Thermotolerant coral-algal mutualisms maintain high rates of nutrient transfer while exposed to heat stress.

Symbiotic mutualisms are essential to ecosystems and numerous species across the tree of life. For reef-building corals, the benefits of their association with endosymbiotic dinoflagellates differ within and across taxa, and nutrient exchange between these partners is influenced by environmental conditions. Furthermore, it is widely assumed that corals associated with symbionts in the genus Durusdinium tolerate high thermal stress at the expense of lower nutrient exchange to support coral growth. We traced both inorganic carbon (H13CO3-) and nitrate (15NO3-) uptake by divergent symbiont species and quantified nutrient transfer to the host coral under normal temperatures as well as in colonies exposed to high thermal stress. Colonies representative of diverse coral taxa associated with Durusdinium trenchii or Cladocopium spp. exhibited similar nutrient exchange under ambient conditions. By contrast, heat-exposed colonies with D. trenchii experienced less physiological stress than conspecifics with Cladocopium spp. while high carbon assimilation and nutrient transfer to the host was maintained. This discovery differs from the prevailing notion that these mutualisms inevitably suffer trade-offs in physiological performance. These findings emphasize that many host-symbiont combinations adapted to high-temperature equatorial environments are high-functioning mutualisms; and why their increased prevalence is likely to be important to the future productivity and stability of coral reef ecosystems.

High physiological function for corals with thermally tolerant, host-adapted symbionts.

The flexibility to associate with more than one symbiont may considerably expand a host's niche breadth. Coral animals and dinoflagellate micro-algae represent one of the most functionally integrated and widespread mutualisms between two eukaryotic partners. Symbiont identity greatly affects a coral's ability to cope with extremes in temperature and light. Over its broad distribution across the Eastern Pacific, the ecologically dominant branching coral, Pocillopora grandis, depends on mutualisms with the dinoflagellates Durusdinium glynnii and Cladocopium latusorum. Measurements of skeletal growth, calcification rates, total mass increase, calyx dimensions, reproductive output and response to thermal stress were used to assess the functional performance of these partner combinations. The results show both host-symbiont combinations displayed similar phenotypes; however, significant functional differences emerged when exposed to increased temperatures. Negligible physiological differences in colonies hosting the more thermally tolerant D. glynnii refute the prevailing view that these mutualisms have considerable growth tradeoffs. Well beyond the Eastern Pacific, pocilloporid colonies with D. glynnii are found across the Pacific in warm, environmentally variable, near shore lagoonal habitats. While rising ocean temperatures threaten the persistence of contemporary coral reefs, lessons from the Eastern Pacific indicate that co-evolved thermally tolerant host-symbiont combinations are likely to expand ecologically and spread geographically to dominate reef ecosystems in the future.

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Formal recognition of host-generalist species of dinoflagellate (Cladocopium, Symbiodiniaceae) mutualistic with Indo-Pacific reef corals.

The existence of widespread species with the capacity to endure diverse, or variable, environments are of importance to ecological and genetic research, and conservation. Such "ecological generalists" are more likely to have key adaptations that allow them to better tolerate the physiological challenges of rapid climate change. Reef-building corals are dependent on endosymbiotic dinoflagellates (Family: Symbiodiniaceae) for their survival and growth. While these symbionts are biologically diverse, certain genetic types appear to have broad geographic distributions and are mutualistic with various host species from multiple genera and families in the order Scleractinia that must acquire their symbionts through horizontal transmission. Despite the considerable ecological importance of putative host-generalist symbionts, they lack formal species descriptions. In this study, we used molecular, ecological, and morphological evidence to verify the existence of five new host-generalist species in the symbiodiniacean genus Cladocopium. Their geographic distribution and prevalence among host communities corresponds to prevailing environmental conditions at both regional and local scales. The influence that each species has on host physiology may partially explain regional differences in thermal sensitivities among coral communities. The potential increased prevalence of a generalist species that endures environmental instability is a consequential ecological response to warming oceans. Large-scale shifts in symbiont dominance could ensure reef coral persistence and productivity in the near term. Ultimately, these formal designations should advance scientific communication and generate informed research questions on the physiology and ecology of coral-dinoflagellate mutualisms.

Acclimatization of a coral-dinoflagellate mutualism at a CO2 vent.

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral-dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios.

Similarities in biomass and energy reserves among coral colonies from contrasting reef environments.

