Range‐wide population genetic structure of the Caribbean sea fan coral,Gorgonia ventalina

The population structure of benthic marine organisms is of central relevance to the conservation and management of these often threatened species, as well as to the accurate understanding of their ecological and evolutionary dynamics. A growing body of evidence suggests that marine populations can be structured over short distances despite theoretically high dispersal potential. Yet the proposed mechanisms governing this structure vary, and existing empirical population genetic evidence is of insufficient taxonomic and geographic scope to allow for strong general inferences. Here, we describe the range‐wide population genetic structure of an ecologically important Caribbean octocoral, Gorgonia ventalina. Genetic differentiation was positively correlated with geographic distance and negatively correlated with oceanographically modelled dispersal probability throughout the range. Although we observed admixture across hundreds of kilometres, estimated dispersal was low, and populations were differentiated across distances <2 km. These results suggest that populations of G. ventalina may be evolutionarily coupled via gene flow but are largely demographically independent. Observed patterns of differentiation corroborate biogeographic breaks found in other taxa (e.g. an east/west divide near Puerto Rico), and also identify population divides not discussed in previous studies (e.g. the Yucatan Channel). High genotypic diversity and absence of clonemates indicate that sex is the primary reproductive mode for G. ventalina. A comparative analysis of the population structure of G. ventalina and its dinoflagellate symbiont, Symbiodinium, indicates that the dispersal of these symbiotic partners is not coupled, and symbiont transmission occurs horizontally.     

Spatial and temporal genetic structure of Symbiodinium populations within a common reef‐building coral on the Great Barrier Reef

The dinoflagellate photosymbiont Symbiodinium plays a fundamental role in defining the physiological tolerances of coral holobionts, but little is known about the dynamics of these endosymbiotic populations on coral reefs. Sparse data indicate that Symbiodinium populations show limited spatial connectivity; however, no studies have investigated temporal dynamics for in hospite Symbiodinium populations following significant mortality and recruitment events in coral populations. We investigated the combined influences of spatial isolation and disturbance on the population dynamics of the generalist Symbiodinium type C2 (ITS1 rDNA) hosted by the scleractinian coral Acropora millepora in the central Great Barrier Reef. Using eight microsatellite markers, we genotyped Symbiodinium in a total of 401 coral colonies, which were sampled from seven sites across a 12‐year period including during flood plume–induced coral bleaching. Genetic differentiation of Symbiodinium was greatest within sites, explaining 70–86% of the total genetic variation. An additional 9–27% of variation was explained by significant differentiation of populations among sites separated by 0.4–13 km, which is consistent with low levels of dispersal via water movement and historical disturbance regimes. Sampling year accounted for 6–7% of total genetic variation and was related to significant coral mortality following severe bleaching in 1998 and a cyclone in 2006. Only 3% of the total genetic variation was related to coral bleaching status, reflecting generally small (8%) reductions in allelic diversity within bleached corals. This reduction probably reflected a loss of genotypes in hospite during bleaching, although no site‐wide changes in genetic diversity were observed. Combined, our results indicate the importance of disturbance regimes acting together with limited oceanographic transport to determine the genetic composition of Symbiodinium types within reefs.     

Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly‐isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral‐associated Symbiodinium had higher protein content than did cultured and sea anemone‐associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone‐associated Symbiodinium had a distinct pattern compared coral‐associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral‐associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts.     

Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing

Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000‐fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral‐algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine‐scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral‐algal symbiosis.     

Symbiodinium population genetics: testing for species boundaries and analysing samples with mixed genotypes

Population genetic markers are increasingly being used to study the diversity, ecology and evolution of Symbiodinium, a group of eukaryotic microbes that are often mutualistic with reef‐building corals. Population genetic markers can resolve individual clones, or strains, from samples of host tissue; however, samples may comprise different species that may confound interpretations of gene flow and genetic structure. Here, we propose a method for resolving species from population genetic data using tests for genetic recombination. Assigning individuals to genetically recombining populations prior to further analyses avoids critical errors in the interpretation of gene flow and dispersal. To demonstrate the effectiveness of the approach, we first apply this method to a simulated data set. We then use the method to resolve two species of host generalist Symbiodinium that commonly co‐occur in reef‐building corals collected from Indo‐West Pacific reefs. We demonstrate that the method is robust even when some hosts contain genotypes from two distinct species. Finally, we examine population genetic data sets from two recently published papers in Molecular Ecology. We show that each strongly supports a two species interpretation, which significantly changes the original conclusions presented in these studies. When combined with available phylogenetic and ecological evidence, the use of population genetic data offers a robust method for unambiguously delimiting morphologically cryptic species.     

