Development of a quantitative PCR-high-resolution melting assay for absolute measurement of coral-Symbiodiniaceae associations and its application to investigating variability at three spatial scales

Previous molecular studies have shown that many corals host a dynamic consortium of dominant and background populations of Symbiodiniaceae genotypes with putatively distinct physiological traits. In the present study, we developed a quantitative PCR assay combined with high-resolution melting analysis (qPCR-HRM) to distinguish which Symbiodiniaceae clades are present in a sample. Because the qPCR-HRM used in isolation yielded identical melt profiles for both clades A and C, this analysis was then coupled with further specific qPCR assays to enable the absolute quantification of all Symbiodiniaceae clades and host cells. When the assays were applied to in hospite samples, they had an absolute quantification level corresponding to one coral embryo of two cells and 1000 symbiont cells. The assays were successful on coral fragments from twelve species (eight families). We then used the qPCR-HRM assay in an ecological survey of Acropora pulchra at different spatial scales (within colony, between colonies and between sites). Differences in abundance and composition of Symbiodiniaceae clades in A. pulchra were observed at all spatial scales, suggesting that various environmental factors drove changes in Symbiodiniaceae assemblages among and within coral colonies. The qPCR-HRM assay developed in this study is a relatively simple, cost-effective and reproducible tool that can be used to accurately differentiate and quantify endosymbiont Symbiodiniaceae clades in coral in the field. This will provide new insights into coral-symbiont shuffling mechanisms and the resilience of coral colonies to environmental stressors.