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 (ITS2) 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, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 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 ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut-off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field-collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 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 ITS2 diversity in field-based studies.

Keywords: Symbiodinium; coral reefs; dinoflagellates; internal transcribed spacer 2; next-generation sequencing; operational taxonomic unit.

Figure 1

(A) Frequency distribution of sequence reads for the 10 most common ITS2 copies from isoclonal culture samples. (B) Rarefaction curve illustrating ITS2 sequence diversity distribution in isoclonal cultures.

Figure 2

Denaturing gradient gel electrophoresis (DGGE) fingerprint of samples used in this study with clades/subclades indicated.

Figure 3

Reproducibility of pyrosequencing rDNA variants for replicated PCRs (using the same DNA extract) of isoclonal cultures (A) CCMP2467 and (B) rt-147 taking the top 10 most abundant sequence variants into account. The sizes of the circles represent relative abundance; circles with an asterisk represent distinct ITS2 variants.

Figure 4

Comparison of top 10 most common variants found from pyrosequencing ITS2 from (A) culture rt-152 (Symbiodinium goreaui or type C1) and several field-collected samples representing clade C Symbiodinium: (B) C1c (sample P.Waj.D7), (C) C1h (sample P.Maq.R2.19) and (D) C41 (sample A.Af.B6). The size of the circles represents relative abundance, and numbers in circles represent order of distinct ITS2 variants for a given sample. Shared ITS2 variants are depicted in a black outer circle representing ITS2 type C1, and a grey outer circle representing ITS2 subtype c.