Regional‐scale dominance of non‐framework building corals on Caribbean reefs affects carbonate production and future reef growth

Coral cover on Caribbean reefs has declined rapidly since the early 1980's. Diseases have been a major driver, decimating communities of framework building Acropora and Orbicella coral species, and reportedly leading to the emergence of novel coral assemblages often dominated by domed and plating species of the genera Agaricia, Porites and Siderastrea. These corals were not historically important Caribbean framework builders, and typically have much smaller stature and lower calcification rates, fuelling concerns over reef carbonate production and growth potential. Using data from 75 reefs from across the Caribbean we quantify: (i) the magnitude of non‐framework building coral dominance throughout the region and (ii) the contribution of these corals to contemporary carbonate production. Our data show that live coral cover averages 18.2% across our sites and coral carbonate production 4.1 kg CaCO₃ m^?2 yr^?1. However, non‐framework building coral species dominate and are major carbonate producers at a high proportion of sites; they are more abundant than Acropora and Orbicella at 73% of sites; contribute an average 68% of the carbonate produced; and produce more than half the carbonate at 79% of sites. Coral cover and carbonate production rate are strongly correlated but, as relative abundance of non‐framework building corals increases, average carbonate production rates decline. Consequently, the use of coral cover as a predictor of carbonate budget status, without species level production rate data, needs to be treated with caution. Our findings provide compelling evidence for the Caribbean‐wide dominance of non‐framework building coral taxa, and that these species are now major regional carbonate producers. However, because these species typically have lower calcification rates, continued transitions to states dominated by non‐framework building coral species will further reduce carbonate production rates below ‘predecline’ levels, resulting in shifts towards negative carbonate budget states and reducing reef growth potential.     

Unraveling hierarchical genetic structure in a marine metapopulation: A comparison of three high‐throughput genotyping approaches

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high‐throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high‐resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high‐throughput genotyping data can elucidate subtle genetic structure at previously‐undetected scales in a dispersive marine fish.     

Microsatellite allele sizes alone are insufficient to delineate species boundaries in Symbiodinium

Symbiodinium are a diverse group of unicellular dinoflagellates that are important nutritional symbionts of reef‐building corals. Symbiodinium putative species (‘types’) are commonly identified with genetic markers, mostly nuclear and chloroplast encoded ribosomal DNA regions. Population genetic analyses using microsatellite loci have provided insights into Symbiodinium biogeography, connectivity and phenotypic plasticity, but are complicated by: (i) a lack of consensus criteria used to delineate inter‐ vs. intragenomic variation within species; and (ii) the high density of Symbiodinium in host tissues, which results in single samples comprising thousands of individuals. To address this problem, Wham & LaJeunesse (2016) present a method for identifying cryptic Symbiodinium species from microsatellite data based on correlations between allele size distributions and nongeographic genetic structure. Multilocus genotypes that potentially do not recombine in sympatry are interpreted as secondary ‘species’ to be discarded from downstream population genetic analyses. However, Symbiodinium species delineations should ideally incorporate multiple physiological, ecological and molecular criteria. This is because recombination tests may be a poor indicator of species boundaries in Symbiodinium due to their predominantly asexual mode of reproduction. Furthermore, discontinuous microsatellite allele sizes in sympatry may be explained by secondary contact between previously isolated populations and by mutations that occur in a nonstepwise manner. Limitations of using microsatellites alone to delineate species are highlighted in earlier studies that demonstrate occasional bimodal distributions of allele sizes within Symbiodinium species and considerable allele size sharing among Symbiodinium species. We outline these issues and discuss the validity of reinterpretations of our previously published microsatellite data from Symbiodinium populations on the Great Barrier Reef (Howells et al. 2013).     

