in 67% of colonies over 3 years Orbicella faveolata (h) 7% (n = 26) 46% (n = 101)* 6% Structure at 10s of km and less. Temporal genotypic stability in 54% of colonies over 3 years Plexaura kuna (h/v) 0% (n = 94) 100% No structure at 10s of m scales investigated. No temporal or habitat variation in DNA fingerprints among clone-mates of the host 3 microsatellites (m); Thornhill et al. (2009), Thornhill et al. (2010) DNA fingerprints; Goulet & Coffroth (2003a,b) Pseudopterogorgia elisabethae (h) 4% (n = 575) 3% Structure at 10s of km 2 microsatellites (m); Santos et al. 2003; Exaiptasia pallida (v/h) 21% (n = 323) 6% Structure between some locations at distances 6 microsatellites (m); Thornhill et al. (2013) of 1800 km but no structure between other localities separated by > km Pocillopora meandrina (v) 85% (n = 279) n/a Structure at 100s of km, but not 10s of km 2 microsatellites (m); Magalon et al. (2006) Sinularia flexibilis (h) 94% (n = 424) n/a Structure at 10s of km 4 microsatellites (s); Howells et al. (2009) Acropora millepora (h) 100% (n = 401) 87% Structure at 10s of km and less. Temporal differentiation at sites resampled at 5- to 7-year intervals 8 microsatellites (s); Howells et al. (2013)

6 Table 1 Continued Symbiodinium type and region Host species and mode of symbiont transmission* Within-host occurrence of multiple genotypes Occurrence of unique genotypes among hosts Spatial and temporal genetic structure Data type ; reference C3Siderastrea Caribbean Siderastrea siderea (h) 10.4% (n = 67) 83% Endemic genotypes at every location, including structure at 100s of km, but not necessarily 10s of km. Genetically isolated from sympatric C7 or C7a/C12 C7 Caribbean Orbicella spp. (h) 7.2% (n = 83) 30% Endemic genotypes at every location, including structure at 100s of km, but not necessarily 10s of km. Genetically isolated from sympatric C7a/C12 or C3 Siderastrea C7a/C12 Caribbean D1 Eastern Pacific D1a/D1-4 S. trenchii Global Orbicella spp. (h) 30.2% (n = 43) 53% Endemic genotypes at every location, including structure at 100s of km, but not necessarily 10s of km. Genetically isolated from sympatric C7 or C3 Siderastrea Pocillopora sp. type I (v) 13% (n = 99) 55% (n = 196)* 8 Caribbean, 22 Pacific and 29 Indian Ocean host taxa n/a No structure at 10s of km. Temporal genotypic stability in 75% of sampled branches and 66% of whole colonies over 9 months n/a (n = 402) 53% No structure at >1000 km among populations within the exception of the Gulf of California vs. Eastern Tropical populations 10 20% in all regions 25% Caribbean, 87% Pacific, and 91% Indian Ocean Structure across 10s of km in the Pacific and Indian Oceans. No structure across the Caribbean where S. trenchii is evidently an introduced species 10 microsatellites (m); Thornhill et al. (2014) 10 microsatellites (m); Thornhill et al. (2014) 10 microsatellites (m); Thornhill et al. (2014) 9 microsatellites (m); Pettay et al. (2011) 11 microsatellites (m); Pettay & LaJeunesse (2013) 12 microsatellites (m); Pettay et al. (2015) n/a, data not available. *Horizontal (h) or vertical (v) symbiont transmission. Multi (m)- or single (s)-locus genotypes. Prada et al. (2014) reported issues with the host identification in this study that confound data interpretations. More than one sample per host colony. SYMBIODINIUM POPULATION GENETICS 2645

7 2646 D. J. THORNHILL ET AL. (a) (b) found in Orbicella spp. as well as C3 Siderastrea from Siderastrea siderea found evidence of recombination within, but not between, Symbiodinium types (Thornhill et al. 2014). These three clade C symbionts each exhibited greater similarity with members of the same lineage from hundreds of kilometres away than with different types found in sympatry in Curacßao and other locations. Thus, types C7, C7a/C12 and C3 Siderastrea behave as distinct species (Thornhill et al. 2014) and similar analyses could be applied to other groups of Symbiodinium as well as micro-eukaryotes in general to provide important insight into species boundaries (LaJeunesse et al. 2012, 2014; Wham et al. 2013; Jeong et al. 2014). As secondary contact between divergent Symbiodinium populations can also produce allelic signals of reproductive isolation, species delimitations are most powerful when multimarker