Deep-sequencing method for quantifying background abundances of symbiodinium types: exploring the rare symbiodinium biosphere in reef-building corals.

Kate M Quigley,Sarah W Davies,Carly D Kenkel,Line K Bay,Bette L Willis,Mikhail V Matz,Mónica Medina

doi:10.1371/journal.pone.0094297

Abstract

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.

Highlights

Coral reefs are one of the most biodiverse ecosystems on earth [1], largely as a consequence of the symbiosis that exists between scleractinian corals and endosymbiotic dinoflagellates within the genus Symbiodinium [2]
Sequences grouped into 1038 clusters, which varied in size from clusters containing more than ten identical sequences (N = 499 clusters) to three clusters containing over 2000 identical sequences (Figure 1A)
This latter pattern, of a few clusters containing a large number of reads, is consistent with expectations for dilution series samples that were dominated by one Symbiodinium type

Summary

Introduction

Coral reefs are one of the most biodiverse ecosystems on earth [1], largely as a consequence of the symbiosis that exists between scleractinian corals and endosymbiotic dinoflagellates within the genus Symbiodinium [2]. The physiology and health of the coral host relies heavily on carbon translocation from these symbionts [3,4], which enhances calcification of the coral host and leads to accretion of present day coral reefs [5] The stability of this symbiosis is threatened by many factors, such as chronic and acute changes in CO2 [6], temperature [7], and irradiance [8]. Uptake of novel types from the environment by adult corals (‘‘symbiont switching’’ [2]) has received little experimental support (but see [17]), the relative abundances of pre-existing symbiont types can change substantially within the coral host as a result of environmental stressors (‘‘symbiont shuffling’’) [18,19,20] These changes in Symbiodinium type complements can strongly influence holobiont fitness characteristics. The consequences of hosting functionally different clades, types and populations for coral physiology are increasingly appreciated [28], the full extent of this variation and its impacts on holobiont health and resilience remain open questions [29]

Methods

Results

Discussion

Conclusion