Higher genotypic diversity and distinct assembly mechanism of free-living Symbiodiniaceae assemblages than sympatric coral-endosymbiotic assemblages in a tropical coral reef.

While in hospite Symbiodiniaceae dinoflagellates are essential for coral health, ambient free-living counterparts are crucial for coral recruitment and resilience. Comparing free-living and in hospite Symbiodiniaceae communities can potentially provide insights into endosymbiont acquisition and recurrent recruitment in bleaching recovery. In this study, we studied coral-endosymbiotic and ambient free-living Symbiodiniaceae communities in the South China Sea. We collected samples from 183 coral and ambient plankton samples and conducted metabarcoding to investigate the diversity distribution, driving factors, and assembly mechanisms of the two groups of Symbiodiniaceae. Results revealed Cladocopium C1 and Durusdinium D1 as dominant genotypes. We detected a higher genotypic diversity in free-living than in hospite symbiodiniacean communities, but with shared dominant genotypes. This indicates a genetically diverse pool of Symbiodiniaceae available for recruitment by corals. Strikingly, we found that the cooler area had more Symbiodiniaceae thermosensitive genotypes, whereas the warmer area had more Symbiodiniaceae thermotolerant genotypes. Furthermore, in hospite and free-living Symbiodiniaceae communities were similarly affected by environmental factors, but shaped by different assembly mechanisms. The in hospite communities were controlled mainly by deterministic processes, whereas the ambient communities by stochastic processes. This study sheds light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms influencing Symbiodiniaceae inside and outside corals.IMPORTANCESymbiodiniaceae dinoflagellates play a pivotal role as key primary producers within coral reef ecosystems. Coral-endosymbiotic Symbiodiniaceae communities have been extensively studied, but relatively little work has been reported on the free-living Symbiodiniaceae community. Conducting a comparative analysis between sympatric coral-endosymbiotic and free-living Symbiodiniaceae communities can potentially enhance the understanding of how endosymbiont communities change in response to changing environments and the mechanisms driving these changes. Our findings shed light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms shaping free-living and in hospite Symbiodiniaceae communities, with implications in evaluating the adaptive and resilient capacity of corals in response to future climate change.

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.

Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae.

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8-99.3%; R 2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.