genetic data are combined with morphological, physiological and/or ecological traits (Howells et al. 2016a). Based on these findings, we advance the following testable hypotheses: Asexual division is, by far, the dominant reproductive mode of Symbiodinium. Asexual reproduction greatly elevates levels of clonality within Symbiodinium populations. Despite this, sexual recombination is an infrequent, yet routine, aspect of Symbiodinium life history. Scores of Symbiodinium species exist in nature; one method of delimiting these species is through the presence or absence of sympatric sexual recombination and allele exchange. Fig. 3 Histograms of pairwise differences among genotypes observed in S. trenchii (type D1a or D1-4) collected in Thailand (LaJeunesse et al. 2014) and simulated sexual and asexual populations. Panel (a): Simulated data showing pairwise differences between genotypes in an asexual population (orange) and sexual population (blue). Panel (b): Pairwise differences between genotypes in the observed population (green). Note that the observed pairwise differences among genotypes exhibit a bimodel pattern. A value of (95% credibility interval [CI] = ; Appendix S1) is estimated for this population as the most likely rate of sexual recombination (with being the rate for asexual reproduction) from a coalescentbased MCMC parameter inference from the observed data. This value of suggests the average pair of genotypes has >200 sexual recombination events in the genealogy between them and their most recent common ancestor. nonoverlapping nature of allelic variation among horizontally transmitted Symbiodinium type B1 populations from two sympatric Caribbean octocoral hosts (Santos et al. 2004). Likewise, population genetic analyses of three sympatric symbiont types C7 and C7a/C12 Free-living Symbiodinium Symbiodinium are well-known symbionts, yet many aspects of their biology suggest populations can exist in a free-living state. For example, some Symbiodinium types will persist indefinitely in the laboratory as cultures grown in nutrient-rich media (Santos et al. 2001). When in culture, Symbiodinium alternates between a nonmotile coccoid stage and a motile mastigote capable of rudimentary swimming via flagella, implying a freeliving stage is important in the life history of these dinoflagellates (Freudenthal 1962; Fitt & Trench 1983). Moreover, when asymbiotic hosts are introduced to a coral reef environment, they rapidly establish a relationship with Symbiodinium, suggesting viable symbiont cells are continuously present in the water column or on benthic substrates (Coffroth et al. 2001; Kinzie et al. 2001; Thornhill et al. 2006c; Abrego et al. 2009; Adams et al. 2009; Cumbo et al. 2013), with support from assays of reef water, benthic sediments and the surface of reef-

8 SYMBIODINIUM POPULATION GENETICS 2647 dwelling organisms readily detecting Symbiodinium DNA (Carlos et al. 1999; Gou et al. 2003; Coffroth et al. 2006; Littman et al. 2008; Manning & Gates 2008; Porto et al. 2008; Adams et al. 2009; Pochon et al. 2010). Lastly but of particular interest, Symbiodinium isolates recovered from hosts vs. the external environment are often genetically distinct (LaJeunesse 2001; Santos et al. 2001; Coffroth et al. 2006; Porto et al. 2008; Pochon et al. 2010; Mordret et al. 2016). In fact, some culturable Symbiodinium species including Symbiodinium pilosum (A2), Symbiodinium voratum (E1) and Symbiodinium kawagutii (F1) have never been detected as intracellular symbionts (LaJeunesse 2001; Jeong et al. 2014). These latter observations suggest certain Symbiodinium lineages have potentially evolved an entirely free-living existence. Although Symbiodinium has an apparently ubiquitous presence in the reef environment outside hosts, no studies to date have unequivocally demonstrated the persistence of perpetually free-living individuals or populations of these symbionts. Instead, the life history of Symbiodinium suggests the continual production of short-lived cells that function principally to seek out and invade new host individuals (Freudenthal 1962; Fitt et al. 1981; Fitt & Trench 1983; Fitt 1985; Pasternak et al. 2004; Magalon et al. 2006). For instance, many scleractinian corals and other symbiotic hosts