Symbiodinium (Dinophyceae) diversity in reef‐invertebrates along an offshore to inshore reef gradient near Lizard Island,Great Barrier Reef

Despite extensive work on the genetic diversity of reef invertebrate‐dinoflagellate symbioses on the Great Barrier Reef (GBR; Australia), large information gaps exist from northern and inshore regions. Therefore, a broad survey was done comparing the community of inshore, mid‐shelf and outer reefs at the latitude of Lizard Island. Symbiodinium (Freudenthal) diversity was characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Thirty‐nine distinct Symbiodinium types were identified from four subgeneric clades (B, C, D, and G). Several Symbiodinium types originally characterized from the Indian Ocean were discovered as well as eight novel types (C1kk, C1LL, C3nn, C26b, C161a, C162, C165, C166). Multivariate analyses on the Symbiodinium species diversity data showed a strong link with host identity, consistent with previous findings. Of the four environmental variables tested, mean austral winter sea surface temperature (SST) influenced Symbiodinium distribution across shelves most significantly. A similar result was found when the analysis was performed on Symbiodinium diversity data of genera with an open symbiont transmission mode separately with chl a and PAR explaining additional variation. This study underscores the importance of SST and water quality related variables as factors driving Symbiodinium distribution on cross‐shelf scales. Furthermore, this study expands our knowledge on Symbiodinium species diversity, ecological partitioning (including host‐specificity) and geographic ranges across the GBR. The accelerating rate of environmental change experienced by coral reef ecosystems emphasizes the need to comprehend the full complexity of cnidarian symbioses, including the biotic and abiotic factors that shape their current distributions.     

Symbiont physiology and population dynamics before and during symbiont shifts in a flexible algal‐cnidarian symbiosis

For cnidarians that can undergo shifts in algal symbiont relative abundance, the underlying algal physiological changes that accompany these shifts are not well known. The sea anemone Anthopleura elegantissima associates with the dinoflagellate Symbiodinium muscatinei and the chlorophyte Elliptochloris marina, symbionts with very different tolerances to light and temperature. We compared the performance of these symbionts in anemones maintained in an 8–11.5 month outdoor common garden experiment with simulated intertidal conditions and three levels of shading (2, 43, and 85% ambient irradiance). Symbiont densities, mitotic indices, photophysiology and pigments were assessed at three time points during the summer, a period of high irradiance and solar heating during aerial exposure. Whereas S. muscatinei was either neutrally or positively affected by higher irradiance treatments, E. marina responded mostly negatively to high irradiance. E. marina in the 85% irradiance treatment exhibited significantly reduced P_max and chlorophyll early in the summer, but it was not until nearly 3 months later that a shift in symbiont relative abundance toward S. muscatinei occurred, coincident with bleaching. Symbiont densities and proportions remained largely stable in all other treatments over time, and displacement of S. muscatinei by E. marina was not observed in the 2% irradiance treatment despite the potentially better performance of E. marina. While our results support the view that rapid changes in symbiont relative abundance are typically associated with symbiont physiological dysfunction and bleaching, they also show that significant temporal lags may occur between the onset of symbiont stress and shifts in symbiont relative abundances.     

Vectored dispersal of Symbiodinium by larvae of a Caribbean gorgonian octocoral

The ability of coral reefs to recover from natural and anthropogenic disturbance is difficult to predict, in part due to uncertainty regarding the dispersal capabilities and connectivity of their reef inhabitants. We developed microsatellite markers for the broadcast spawning gorgonian octocoral Eunicea (Plexaura) flexuosa (four markers) and its dinoflagellate symbiont, Symbiodinium B1 (five markers), and used them to assess genetic connectivity, specificity and directionality of gene flow among sites in Florida, Panama, Saba and the Dominican Republic. Bayesian analyses found that most E. flexuosa from the Florida reef tract, Saba and the Dominican Republic were strongly differentiated from many E. flexuosa in Panama, with the exception of five colonies from Key West that clustered with colonies from Panama. In contrast, Symbiodinium B1 was more highly structured. At least seven populations were detected that showed patterns of isolation by distance. The symbionts in the five unusual Key West colonies also clustered with symbionts from Panama, suggesting these colonies are the result of long‐distance dispersal. Migration rate tests indicated a weak signal of northward immigration from the Panama population into the lower Florida Keys. As E. flexuosa clonemates only rarely associated with the same Symbiodinium B1 genotype (and vice versa), these data suggest a dynamic host–symbiont relationship in which E. flexuosa is relatively well dispersed but likely acquires Symbiodinium B1 from highly structured natal areas prior to dispersal. Once vectored by host larvae, these symbionts may then spread through the local population, and/or host colonies may acquire different local symbiont genotypes over time.     

