The genome of a giant clam zooxanthella (Cladocopium infistulum) offers few clues to adaptation as an extracellular symbiont with high thermotolerance.

BACKGROUND: Cladocopium infistulum (Symbiodiniaceae) is a dinoflagellate specialized to live in symbiosis with western Pacific giant clams (Tridacnidae). Unlike coral-associated symbionts, which reside within the host cells, C. infistulum inhabits the extracellular spaces of the clam's digestive diverticula. It is phylogenetically basal to a large species complex of stress-tolerant Cladocopium, many of which are associated with important reef-building corals in the genus Porites. This close phylogenetic relationship may explain why C. infistulum exhibits high thermotolerance relative to other tridacnid symbionts. Moreover, past analyses of microsatellite loci indicated that Cladocopium underwent whole-genome duplication prior to the adaptive radiations that led to its present diversity. RESULTS: A draft genome assembly of C. infistulum was produced using long- and short-read sequences to explore the genomic basis for adaptations underlying thermotolerance and extracellular symbiosis among dinoflagellates and to look for evidence of genome duplication. Comparison to three other Cladocopium genomes revealed no obvious over-representation of gene groups or families whose functions would be important for maintaining C. infistulum's unique physiological and ecological properties. Preliminary analyses support the existence of partial or whole-genome duplication among Cladocopium, but additional high-quality genomes are required to substantiate these findings. CONCLUSION: Although this investigation of Cladocopium infistulum revealed no patterns diagnostic of heat tolerance or extracellular symbiosis in terms of overrepresentation of gene functions or genes under selection, it provided a valuable genomic resource for comparative analyses. It also indicates that ecological divergence among Cladocopium species, and potentially among other dinoflagellates, is partially governed by mechanisms other than gene content. Thus, additional high-quality, multiomic data are needed to explore the molecular basis of key phenotypes among symbiotic microalgae.

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.

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.

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.

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.

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.

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.

Microbial invasion of the Caribbean by an Indo-Pacific coral zooxanthella.

Human-induced environmental changes have ushered in the rapid decline of coral reef ecosystems, particularly by disrupting the symbioses between reef-building corals and their photosymbionts. However, escalating stressful conditions enable some symbionts to thrive as opportunists. We present evidence that a stress-tolerant "zooxanthella" from the Indo-Pacific Ocean, Symbiodinium trenchii, has rapidly spread to coral communities across the Greater Caribbean. In marked contrast to populations from the Indo-Pacific, Atlantic populations of S. trenchii contained exceptionally low genetic diversity, including several widespread and genetically similar clones. Colonies with this symbiont tolerate temperatures 1-2 °C higher than other host-symbiont combinations; however, calcification by hosts harboring S. trenchii is reduced by nearly half, compared with those harboring natives, and suggests that these new symbioses are maladapted. Unforeseen opportunism and geographical expansion by invasive mutualistic microbes could profoundly influence the response of reef coral symbioses to major environmental perturbations but may ultimately compromise ecosystem stability and function.