Host specificity of symbiodinium variants revealed by an its2 metahaplotype approach

ABSTRACT Analysis of the widely used ITS region is confounded by the presence of intragenomic variants (IGVs). In _Symbiodinium_, the algal symbionts of reef building corals, deep-sequencing

analyses are used to characterise communities within corals, yet these analyses largely overlook IGVs. Here we consider that distinct ITS2 sequences could represent IGVs rather than

distinct symbiont types and argue that symbionts can be distinguished by their proportional composition of IGVs, described as their ITS2 metahaplotype. Using our metahaplotype approach on

Minimum Entropy Decomposition (MED) analysis of ITS2 sequences from the corals _Acropora downingi_, _Cyphastrea microphthalma_ and _Playgyra daedalea_, we show the dominance of a single

species-specific _Symbiodinium_ C3 variant within each coral species. We confirm the presence of these species-specific symbionts using the psbA non-coding region. Our findings highlight the

importance of accounting for IGVs in ITS2 analyses and demonstrate their capacity to resolve biological patterns that would otherwise be overlooked. SIMILAR CONTENT BEING VIEWED BY OTHERS

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PHYLOGENETIC HAPLOTYPE NETWORKS MEET GLOBAL DNA METABARCODING DATASETS Article Open access 15 February 2021 MAIN The taxonomy of diverse microbial groups (for example, fungi, green algae and

dinoflagellates) is frequently distinguished using the ITS region of rDNA (LaJeunesse, 2001; Mai and Coleman, 1997; Schoch et al., 2012; Stern et al., 2012). However, there are multiple

copies of rDNA present in genomes, with differing forms known as intragenomic variants (IGV). Such IGVs can confound ITS-based analyses (Álvarez and Wendel, 2003; Thornhill et al., 2007;

Stat et al., 2011; Stern et al., 2012), but in the absence of more suitable markers, the ITS region remains a dominant means of characterising diverse microbial communities. The ITS2 region

underpins the taxonomy of the dinoflagellate _Symbiodinium_ (LaJeunesse, 2005), a symbiont widely associated with cnidarians, most notably reef building corals. _Symbiodinium_ is fundamental

to the coral holobiont, contributing >90% of the energy budget and influencing corals’ capacity to respond to environmental stress (Muscatine et al., 1984; Berkelmans and Van Oppen

2006). Deep sequencing of _Symbiodinium_ communities typically use the ITS2 region and are processed using OTU or phylotyping approaches (for example, Arif et al., 2014; Cunning et al.,

2015). These approaches either incorporate IGVs within OTUs or do not consider them in analyses. Instead of considering IGVs as incidental, here we show that the composition of ITS2 IGVs can

be used to differentiate distinct taxonomic sub-groups, revealing biological patterns consistent with a highly polymorphic marker. We compared symbiont communities among three coral species

(_Acropora downingii_, _Cyphastrea microphthalma_ and _Platygyra deadalea_) focusing on clade C as it is the most numerically dominant clade among these species at our sampling location

(Hume et al., 2015; Supplementary Table 1). We assessed the _Symbiodinium_ ITS2 sequence composition using an amplicon sequencing approach (see Supplementary Methods; _N_=13–15 ind./coral

sp.). Briefly, amplification of ITS2 and psbA non-coding region (psbAncr) was performed with modified Sym_Var primers (Hume et al., 2015). Normalised libraries were then sequenced on the

MiSeq Platform (Illumina) using the MiSeq v3 600-cycle kit. Demultiplexing, quality filtering, adapter removal, chimera removal and rarefaction (Supplementary Figure 1) were performed prior

to analysis with the Minimum Entropy Decomposition (MED) pipeline (Eren et al., 2015). MED nodes were analysed using _vegan_ (in R) and permutation multivariate analysis of variance

(_adonis_ in _vegan_). We compared our IGV approach with the OTU approach processed using the _mothur_ pipeline for _Symbiodinium_ (Schloss et al., 2009; Arif et al., 2014). Symbiont

communities from each species were initially analysed using the traditional OTU-based approach, with a 97% cutoff; this showed the presence of a single OTU, identified as a C3-type symbiont,

accounting for 100% of all ITS2 sequences. In essence, the OTU approach characterised all coral species as hosting the same host-generalist C3 symbiont type. As oligotyping (including

minimum entropy decomposition, MED) has been shown to reveal biologically relevant patterns masked by OTU approaches (Eren et al., 2014, Eren et al., 2015), we then applied MED analyses to

these symbiont communities. MED is ideally suited to analysing the extreme abundance of rare ITS2 variants in _Symbiodinium_ (Arif et al., 2014) by identifying the biologically informative

positions and therefore removing methodological and biological noise. Despite collapsing the data into distinct nodes, MED analyses retain the patterns observed in haplotype-based analyses