harbour millions of clonal Symbiodinium cells in their tissues and enormous numbers of symbiont cells are released from these hosts in a viable state on a routine basis (Steele 1977; Muscatine & Pool 1979; Stimson & Kinzie 1991; Jones & Yellowlees 1997; Maruyama & Heslinga 1997; Thornhill et al. 2011). Along with this, corallivorous fishes as well as invertebrates themselves release viable Symbiodinium in their faecal matter (Muller-Parker 1984; Porto et al. 2008; Castro-Sanguino & Sanchez 2012). Avenues such as these provide numerous and steady inputs of symbiont cells into the surrounding environment, contributing to an abundance of free-living Symbiodinium. Once in the environment, however, Symbiodinium cells do not appear to travel long distances. Utilizing cultured isolates, Fitt & Trench (1983) estimated that individual cells swim just 3 10 m over the course of 1 day before shedding their flagella and settling to the benthos. Following settlement, these nonmotile cells do not resume swimming until their next division cycle, which requires access to nutrients. Furthermore, dinoflagellate cells are negatively buoyant, which presumably causes free-living Symbiodinium to migrate over short distances or to be locally retained (Howells et al. 2009). Indeed, microscopic and molecular evidence suggests Symbiodinium are more abundant in reef sediments or macroalgal surfaces than in the water column and exhibit declining abundances with distance from a reef environment (Littman et al. 2008; Porto et al. 2008). When the opportunity arises, however, Symbiodinium cells can actively swarm towards and enter a potential host s mouth, apparently attracted by chemical cues or the availability of nutrients (Fitt 1984). The expulsion from hosts of numerous clonal Symbiodinium cells, under otherwise normal conditions, has been purported to be a means of density regulation (Jones & Yellowlees 1997; Wooldridge 2010; Cunning et al. 2015). But this interpretation assumes hosts exert predominant control over the relationship, possibly through access to the inorganic nutrients supplied to its symbionts (Rands et al. 1993; Hooper et al. 2002; Wooldridge 2010). By contrast, hosts have little ability to influence and regulate symbiont density in many relationships involving parasites like apicomplexans (i.e. Amo et al. 2005; Field & Michiels 2005). For cnidarian Symbiodinium associations, notably little change occurs in host gene expression during the successful establishment of a symbiosis, implying endosymbionts evade detection by the host (Trench 1993; Weis 2008; Voolstra et al. 2009; Hill & Hill 2012). But this raises intriguing questions: If this is the case, then why and how are appreciable numbers of symbionts being expelled if the host does not recognize them in the first place? When viewed from a symbiont-centric perspective, one can posit Symbiodinium employs a strategy of pathogen-like shedding, wherein cells actively released from current hosts are not related to density regulation but rather a means to reach new habitats, or in this case, additional host individuals capable of establishing a symbiosis (reviewed by Holt & Barfield 2006). Under this hypothesis, it is improbable that released cells need to be indefinitely free-living, which is consistent with longevity estimates for Symbiodinium of ~7 days in the environment (Nitschke 2015). Instead, Symbiodinium sources are continually renewed and survival of individual cells is likely diminished due to decreased habitat suitability, increased uncertainty and limited access to nutrients. Importantly, the loss of individual cells is a low-risk endeavour for highly clonal Symbiodinium populations because if just one daughter cell among millions infects a new host and successfully establishes a symbiosis, then the result is a doubling of the number of habitats occupied by that clonal genotype. This scenario provides the following testable hypotheses: Symbiodinium cells have short lifespans and low proliferation rates in the free-living reef environment compared to within-host individuals. Abundances of Symbiodinium cells in the environment are determined by the release rate of viable cells from hosts and thus decline with increasing distance from host individuals.