Coral reefs are declining worldwide, yet some coral populations are better adapted to withstand reductions in pH and the rising frequency of marine heatwaves. The nearshore reef habitats of Palau, Micronesia are a proxy for a future of warmer, more acidic oceans. Coral populations in these habitats can resist, and recover from, episodes of thermal stress better than offshore conspecifics. To explore the physiological basis of this tolerance, we compared tissue biomass (ash-free dry weight cm-2), energy reserves (i.e., protein, total lipid, carbohydrate content), and several important lipid classes in six coral species living in both offshore and nearshore environments. In contrast to expectations, a trend emerged of many nearshore colonies exhibiting lower biomass and energy reserves than colonies from offshore sites, which may be explained by the increased metabolic demand of living in a warmer, acidic, environment. Despite hosting different dinoflagellate symbiont species and having access to contrasting prey abundances, total lipid and lipid class compositions were similar in colonies from each habitat. Ultimately, while the regulation of colony biomass and energy reserves may be influenced by factors, including the identity of the resident symbiont, kind of food consumed, and host genetic attributes, these independent processes converged to a similar homeostatic set point under different environmental conditions.

Mutualistic microalgae co-diversify with reef corals that acquire symbionts during egg development.

The application of molecular genetics has reinvigorated and improved how species are defined and investigated scientifically, especially for morphologically cryptic micro-organisms. Here we show how species recognition improves understanding of the ecology and evolution of mutualisms between reef-building corals and their mutualistic dinoflagellates (i.e. Symbiodiniaceae). A combination of genetic, ecological, and morphological evidence defines two sibling species of Cladocopium (formerly Symbiodinium Clade C), specific only to host corals in the common genus Pocillopora, which transmit their obligate symbionts during oogenesis. Cladocopium latusorum sp. nov. is symbiotic with P. grandis/meandrina while the smaller-celled C. pacificum sp. nov. associates with P. verrucosa. Both symbiont species form mutualisms with Pocillopora that brood their young. Populations of each species, like their hosts, are genetically well connected across the tropical and subtropical Pacific Ocean, indicating a capacity for long-range dispersal. A molecular clock approximates their speciation during the late Pliocene or early Pleistocene as Earth underwent cycles of precipitous cooling and warming; and corresponds to when their hosts were also diversifying. The long temporal and spatial maintenance of high host fidelity, as well as genetic connectivity across thousands of kilometers, indicates that distinct ecological attributes and close evolutionary histories will restrain the adaptive responses of corals and their specialized symbionts to rapid climate warming.

Miliolidium n. gen, a New Symbiodiniacean Genus Whose Members Associate with Soritid Foraminifera or Are Free-Living.

The dinoflagellate family Symbiodiniaceae comprises numerous divergent genera containing species whose ecologies range from endosymbiotic to free-living. While many associate with invertebrates including corals, sea anemones, jellyfish, giant clams, and flatworms, others occur within the cytoplasm of large protists, most notably benthic foraminifera in the sub-family Soritinae. Recent systematic revisions to the Symbiodiniaceae left out formal naming of some divergent lineages because each lacked a representative type species to erect new genus names. Here we provide genetic, morphological and ecological evidence to describe a new genus and species. Miliolidium n. gen. is closely related to the genus Durusdinium and contains several genetically divergent ecologically distinct lineages found in distant geographic locations indicating an Indo-Pacific wide distribution. One of these, Miliolidium leei n. sp., is represented by an isolate cultured from Amphisorus sp. originally collected in the Gulf of Eilat, northern Red Sea. Its peripheral chloroplast extensions are uniquely petal- or lobe-shaped, and cells possess a pyrenoid with three stalks connecting to chloroplasts, and without thylakoid intrusions. It is related to an isolate cultured from an azooxanthellate sponge from Palau and another that is commonly harbored by the soritid Marginopora vertebralis in shallow reef habitats from Guam. Research on Symbiodiniaceae diversity including free-living species in benthic habitats and those mutualistic with soritid foraminifera remains extremely limited as does our knowledge of their diversity, physiology, biogeography, and ecology.

Iron Availability Modulates the Response of Endosymbiotic Dinoflagellates to Heat Stress.

