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

Chatchanit Arif,Till Bayer,Camille Daniels,Adrian Barbrook,Eulalia Banguera‐Hinestroza,Christian R Voolstra,Christopher J Howe,Todd C Lajeunesse

doi:10.1111/mec.12869

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 SymbiodiniumITS2 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.

Highlights

Coral reef communities depend critically on the relationship between scleractinian corals and photosynthetic endosymbionts in the genus Symbiodinium
We applied 454-based amplicon sequencing of the internal transcribed spacer 2 (ITS2) rDNA region from Symbiodinium to a collection of replicated isoclonal cultures (n = 7), pooled cultures (n = 2) and environmental samples (n = 6) (Table 1)
Taking only isoclonal culture samples into account, we identified on average 230.86 ITS2 sequence variants per culture, indicating that there is a substantial number of distinct ITS2 sequence variants found within Symbiodinium genomes (Table 1)

Summary

Introduction

Rowan & Powers (1991) were the first to examine Symbiodinium evolutionary relationships using restriction fragment length polymorphisms (RFLPs) of the small ribosomal subunit RNA (SSU) gene from dinoflagellates from various marine invertebrate hosts. Their phylogeny identified divergent lineages within this genus, which exhibited sequence differences comparable to those observed among dinoflagellates from different taxonomic families and orders (Rowan & Powers 1992). A systematic survey by Sampayo et al (2009) targeting ribosomal, mitochondrial and chloroplast genes employing 13 distinct genetic analyses found that different markers showed remarkable concordance, but differed in their relative ability to resolve ecologically distinct units. The ITS2 region is still the most commonly employed DNA marker used to assess Symbiodinium diversity from a diverse array of hosts and over large geographical distances (LaJeunesse et al 2010; Wicks et al 2010; Silverstein et al 2011; Putnam et al 2012; Tonk et al 2013a)

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Conclusion