9 2648 D. J. THORNHILL ET AL. Symbiodinium distribution in the reef environment is determined by the dinoflagellate cell s intrinsic dispersal capacity, transit time from their hosts and extrinsic oceanographic conditions (either singularly or in combination). Taken together, the origin and availability of free-living Symbiodinium significantly influences not only broader spatial patterns of population genetic structure of these symbionts among reefs, but the diversity of symbiont genotypes within hosts as well. We elaborate on this latter situation in the next section. Symbiodinium diversity within individual hosts Thousands to millions of Symbiodinium cells can reside within reef-dwelling invertebrates and while some hosts may harbour two or more symbiont species simultaneously, many host individuals associate with just one species of Symbiodinium (Goulet & Coffroth 2003a; Ulstrup & van Oppen 2003; Chen et al. 2005; Thornhill et al. 2006a,b, 2011; Sampayo et al. 2008; Stat et al. 2009; Byler et al. 2013; Ziegler et al. 2015). At the population level, analogous patterns of low within-host diversity are commonly observed. For example, in Symbiodinium type B1 from the Caribbean, 73% of individual scleractinian, octocoral and anemone hosts associate with just one multilocus microsatellite genotype of symbiont (Table 1). Similarly, scleractinian corals hosting Symbiodinium types A3, C7, C7a/C12 and C3 Siderastrea in the Caribbean, as well as Symbiodinium type D1 in the Eastern Pacific, typically associate with a single microsatellite genotype ( 70% of samples; Pettay et al. 2011; Pinzon 2011; Pettay & LaJeunesse 2013; Baums et al. 2014; Thornhill et al. 2014). In contrast, for Symbiodinium Clade C from the West Pacific, 85% of sampled scleractinian and octocoral hosts harbour populations with multiple alleles at individual microsatellite loci, signifying coexistence of closely related genotypes (Table 1; Magalon et al. 2006; Howells et al. 2009, 2013). Higher within-host population diversity can also be detected with increased survey effort, either by genotyping biopsies from different areas, or by repeated temporal sampling, of the same host individual (Thornhill et al. 2009; Howells 2011; Pettay et al. 2011; but see Goulet & Coffroth 2003a; Baums et al. 2014). Although such spatial and temporal heterogeneity in Symbiodinium genotypes within a host does occur, a unifying feature is that clonality dominates within-host populations, regardless of whether they are composed of one or a few individual symbiont genotypes. The clonal composition of within-host Symbiodinium populations is likely influenced by factors reducing genetic diversity at, and following, establishment of the symbiosis. Initially, within-host symbiont populations are influenced by the local availability of free-living Symbiodinium genotypes that are ingestible by a host and capable of successfully forming a viable symbiosis, or those that are transmitted from the maternal parent in vertically transmitted cases (Coffroth et al. 2001; Andras et al. 2011; Howells et al. 2013). Notably, the identity of viable symbionts available from environmental pools may change temporally, as reflected by variability in symbiont genotypes harboured by different cohorts of juvenile hosts (Andras et al. 2011; Howells et al. 2013). Over time, symbiont populations are unlikely to mirror those at the initial uptake event due to selection, competitive processes and differing rates of asexual proliferation (Fitt & Trench 1983; Wilkerson et al. 1988). Such processes would ultimately result in the dominance of one symbiont genotype across the host or, as seen in some cases, potential niche partitioning of genotypes into distinct microhabitats within a host (Rowan et al. 1997). Similarly, winnowing of the symbiosis by the host or environmental conditions could also select for particular symbiont genotypes over others (Nyholm & McFall-Ngai 2004; Abrego et al. 2009, 2012). Temporal stability is also a shared feature of withinhost Symbiodinium populations (Goulet & Coffroth 2003b; Kirk et al. 2005, 2009; Thornhill et al. 2009; Pettay et al. 2011), and such patterns are common and well documented in laboratory, field and modelling studies of eukaryotic phytoplankton (Hutchinson 1961) as well as marine apicomplexans (reviewed in Kirk et al. 2013). This is not surprising given that once a clonal lineage of Symbiodinium is established within a host, it would have a substantial numerical