(Supplementary Figures 2 and 3). In contrast to the OTU approach, MED analysis revealed 44 distinct MED nodes (sequences grouped together exclusively by informative positions), characterised

by sequence variants of ITS2 types C3 and C3 Gulf (Supplementary Figure 4). The most abundant ITS2 nodes were common to all coral species, with the most abundant node accounting for 30–50%

of ITS2 sequences in all three coral species. Although the relative proportion of nodes varied among species, they were remarkably consistent within species. Species-specific grouping was

confirmed by multivariate analysis (Adonis, ITS2, F=213.52, _r_2=0.918, _P_=0.000999). Without an additional marker, interpretation of these data is complex as each MED node could either

represent a distinct symbiont or an intragenomic variant (IGV). There are two biologically distinct scenarios in which these data could be interpreted. These coral species could host a mixed

community of symbionts (for example, Sampayo et al., 2007), kept in relatively consistent proportions within species, although the most abundant symbiont taxa would be shared across all

coral species. Alternatively, each coral species could host a single symbiont taxon, which is distinguished only by the species-specific composition of its IGVs, herein referred to as an

‘ITS2 metahaplotype’. Here we will describe these two scenarios using the terms ‘mixed community’ and ‘single taxon’ (an unresolved taxonomic unit), respectively. In order to distinguish

between these scenarios, we performed MED analysis on psbAncr, a marker that provides high phylogenetic resolution in _Symbiodinium_ and although multi-copy, is typically present as a single

dominant sequence with low abundances of IGVs (LaJeunesse and Thornhill, 2011). Under the mixed community scenario, we would predict a mixture of psbAncr MED nodes in each individual coral.

For example, in _A. downingi_, we observed four abundant ITS2 MED nodes and if each node represents a separate symbiont, we should expect to see four correspondingly abundant psbAncr MED

nodes. Furthermore, the abundant nodes should be shared across coral species. Our analyses shows this is not the case. Instead, each individual coral was dominated by a single psbAncr MED

node, which is indicative of the presence of a single symbiont type (Figure 1d). In agreement with the ITS2 data, the psbAncr MED node composition results in species-specific clustering

(Figures 1e and f—Adonis, psbAncr, F=38.51, _r_2=0.670, _P_=0.000999). Overall, these results support the single taxon scenario: that each coral species hosts a single species-specific

symbiont characterised by its unique metahaplotype. These observations highlight the important role that IGVs could play in improving the taxonomic characterisation of _Symbiodinium_ and

other microbial groups, by providing insights into unique species-specific symbiont signatures that would have gone unnoticed by traditional ITS2 approaches. _Symbiodinium_ C3 was initially

considered a widespread generalist (LaJeunesse, 2005). However, our observation of diverse variants, which are coral host species-specific, within C3 support previous work that shows that

there is substantial cryptic diversity that is currently unresolved (Thornhill et al., 2014). This has important implications for our understanding of specificity and diversity of

coral–algal symbioses, and approaches that make use of IGVs provide the opportunity to shed light on these relationships. We have demonstrated the applicability of the metahaplotype approach

for _Symbiodinium_ clade C and in principle, it should be broadly applicable to other clades. Combining ITS2 and psbAncr amplicon sequencing analyses provides a framework whereby mixed ITS2

communities found elsewhere (for example, Sampayo et al., 2007) can be tested to identify whether monotypic communities and host specificity are found in other regions and species. This

will provide a greater appreciation of the different symbiotic strategies employed by corals and help identify the environmental and biological drivers behind the single taxon and mixed

community approaches. The power of such IGV approaches is not limited to coral symbionts, and can be applied to the diverse range of microbial organisms that are currently classified by

traditional ITS2 methods (for example, Schoch et al., 2012; Stern et al., 2012), particularly in metagenomics studies where IGVs could inflate diversity estimates. Our analyses demonstrate

the applicability of our MED-based metahaplotype approach to characterizing relatively simple communities but it could be transferred to more complex communities. However, analysis of mixed

communities would greatly benefit from the development of approaches that could identify individual metahaplotypes within a heterogeneous dataset. Transferring the metahaplotype approach to

mixed communities would be a worthwhile endeavor as we clearly show that incorporating ITS2 IGVs into analyses of microbial communities reveals fundamental ecological processes that would

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New York University Abu Dhabi Institute for funding and the Environment Agency Abu Dhabi for permitting. This work was supported by the New York University Core Sequencing and

Bioinformatics Groups. Data used in this study are deposited in the Dryad Digital Repository. (http://dx.doi.org/10.5061/dryad.h6s54). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Center

for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, UAE Edward G Smith, Remi N Ketchum & John A Burt Authors * Edward G Smith View author publications You can

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publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Edward G Smith. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Smith, E., Ketchum, R. & Burt, J. Host specificity of _Symbiodinium_ variants revealed by an ITS2 metahaplotype approach. _ISME J_ 11,

1500–1503 (2017). https://doi.org/10.1038/ismej.2016.206 Download citation * Received: 15 June 2016 * Revised: 20 October 2016 * Accepted: 25 November 2016 * Published: 17 February 2017 *

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