Zoantharian Endosymbiont Community Dynamics During a Stress Event.

Coral reefs are complex ecosystems composed of many interacting species. One ecologically important group consists of zoantharians, which are closely related to reef-building corals. Like corals, zoantharians form mutualistic symbioses with dinoflagellate micro-algae (family Symbiodiniaceae), but their associations remain underexplored. To examine the degree to which zoantharians exhibit altered symbiont dynamics under changing environmental conditions, we reciprocally transplanted colonies of Zoanthus sansibaricus between intertidal (2 m) and subtidal (26 m) depths within a reef in Okinawa, Japan. At this location, Z. sansibaricus can associate with three Symbiodiniaceae species from two genera distributed along a light and depth gradient. We developed species-specific molecular assays and sampled colonies pre- and post-transplantation to analyze symbiont community diversity. Despite large environmental differences across depths, we detected few symbiont compositional changes resulting from transplantation stress. Colonies sourced from the intertidal zone associated with mixtures of a "shallow" Symbiodinium sp. and a "shallow" Cladocopium sp. independent of whether they were transplanted to shallow or deep waters. Colonies sourced from the subtidal zone were dominated by a "deep" Cladocopium sp. regardless of transplant depth. Subtidal colonies brought to shallow depths did not transition to the presumably high-light adapted shallow symbionts present in the new environment, but rather bleached and died. These patterns mirror observations of highly stable coral-algal associations subjected to depth transplantation. Our results indicate that Zoanthus-Symbiodiniaceae symbioses remain stable despite stress, suggesting these important reef community members have relatively low capacity to shuffle to more stress-tolerant micro-algae in response to ongoing climate change.

Host and Symbiont Cell Cycle Coordination Is Mediated by Symbiotic State, Nutrition, and Partner Identity in a Model Cnidarian-Dinoflagellate Symbiosis.

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G2/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

N-Linked Surface Glycan Biosynthesis, Composition, Inhibition, and Function in Cnidarian-Dinoflagellate Symbiosis.

The success of symbioses between cnidarian hosts (e.g., corals and sea anemones) and micro-algal symbionts hinges on the molecular interactions that govern the establishment and maintenance of intracellular mutualisms. As a fundamental component of innate immunity, glycan-lectin interactions impact the onset of marine endosymbioses, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. In this study, we examined the canonical N-glycan biosynthesis pathway in the genome of the dinoflagellate symbiont Breviolum minutum (family Symbiodiniaceae) and found it to be conserved with the exception of the transferase GlcNAc-TII (MGAT2). Using coupled liquid chromatography-mass spectrometry (LC-MS/MS), we characterized the cell surface N-glycan content of B. minutum, providing the first insight into the molecular composition of surface glycans in dinoflagellates. We then used the biosynthesis inhibitors kifunensine and swainsonine to alter the glycan composition of B. minutum. Successful high-mannose enrichment via kifunensine treatment resulted in a significant decrease in colonization of the model sea anemone Aiptasia (Exaiptasia pallida) by B. minutum. Hybrid glycan enrichment via swainsonine treatment, however, could not be confirmed and did not impact colonization. We conclude that functional Golgi processing of N-glycans is critical for maintaining appropriate cell surface glycan composition and for ensuring colonization success by B. minutum.

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.

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.

A preliminary survey of zoantharian endosymbionts shows high genetic variation over small geographic scales on Okinawa-jima Island, Japan.

Symbiotic dinoflagellates (genus Symbiodinium) shape the responses of their host reef organisms to environmental variability and climate change. To date, the biogeography of Symbiodinium has been investigated primarily through phylogenetic analyses of the ribosomal internal transcribed spacer 2 region. Although the marker can approximate species-level diversity, recent work has demonstrated that faster-evolving genes can resolve otherwise hidden species and population lineages, and that this diversity is often distributed over much finer geographical and environmental scales than previously recognized. Here, we use the noncoding region of the chloroplast psbA gene (psbAncr) to examine genetic diversity among clade C Symbiodinium associating with the common reef zoantharian Palythoa tuberculosa on Okinawa-jima Island, Japan. We identify four closely related Symbiodinium psbAncr lineages including one common generalist and two potential specialists that appear to be associated with particular microhabitats. The sea surface temperature differences that distinguish these habitats are smaller than those usually investigated, suggesting that future biogeographic surveys of Symbiodinium should incorporate fine scale environmental information as well as fine scale molecular data to accurately determine species diversity and their distributions.