advantage over conspecifics. Barring entry of a superior competitor or environmental shifts capable of perturbing the stability of the established relationship, competitive exclusion and saturation of competent host cells likely limit temporal change. However, the spread of novel mutations (Correa & Baker 2011; van Oppen et al. 2011), sexual recombination (Chi et al. 2014), ingestion and phagocytosis of new genotypes (Coffroth et al. 2010) and environmental shifts altering the selective environment could introduce genetic diversity to within-host populations of Symbiodinium, thus contributing to mixed symbiont populations (Table 1). From the perspective of the host, having genetic diversity within its Symbiodinium population may be beneficial. This possibility has been contentiously debated since the inception of the Adaptive Bleaching Hypothesis (Buddemeier & Fautin 1993), which proposes that shifts in Symbiodinium communities occur in response to environmental perturbations (Baker 2001; Thornhill et al. 2006a; Jones et al. 2008; LaJeunesse et al. 2009; Howells et al. 2013). Harbouring a diverse symbiont community potentially provides physiological

10 SYMBIODINIUM POPULATION GENETICS 2649 versatility, offering hosts a buffer when facing a changing environment (e.g. Silverstein et al. 2012; Howells et al. 2013). However and in contrast, incoming symbiont genotypes could be deleterious rather than beneficial to the host (Sachs & Wilcox 2006) and competition between symbiont genotypes can reduce the energy available for translocation to the host (Frank 1996; Douglas 1998; Hill 2014). Thus, higher within-host Symbiodinium diversity could paradoxically result in a fitness penalty to the host and its symbionts. Based on the above literature and interpretations, we advance the following hypotheses regarding within-host diversity at the population level: Within-host Symbiodinium populations are composed of primarily one (or a few) clonal genotype(s) shaped by processes of asexual proliferation, competition and winnowing. Secondary uptake of Symbiodinium cells, mutations and recombination, and environmental change result in the presence of additional Symbiodinium genotypes within a host individual. Regardless of the role diverse Symbiodinium communities play for hosts, there is enormous evolutionary potential for local adaptation in these symbiont populations themselves. We next examine patterns of connectivity among Symbiodinium populations in this context. Symbiodinium population structure among reefs and regions Vertical symbiont transmission The most direct way Symbiodinium can associate with new generations of hosts is by being vertically transmitted from an individual to its sexual offspring (i.e. within eggs or larvae) or clonal propagules (i.e. tissue fragments arising from host damage or fission). This strategy ensures host offspring are provisioned with a compatible Symbiodinium species with immediate autotrophic nutritional benefit (Douglas 1998). Such transmission in host tissues appears to convey Symbiodinium populations with substantially greater dispersal potential and connectivity among reef environments when compared to horizontal transmission. For instance, genetic structure for the scleractinian coral Pocillopora sp. type 1 and its maternally transmitted Symbiodinium type D1 were correlated in the Eastern Tropical Pacific, with host corals and their symbionts forming single, undifferentiated populations throughout thousands of square kilometres (excluding the Gulf of California; Pettay et al. 2013). Elsewhere in the South Pacific, a strong relationship was detected between island groups for the genetic structure of Pocillopora meandrina and its maternally inherited Symbiodinium type C1 populations (Magalon et al. 2005, 2006). The occurrence of the same symbiont genotype across ocean basins has also been documented for the anemone Exaiptasia pallida (formerly Aiptasia pallida; Grajales & Rodriquez 2014), with an identical Symbiodinium minutum (within type B1) genotype recovered from Japan, Hawaii and Mexico (Thornhill et al. 2013). As this host is considered weedy and primarily reproduces via asexual fragmentation of pedal tissue with vertical inheritance of symbionts, anthropogenic vectoring likely explains this homogeneous distribution across over km (Thornhill et al. 2013). Horizontal symbiont transmission While vertical Symbiodinium transmission guarantees successful establishment of a symbiosis in offspring, a majority of hosts, including 75% of scleractinian corals, acquire their dinoflagellate symbionts from the environment anew at each generation (Baird et al. 2009). 