Warming and nutrient limitation are stressors known to weaken the health of microalgae. In situations of stress, access to energy reserves can minimize physiological damage. Because of its widespread requirements in biochemical processes, iron is an important trace metal, especially for photosynthetic organisms. Lowered iron availability in oceans experiencing rising temperatures may contribute to the thermal sensitivity of reef-building corals, which rely on mutualisms with dinoflagellates to survive. To test the influence of iron concentration on thermal sensitivity, the physiological responses of cultured symbiotic dinoflagellates (genus Breviolum; family Symbiodiniaceae) were evaluated when exposed to increasing temperatures (26 to 30°C) and iron concentrations ranging from replete (500 pM Fe') to limiting (50 pM Fe') under a diurnal light cycle with saturating radiance. Declines in photosynthetic efficiency at elevated temperatures indicated sensitivity to heat stress. Furthermore, five times the amount of iron was needed to reach exponential growth during heat stress (50 pM Fe' at 26-28°C vs. 250 pM Fe' at 30°C). In treatments where exponential growth was reached, Breviolum psygmophilum grew faster than B.minutum, possibly due to greater cellular contents of iron and other trace metals. The metal composition of B.psygmophilum shifted only at the highest temperature (30°C), whereas changes in B.minutum were observed at lower temperatures (28°C). The influence of iron availability in modulating each alga's response to thermal stress suggests the importance of trace metals to the health of coral-algal mutualisms. Ultimately, a greater ability to acquire scarce metals may improve the tolerance of corals to physiological stressors and contribute to the differences in performance associated with hosting one symbiont species over another.

Zooxanthellae.

LaJeunesse introduces the group of unicellular microalgae referred to as 'zooxanthellae'.

Host-symbiont combinations dictate the photo-physiological response of reef-building corals to thermal stress.

High sea surface temperatures often lead to coral bleaching wherein reef-building corals lose significant numbers of their endosymbiotic dinoflagellates (Symbiodiniaceae). These increasingly frequent bleaching events often result in large scale coral mortality, thereby devasting reef systems throughout the world. The reef habitats surrounding Palau are ideal for investigating coral responses to climate perturbation, where many inshore bays are subject to higher water temperature as compared with offshore barrier reefs. We examined fourteen physiological traits in response to high temperature across various symbiotic dinoflagellates in four common Pacific coral species, Acropora muricata, Coelastrea aspera, Cyphastrea chalcidicum and Pachyseris rugosa found in both offshore and inshore habitats. Inshore corals were dominated by a single homogenous population of the stress tolerant symbiont Durusdinium trenchii, yet symbiont thermal response and physiology differed significantly across coral species. In contrast, offshore corals harbored specific species of Cladocopium spp. (ITS2 rDNA type-C) yet all experienced similar patterns of photoinactivation and symbiont loss when heated. Additionally, cell volume and light absorption properties increased in heated Cladocopium spp., leading to a greater loss in photo-regulation. While inshore coral temperature response was consistently muted relative to their offshore counterparts, high physiological variability in D. trenchii across inshore corals suggests that bleaching resilience among even the most stress tolerant symbionts is still heavily influenced by their host environment.

Considerations for maximizing the adaptive potential of restored coral populations in the western Atlantic.

Active coral restoration typically involves two interventions: crossing gametes to facilitate sexual larval propagation; and fragmenting, growing, and outplanting adult colonies to enhance asexual propagation. From an evolutionary perspective, the goal of these efforts is to establish self-sustaining, sexually reproducing coral populations that have sufficient genetic and phenotypic variation to adapt to changing environments. Here, we provide concrete guidelines to help restoration practitioners meet this goal for most Caribbean species of interest. To enable the persistence of coral populations exposed to severe selection pressure from many stressors, a mixed provenance strategy is suggested: genetically unique colonies (genets) should be sourced both locally as well as from more distant, environmentally distinct sites. Sourcing three to four genets per reef along environmental gradients should be sufficient to capture a majority of intraspecies genetic diversity. It is best for practitioners to propagate genets with one or more phenotypic traits that are predicted to be valuable in the future, such as low partial mortality, high wound healing rate, high skeletal growth rate, bleaching resilience, infectious disease resilience, and high sexual reproductive output. Some effort should also be reserved for underperforming genets because colonies that grow poorly in nurseries sometimes thrive once returned to the reef and may harbor genetic variants with as yet unrecognized value. Outplants should be clustered in groups of four to six genets to enable successful fertilization upon maturation. Current evidence indicates that translocating genets among distant reefs is unlikely to be problematic from a population genetic perspective but will likely provide substantial adaptive benefits. Similarly, inbreeding depression is not a concern given that current practices only raise first-generation offspring. Thus, proceeding with the proposed management strategies even in the absence of a detailed population genetic analysis of the focal species at sites targeted for restoration is the best course of action. These basic guidelines should help maximize the adaptive potential of reef-building corals facing a rapidly changing environment.

SymPortal: A novel analytical framework and platform for coral algal symbiont next-generation sequencing ITS2 profiling.