A citizen science approach to monitoring bleaching in the zoantharian Palythoa tuberculosa.

Coral reef bleaching events are expected to become more frequent and severe in the near future as climate changes. The zoantharian Palythoa tuberculosa bleaches earlier than many scleractinian corals and may serve as an indicator species. Basic monitoring of such species could help to detect and even anticipate bleaching events, especially in areas where more sophisticated approaches that rely on buoy or satellite measurements of sea surface temperature are unavailable or too coarse. One simple and inexpensive monitoring method involves training volunteers to record observations of host color as a proxy for symbiosis quality. Here, we trained university students to take the 'color fingerprint' of a reef by assessing the color of multiple randomly selected colonies of P. tuberculosa at one time point in Okinawa Island, Japan. We tested the reliability of the students' color scores and whether they matched expectations based on previous monthly monitoring of tagged colonies at the same locations. We also measured three traditional metrics of symbiosis quality for comparison: symbiont morphological condition, cell density, and chlorophyll a content. We found that P. tuberculosa color score, although highly correlated among observers, provided little predictive power for the other variables. This was likely due to inherent variation in colony color among generally healthy zoantharians in midwinter, as well as low sample size and brief training owing to the course structure. Despite certain limitations of P. tuberculosa as a focal organism, the citizen science approach to color monitoring has promise, and we outline steps that could improve similar efforts in the future.

Intraspecific diversity among partners drives functional variation in coral symbioses.

The capacity of coral-dinoflagellate mutualisms to adapt to a changing climate relies in part on standing variation in host and symbiont populations, but rarely have the interactions between symbiotic partners been considered at the level of individuals. Here, we tested the importance of inter-individual variation with respect to the physiology of coral holobionts. We identified six genetically distinct Acropora palmata coral colonies that all shared the same isoclonal Symbiodinium 'fitti' dinoflagellate strain. No other Symbiodinium could be detected in host tissues. We exposed fragments of each colony to extreme cold and found that the stress-induced change in symbiont photochemical efficiency varied up to 3.6-fold depending on host genetic background. The S. 'fitti' strain was least stressed when associating with hosts that significantly altered the expression of 184 genes under cold shock; it was most stressed in hosts that only adjusted 14 genes. Key expression differences among hosts were related to redox signaling and iron availability pathways. Fine-scale interactions among unique host colonies and symbiont strains provide an underappreciated source of raw material for natural selection in coral symbioses.

New species of Clade B Symbiodinium (Dinophyceae) from the greater Caribbean belong to different functional guilds: S. aenigmaticum sp. nov., S. antillogorgium sp. nov., S. endomadracis sp. nov., and S. pseudominutum sp. nov.

Molecular approaches have begun to supersede traditional morphometrics in the species delineation of micro-eukaryotes. In addition to fixed differences in DNA sequences, recent genetics-based descriptions within the dinoflagellate genus Symbiodinium have incorporated confirmatory morphological, physiological, and ecological evidence when possible. However, morphological and physiological data are difficult to collect from species that have not been cultured, while the natural ecologies of many cultured species remain unknown. Here, we rely on genetic evidence-the only data consistently available among all taxa investigated-to describe four new Clade B Symbiodinium species. The 'host-specialized' species (S. antillogorgium sp. nov. and S. endomadracis sp. nov.) engage in mutualisms with specific cnidarian hosts, but exhibit differences in our ability to culture them in vitro. The ecologically 'cryptic' species (S. aenigmaticum sp. nov. and S. pseudominutum sp. nov.) thrive in culture, but their roles or functions in the ecosystem (i.e., niches) are yet to be documented. These new species call further attention to the spectrum of ecological guilds among Symbiodinium.