2 This strategy likely allows hosts to form partnerships with Symbiodinium genotypes that are best suited to the environmental conditions (Douglas 1998; Howells et al. 2012). In these cases, patterns of genetic structure are remarkably consistent, with significant differentiation between populations detected at distances as low as 10s of kilometres regardless of oceanic basin, host species or Symbiodinium type (Table 1). In general, the connectivity among horizontally transmitted Symbiodinium populations is low when compared to scleractinian coral hosts (e.g. Thornhill et al. 2009, 2014; Foster et al. 2012), implying population structure is decoupled among the partners. For instance, Baums et al. (2014) inferred an order of magnitude difference in gene flow between populations of the Caribbean coral Acropora palmata and its symbionts belonging to Symbiodinium type A3. In this case, the range wide distribution of A. palmata resolved to just two populations, one each in the eastern and western Caribbean Sea, respectively (Baums et al. 2005, 2014). While eastern and western subdivisions also occurred for Symbiodinium type A3, each could be further subdivided into as many as seven regionally distinct populations (Baums et al. 2014). Similarly, there was no correlation between the genetic structure of the soft coral host Sinularia flexibilis and its horizontally acquired Symbiodinium type C1:3a (sensu 2 Whereas vertical and horizontal transmissions are generally considered to be a strict dichotomy in corals, recent research has demonstrated that some vertically transmitting corals also acquire symbionts from the environment (Byler et al. 2013; Boulotte et al. 2016).

11 2650 D. J. THORNHILL ET AL. Goulet et al. 2008) populations along the Great Barrier Reef (Bastidas et al. 2001; Howells et al. 2009). To date, the most commonly offered explanation for the highly structured nature of many horizontally transmitted Symbiodinium populations is limited dispersal and recruitment potential (e.g. Santos et al. 2003; Howells et al. 2009; Thornhill et al. 2009), with microevolutionary forces such as genetic drift and mutation leading to divergence among populations over time. Assuming the likelihood that a competent host acquires a particular symbiont genotype is proportional to that genotype s relative abundance in the environment, resident Symbiodinium would frequently outcompete any migrants (a density-dependent barrier, hypothesized in Thornhill et al. 2009) when it comes to establishing relationships with new host individuals. Consequently, the high F ST values observed between these Symbiodinium populations may reflect the continual release of resident cells into the external environment vastly outnumbering the influx of immigrant cells, whose abundances are predicted to be low due to limited migration capacity. Along with this, we discuss below alternative hypotheses that could also drive horizontally transmitted Symbiodinium populations to be highly structured. Reanalysis of horizontal symbiont transmission To further explore the high prevalence of, and the extreme structure associated with, horizontally transmitted Symbiodinium populations, we examined the relationship between genetic and geographic distances of the Caribbean-wide data from Symbiodinium type A3 (Fig. 4a; Appendix S1; data from Baums et al. 2014). After taking into account the strong genetic structure (a) (b) Fig. 4 Relationship between genetic distance and geographic distance in Symbiodinium type A3 based on Baums et al. (2014). Black data points are pairwise comparisons between locations, with colour shading representing inferred data point density. Dotted lines represent statistically significant linear regressions of the data. Panel (a): Nei s genetic distance and distance in km with data grouped by collection site from Baums et al. (2014). Note the mean genetic distance does not increase with distance. Panel (b): The absolute value of the residuals from the linear model of Panel (a). Note the data show a very weak negative relationship between variance in pairwise genetic distance and geographic distance, rather than the expected positive relationship, suggesting that phenomena other than isolation by distance may be shaping patterns of genetic divergence between populations.

