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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.

Black Band Disease (BBD) is a widely distributed and destructive coral disease that has been studied on a global scale, but baseline data on coral diseases is missing from many areas of the Arabian Seas. Here we report on the broad distribution and prevalence of BBD in the Red Sea in addition to documenting a bleaching-associated outbreak of BBD with subsequent microbial community characterization of BBD microbial mats at this reef site in the southern central Red Sea. Coral colonies with BBD were found at roughly a third of our 22 survey sites with an overall prevalence of 0.04%. Nine coral genera were infected including Astreopora, Coelastrea, Dipsastraea, Gardineroseris, Goniopora, Montipora, Pavona, Platygyra, and Psammocora. For a southern central Red Sea outbreak site, overall prevalence was 40 times higher than baseline (1.7%). Differential susceptibility to BBD was apparent among coral genera with Dipsastraea (prevalence 6.1%), having more diseased colonies than was expected based on its abundance within transects. Analysis of the microbial community associated with the BBD mat showed that it is dominated by a consortium of cyanobacteria and heterotrophic bacteria. We detected the three main indicators for BBD (filamentous cyanobacteria, sulfate-reducing bacteria (SRB), and sulfide-oxidizing bacteria (SOB)), with high similarity to BBD-associated microbes found worldwide. More specifically, the microbial consortium of BBD-diseased coral colonies in the Red Sea consisted of Oscillatoria sp. (cyanobacteria), Desulfovibrio sp. (SRB), and Arcobacter sp. (SOB). Given the similarity of associated bacteria worldwide, our data suggest that BBD represents a global coral disease with predictable etiology. Furthermore, we provide a baseline assessment of BBD disease prevalence in the Red Sea, a still understudied region.

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.