FISH-Flow: a quantitative molecular approach for describing mixed clade communities of Symbiodinium

Our understanding of reef corals and their fate in a changing climate is limited by our ability to monitor the diversity and abundance of the dinoflagellate endosymbionts that sustain them. This study combined two well-known methods in tandem: fluorescent in situ hybridization (FISH) for genotype-specific labeling of Symbiodinium and flow cytometry to quantify the abundance of each symbiont clade in a sample. This technique (FISH-Flow) was developed with cultured Symbiodinium representing four distinct clades (based on large subunit rDNA) and was used to distinguish and quantify these types with high efficiency and few false positives. This technique was also applied to freshly isolated symbionts of Orbicella faveolata and Orbicella annularis. Isolates from acutely bleached coral tissues had significantly lower labeling efficiency; however, isolates from healthy tissue had efficiencies comparable to cultured Symbiodinium trials. RNA degradation in bleaching samples may have interfered with labeling of cells. Nevertheless, we were able to determine that, with and without thermal stress, experimental columns of the coral O. annularis hosted a majority of clade B and B/C symbionts on the top and side of the coral column, respectively. We demonstrated that, for cultured Symbiodinium and Symbiodinium freshly isolated from healthy host tissues, the relative ratio of clades could be accurately determined for clades present at as low as 7 % relative abundance. While this method does not improve upon PCR-based techniques in identifying clades at background levels, FISH-Flow provides a high precision, flexible system for targeting, quantifying and isolating Symbiodinium genotypes of interest.