Assessing Symbiodinium diversity in scleractinian corals via next‐generation sequencing‐based genotyping of the ITS2 rDNA region

Abstract

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS 2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS 2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS 2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)‐based pipeline for Symbiodinium ITS 2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut‐off of 0.03 collapsed intragenomic ITS 2 variants of isoclonal cultures into single OTU s. When applied to the analysis of field‐collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS 2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU‐based framework, we can improve objectivity, comparability and simplicity when assessing ITS 2 diversity in field‐based studies.