12 SYMBIODINIUM POPULATION GENETICS 2651 between the eastern and western Caribbean Sea (see Baums et al. 2014), there is no significant relationship between genetic and geographic distances (Mantel test; P = 0.98; Fig. 4a), implying a surprising absence of isolation by distance (IBD) in the data. Instead, a weak but significant negative correlation (P < 0.01) between the variance in genetic distance among reefs and their geographic distance is apparent (Fig. 4b). Although this weak negative relationship might be explained in part by the bias towards short distance comparisons in the data set, it is clear that the relationship between the variance in genetic distance and geographic distance is not positive. This is unexpected as the potential for genetic barriers that may affect dispersal patterns should increase or compound over geographic distance, thus reflecting a positive (rather than negative) relationship between the variance in genetic and geographic distances. Taken together, how can populations separated by only 10s of km exhibit genetic differentiation that is equal to or even sometimes exceeds that for populations separated by 1000s of km? Although there is evidence for IBD playing a role at large scales (Andras et al. 2011), the analyses presented here suggest that a model based solely on IBD through limited dispersal is insufficient for explaining the genetic differentiation among these Symbiodinium populations at small scales (10s of km). In the following sections, we discuss biological processes that might contribute to this pattern. Influence of variance in propagule number on Symbiodinium population structure Rapid asexual proliferation in an organism s life cycle produces populations of individuals that are more related to one another than predicted by the Wright Fisher model (Fisher 1930; Wright 1931) often used to interpret population genetic data. Such populations, dominated by clonal individuals, can exhibit elevated rates of drift and selection (Chang et al. 2013), lower effective to census population size ratios (Hedrick 2005; Eldon & Wakeley 2006) and high estimates of differentiation (i.e. F ST ) despite frequent migration (Eldon & Wakeley 2009). For Symbiodinium, infection of a host and successful establishment of the symbiotic relationship likely facilitates the rapid replication of particular symbiont genotypes that subsequently contribute disproportionately to the gene pool capable of infecting future host individuals. This situation would, in turn, generate patterns of high relatedness on a reef over relatively short timescales and result in significant differences in genetic composition between reefs even if migration rates are high (Eldon & Wakeley 2009). To demonstrate this, we utilized model simulations to depict a series of two populations in which the number of individuals and migration rate are both held constant while changing only the variance in number of clonal propagules between simulations (Fig. 5). When individuals produce similar numbers of clonal daughter cells, the effective population size, N e, remains large and with little genetic differentiation between populations, as measured by F ST. However, increased variation in proliferation success among individuals results in a decline of N e and an increase in F ST, with even modest biases in proliferation success having dramatic effects on inferred genetic patterns. Thus, significant genetic differentiation can occur between clonal populations even when migration rates are high (Hedrick 2005, Eldon & Wakeley 2009). This implies that estimates of F ST and its various derivatives as indicators of population differentiation and migration rate for Symbiodinium may not be appropriate at small oceanographic scales (i.e. <100 km). While the presented models represent a fairly simple exercise, their results and possibilities warrant further consideration as explanations for population genetic patterns in future Symbiodinium studies. Selection and local adaptation of Symbiodinium populations For many horizontally transmitted Symbiodinium populations, migration alone may be insufficient to counteract the effects of genetic drift or natural selection. In the case of drift, population allele frequencies are expected to change randomly with time due to stochastic sampling at each generation. Selection, in contrast, might Effective population size (inferred N e ) F st vs. offspring variance inferred N e vs. offspring variance Variance in number of asexual propagules Fig. 5 Results of simulations of two Symbiodinium populations of individuals with a fixed migration rate but varying offspring number across individuals. Note the increase in F ST and decrease in N e with increasing variance in number of asexual propagules even though migration rate and census size are modelled as constant. Genetic differentiation (F st )