We present SymPortal (SymPortal.org), a novel analytical framework and platform for genetically resolving the algal symbionts of reef corals using next-generation sequencing (NGS) data of the ITS2 rDNA. Although the ITS2 marker is widely used to genetically characterize taxa within the family Symbiodiniaceae (formerly the genus Symbiodinium), the multicopy nature of the marker complicates its use. Commonly, the intragenomic diversity resultant from this multicopy nature is collapsed by analytical approaches, thereby focusing on only the most abundant sequences. In contrast, SymPortal employs logic to identify within-sample informative intragenomic sequences, which we have termed 'defining intragenomic variants' (DIVs), to identify ITS2-type profiles representative of putative Symbiodiniaceae taxa. By making use of this intragenomic ITS2 diversity, SymPortal is able to resolve genetic delineations using the ITS2 marker at a level that was previously only possible by using additional genetic markers. We demonstrate this by comparing this novel approach to the most commonly used alternative approach for NGS ITS2 data, the 97% similarity clustering to operational taxonomic units (OTUs). The SymPortal platform accepts NGS raw sequencing data as input to provide an easy-to-use, standardization-enforced, and community-driven framework that integrates with a database to gain resolving power with increased use. We consider that SymPortal, in conjunction with ongoing large-scale sampling and sequencing efforts, should play an instrumental role in making future sampling efforts more comparable and in maximizing their efficacy in working towards the classification of the global Symbiodiniaceae diversity.

New Species of Closely Related Endosymbiotic Dinoflagellates in the Greater Caribbean have Niches Corresponding to Host Coral Phylogeny.

Symbiotic dinoflagellates in the genus Breviolum (formerly Symbiodinium Clade B) dominate coral communities in shallow waters across the Greater Caribbean. While some formally described species exist, mounting genetic, and ecological evidence indicate that numerous more comprise this genus, many of which are closely related. To test this, colonies of common reef-building corals were sampled across a large geographical range. Phylogenetic and population genetic markers then used to examine evolutionary divergence and delineate boundaries of genetic recombination. Three new candidate species were distinguished by fixed differences in nucleotide sequences from nuclear and chloroplast DNA. Population connectivity was evident within each lineage over thousands of kilometers, however, substantial genetic structure persisted between lineages co-occurring within sampling locations, signifying reproductive isolation. While geographically widespread with overlapping distributions, each species is ecologically distinct, exhibiting specific mutualisms with phylogenetically distinct coral hosts. Moreover, significant differences in mean cell sizes provide some morphological evidence substantiating formal species distinctions. In providing evidence that satisfies the biological, phylogenetic, ecological, and morphological species concepts, we classify and formally name Breviolum faviinorum n. sp., primarily associated with Caribbean corals belonging to the Caribbean subfamily Faviinae; B. meandrinium n. sp., associated with corals belonging to the family Meandrinidae; and B. dendrogyrum n. sp., a symbiont harbored exclusively by the threatened coral Dendrogyra cylindrus. These findings support the primary importance of niche diversification (i.e. host habitat) in the speciation of symbiotic dinoflagellates.

Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts.

The advent of molecular data has transformed the science of organizing and studying life on Earth. Genetics-based evidence provides fundamental insights into the diversity, ecology, and origins of many biological systems, including the mutualisms between metazoan hosts and their micro-algal partners. A well-known example is the dinoflagellate endosymbionts ("zooxanthellae") that power the growth of stony corals and coral reef ecosystems. Once assumed to encompass a single panmictic species, genetic evidence has revealed a divergent and rich diversity within the zooxanthella genus Symbiodinium. Despite decades of reporting on the significance of this diversity, the formal systematics of these eukaryotic microbes have not kept pace, and a major revision is long overdue. With the consideration of molecular, morphological, physiological, and ecological data, we propose that evolutionarily divergent Symbiodinium "clades" are equivalent to genera in the family Symbiodiniaceae, and we provide formal descriptions for seven of them. Additionally, we recalibrate the molecular clock for the group and amend the date for the earliest diversification of this family to the middle of the Mesozoic Era (∼160 mya). This timing corresponds with the adaptive radiation of analogs to modern shallow-water stony corals during the Jurassic Period and connects the rise of these symbiotic dinoflagellates with the emergence and evolutionary success of reef-building corals. This improved framework acknowledges the Symbiodiniaceae's long evolutionary history while filling a pronounced taxonomic gap. Its adoption will facilitate scientific dialog and future research on the physiology, ecology, and evolution of these important micro-algae.

Extensive transcriptional variation poses a challenge to thermal stress biomarker development for endangered corals.

