Deep-Sequencing Method for Quantifying Background Abundances of Symbiodinium Types: Exploring the Rare Symbiodinium Biosphere in Reef-Building Corals

The capacity of reef-building corals to associate with environmentally-appropriate types of endosymbionts from the dinoflagellate genus Symbiodinium contributes significantly to their success at local scales. Additionally, some corals are able to acclimatize to environmental perturbations by shuffling the relative proportions of different Symbiodinium types hosted. Understanding the dynamics of these symbioses requires a sensitive and quantitative method of Symbiodinium genotyping. Electrophoresis methods, still widely utilized for this purpose, are predominantly qualitative and cannot guarantee detection of a background type below 10% of the total Symbiodinium population. Here, the relative abundances of four Symbiodinium types (A13, C1, C3, and D1) in mixed samples of known composition were quantified using deep sequencing of the internal transcribed spacer of the ribosomal RNA gene (ITS-2) by means of Next Generation Sequencing (NGS) using Roche 454. In samples dominated by each of the four Symbiodinium types tested, background levels of the other three types were detected when present at 5%, 1%, and 0.1% levels, and their relative abundances were quantified with high (A13, C1, D1) to variable (C3) accuracy. The potential of this deep sequencing method for resolving fine-scale genetic diversity within a symbiont type was further demonstrated in a natural symbiosis using ITS-1, and uncovered reef-specific differences in the composition of Symbiodinium microadriaticum in two species of acroporid corals (Acropora digitifera and A. hyacinthus) from Palau. The ability of deep sequencing of the ITS locus (1 and 2) to detect and quantify low-abundant Symbiodinium types, as well as finer-scale diversity below the type level, will enable more robust quantification of local genetic diversity in Symbiodinium populations. This method will help to elucidate the role that background types have in maximizing coral fitness across diverse environments and in response to environmental change.