The distribution of the thermally tolerant symbiont lineage (Symbiodinium clade D) in corals from Hawaii: correlations with host and the history of ocean thermal stress

Spatially intimate symbioses, such as those between scleractinian corals and unicellular algae belonging to the genus Symbiodinium, can potentially adapt to changes in the environment by altering the taxonomic composition of their endosymbiont communities. We quantified the spatial relationship between the cumulative frequency of thermal stress anomalies (TSAs) and the taxonomic composition of Symbiodinium in the corals Montipora capitata, Porites lobata, and Porites compressa across the Hawaiian archipelago. Specifically, we investigated whether thermally tolerant clade D Symbiodinium was in greater abundance in corals from sites with high frequencies of TSAs. We recovered 2305 Symbiodinium ITS2 sequences from 242 coral colonies in lagoonal reef habitats at Pearl and Hermes Atoll, French Frigate Shoals, and Kaneohe Bay, Oahu in 2007. Sequences were grouped into 26 operational taxonomic units (OTUs) with 12 OTUs associated with Montipora and 21 with Porites. Both coral genera associated with Symbiodinium in clade C, and these co‐occurred with clade D in M. capitata and clade G in P. lobata. The latter represents the first report of clade G Symbiodinium in P. lobata. In M. capitata (but not Porites spp.), there was a significant correlation between the presence of Symbiodinium in clade D and a thermal history characterized by high cumulative frequency of TSAs. The endogenous community composition of Symbiodinium and an association with clade D symbionts after long‐term thermal disturbance appear strongly dependent on the taxa of the coral host.     

Symbiodinium (Dinophyceae) community patterns in invertebrate hosts from inshore marginal reefs of the southern Great Barrier Reef,Australia

The broad range in physiological variation displayed by Symbiodinium spp. has proven imperative during periods of environmental change and contribute to the survival of their coral host. Characterizing how host and Symbiodinium community assemblages differ across environmentally distinct habitats provides useful information to predict how corals will respond to major environmental change. Despite the extensive characterizations of Symbiodinium diversity found amongst reef cnidarians on the Great Barrier Reef (GBR) substantial biogeographic gaps exist, especially across inshore habitats. Here, we investigate Symbiodinium community patterns in invertebrates from inshore and mid‐shelf reefs on the southern GBR, Australia. Dominant Symbiodinium types were characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Twenty one genetically distinct Symbiodinium types including four novel types were identified from 321 reef‐invertebrate samples comprising three sub‐generic clades (A, C, and D). A range of host genera harbored C22a, which is normally rare or absent from inshore or low latitude reefs in the GBR. Multivariate analysis showed that host identity and sea surface temperature best explained the variation in symbiont communities across sites. Patterns of changes in Symbiodinium community assemblage over small geographic distances (100s of kilometers or less) indicate the likelihood that shifts in Symbiodinium distributions and associated host populations, may occur in response to future climate change impacting the GBR.     

Unexpected cryptic species diversity in the widespread coral Seriatopora hystrix masks spatial‐genetic patterns of connectivity

Mounting evidence of cryptic species in a wide range of taxa highlights the need for careful analyses of population genetic data sets to unravel within‐species diversity from potential interspecies relationships. Here, we use microsatellite loci and hierarchical clustering analysis to investigate cryptic diversity in sympatric and allopatric (separated by 450 km) populations of the widespread coral Seriatopora hystrix on the Great Barrier Reef. Structure analyses delimited unique genetic clusters that were confirmed by phylogenetic and extensive population‐level analyses. Each of four sympatric yet distinct genetic clusters detected within S. hystrix demonstrated greater genetic cohesion across regional scales than between genetic clusters within regions (<10 km). Moreover, the magnitude of genetic differentiation between different clusters (>0.620 G”_ST) was similar to the difference between S. hystrix clusters and the congener S. caliendrum (mean G”_ST 0.720). Multiple lines of evidence, including differences in habitat specificity, mitochondrial identity, Symbiodinium associations and morphology, corroborate the nuclear genetic evidence that these distinct clusters constitute different species. Hierarchical clustering analysis combined with more traditional population genetic methods provides a powerful approach for delimiting species and should be regularly applied to ensure that ecological and evolutionary patterns interpreted for single species are not confounded by the presence of cryptic species.     

Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins

The Aiptasia–Symbiodinium symbiosis is a promising model for experimental studies of cnidarian–dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re‐evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2‐DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine‐scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular‐genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host–symbiont specificity and widely distributed populations in an important model system.