As climate changes, sea surface temperature anomalies that negatively impact coral reef organisms continue to increase in frequency and intensity. Yet, despite widespread coral mortality, genetic diversity remains high even in those coral species listed as threatened. While this is good news in many ways, it presents a challenge for the development of biomarkers that can identify resilient or vulnerable genotypes. Taking advantage of three coral restoration nurseries in Florida that serve as long-term common garden experiments, we exposed over 30 genetically distinct Acropora cervicornis colonies to hot and cold temperature shocks seasonally and measured pooled gene expression responses using RNAseq. Targeting a subset of 20 genes, we designed a high-throughput qPCR array to quantify expression in all individuals separately under each treatment with the goal of identifying predictive and/or diagnostic thermal stress biomarkers. We observed extensive transcriptional variation in the population, suggesting abundant raw material is available for adaptation via natural selection. However, this high variation made it difficult to correlate gene expression changes with colony performance metrics such as growth, mortality and bleaching susceptibility. Nevertheless, we identified several promising diagnostic biomarkers for acute thermal stress that may improve coral restoration and climate change mitigation efforts in the future.

Sibling species of mutualistic Symbiodinium clade G from bioeroding sponges in the western Pacific and western Atlantic oceans.

Dinoflagellates in the genus Symbiodinium associate with a broad array of metazoan and protistian hosts. Symbiodinium-based symbioses involving bioeroding sponge hosts have received less attention than those involving popular scleractinian hosts. Certain species of common Cliona harbor high densities of an ecologically restricted group of Symbiodinium, referred to as Clade G. Clade G Symbiodinium are also known to form stable and functionally important associations with Foraminifera and black corals (Antipatharia) Analyses of genetic evidence indicate that Clade G likely comprises several distinct species. Here, we use nucleotide sequence data in combination with ecological and geographic attributes to formally describe Symbiodinium endoclionum sp. nov. obtained from the Pacific boring sponge Cliona orientalis and Symbiodinium spongiolum sp. nov. from the congeneric western Atlantic sponge Cliona varians. These species appear to be part of an adaptive radiation comprising lineages of Clade G specialized to the metazoan phyla Porifera and Cnidaria, which began prior to the separation of the Pacific and Atlantic Oceans.

Validation and description of Symbiodinium microadriaticum, the type species of Symbiodinium (Dinophyta).

It has been 55 years since Hugo Freudenthal described Symbiodinium microadriaticum (Dinophyceae), the type species of this large and important dinoflagellate genus found commonly in mutualistic symbiosis with cnidarians, other invertebrates, and certain protists. However, no type specimen was designated by Freudenthal, thus S. microadriaticum was invalid, as was Symbiodinium and every species subsequently assigned to the genus. The original culture was lost, but since 1979, a different culture, CCMP2464/rt-061, had been considered to represent S. microadriaticum. From this culture, a preserved specimen is herein designated the holotype of S. microadriaticum, validating the binomial and Symbiodinium. All binary designations previously considered to belong in Symbiodinium also are validated herein.

Biogeography and molecular diversity of coral symbionts in the genus Symbiodinium around the Arabian Peninsula.

AIM: Coral reefs rely on the symbiosis between scleractinian corals and intracellular, photosynthetic dinoflagellates of the genus Symbiodinium making the assessment of symbiont diversity critical to our understanding of ecological resilience of these ecosystems. This study characterizes Symbiodinium diversity around the Arabian Peninsula, which contains some of the most thermally diverse and understudied reefs on Earth. LOCATION: Shallow water coral reefs throughout the Red Sea (RS), Sea of Oman (SO), and Persian/Arabian Gulf (PAG). METHODS: Next-generation sequencing of the ITS2 marker gene was used to assess Symbiodinium community composition and diversity comprising 892 samples from 46 hard and soft coral genera. RESULTS: Corals were associated with a large diversity of Symbiodinium, which usually consisted of one or two prevalent symbiont types and many types at low abundance. Symbiodinium communities were strongly structured according to geographical region and to a lesser extent by coral host identity. Overall symbiont communities were composed primarily of species from clade A and C in the RS, clade A, C, and D in the SO, and clade C and D in the PAG, representing a gradual shift from C- to D-dominated coral hosts. The analysis of symbiont diversity in an Operational Taxonomic Unit (OTU)-based framework allowed the identification of differences in symbiont taxon richness over geographical regions and host genera. MAIN CONCLUSIONS: Our study represents a comprehensive overview over biogeography and molecular diversity of Symbiodinium in the Arabian Seas, where coral reefs thrive in one of the most extreme environmental settings on the planet. As such our data will serve as a baseline for further exploration into the effects of environmental change on host-symbiont pairings and the identification and ecological significance of Symbiodinium types from regions already experiencing 'Future Ocean' conditions.

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.