Improved resolution of reef-coral endosymbiont (Symbiodinium) species diversity, ecology, and evolution through psbA non-coding region genotyping

Abstract

Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbA(ncr)) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbA(ncr) haplotype was recovered in most samples through direct sequencing (~80-90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbA(ncr) sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbA(ncr) from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbA(ncr) sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbA(ncr) data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates.

Figure 1. ITS2-DGGE fingerprints of Symbiodinium from Montipora spp. sampled in Oahu, Hawaii.

Each diagnostic fingerprint comprises high-melting-lower-migrating homoduplexes (open arrowheads) and low-melting-higher-migrating heteroduplexes (solid arrowheads). Homoduplexes apparently correspond to the numerically dominant sequence variant in the ribosomal array of each ITS type [i.e.], . Sample 79 contains two symbiont profiles, C31 and D1-4-6. Samples used in bacterial cloning/sequencing of the ITS2 and psbA ^ncr are labeled on the gel by their psbA^ncr haplotypes (h1, h2, h3 etc…; see Figs. 2a and 3).

Figure 2. Sequence variation recovered by cloning vs. DGGE screening.

(a) Unrooted maximum parsimony of sequence variants recovered from the process of bacterial cloning/sequencing of ITS2 rDNA from four samples of C31/C31c (h2–h5) and one sample of type C26a (h1) from a subset of samples presented in Figure 1 (b) A corresponding and highly simplified phylogeny based on screening the numerically dominant sequence variants using DGGE fingerprinting. Clones from each sample were color coordinated for comparison and apply to other figures throughout the text.

Figure 3. The clade D Symbiodinium sp. harbored by Hawaiian Montipora (a) ITS2-DGGE fingerprinting produced three diagnostic homoduplex bands from each sample containing this Symbiodinium.

Designated D1-4-6, band “1” is ancestral while bands “4” and “6” represent derived sequence variants abundant in the genome. (b) All three dominant sequences were recovered multiple times from the bacterial cloning/sequencing from the sample 79, Figure 1. A profile matching Symbiodinium D1-4-6 was found in a few Pocillopora from the Andaman Sea, northeastern Indian Ocean and was also recovered from Pocillopora colonies in the aquarium trade.

Figure 4. Color morphs of Montipora capitata are dominated by different species of Symbiodinium.

The reddish brown colony on the left harbors the Montipora-specific symbiont C31/C31c, while the orangish-brown colony on the right harbors the Clade D symbiont, D1-4-6.

Figure 5. The analysis of Symbiodinium psbA^ncr acquired from field samples and isoclonal cultures.

(a) Unrooted phylogenetic analysis of partial (∼500 bases) psbA ^ncr sequences recovered from direct sequencing of Symbiodinium from Hawaiian Montipora. The symbiont's ITS2 designation and host species identity are labeled and correspond with phylogenetic groupings based on psbA^ncr sequences. Samples subjected to bacterial cloning and intensive sequencing are shaded in black. Bootstrap values based on 1000 replicates are labeled for branches separating each lineage group. (b) The phylogenetic analysis of bacterially cloned psbA^ncr shows minimal sequence variation within a particular sample, but considerable variation between samples. The number of clones sequenced is indicated for each sample analyzed. (c) The extent of intragenomic variation was examined by the cloning and sequencing of psbA ^ncr amplicons from the isoclonal clade C cultures, rt113, rt152, and rt203 (n = 16 sequences per culture). Sequence divergence among variants was small among strains of Symbiodinium goreaui (type C1; rt113 and rt152), however these were nonalignable with sequence variants recovered from rt203. The re-cloning and sequencing (n = 16) of a single cloned amplicon from each cultured isolate (grey arrows) assessed the frequency of artifacts created by the PCR/cloning process.

Figure 6. Alignment of partial psbA ^ncr sequences comparing D1-4-6 with that of Symbiodinium trenchi (synonymous with D1a or D1-4) obtained from different hosts in geographically separate regions.

Sequences marked by the asterisk are from isolates in culture.

Figure 7. Phylogenetic reconstruction based on partial psbA ^ncr of common Symbiodinium ITS types characterized from locations throughout the Indo-Pacific (see map).

Despite originating from geographically remote locations, psbA ^ncr haplotypes of particular ITS types group to form clades separated by considerable sequence divergence from other ITS types. A diverse monophyletic lineage consisting of different ITS2 types associates exclusively with coral species in the genus Montipora. These are divergent from ‘ancestral’ host-generalist lineages C3, C21, and C27. The inset (shaded grey) is a corresponding ITS2 phylogeny showing how, despite minimal sequence divergence, its topology reflects that of the psbA ^ncr tree.

Figure 8. Average pairwise genetic distances compared among intragenomic, inter-individual, and inter-species sequence variation of the psbA ^ncr (error bars represent ± SD).

Note the minimal overlap of nucleotide differences (Distance) between intragenomic (variation within a culture) and inter-individual variation (variation within an ITS2 lineage). This region is sometimes non-alignable among species lineages within a Symbiodinium Clade.