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.], [ 7,24]. Sample 79 contains two symbiont profiles, C31 and D1-4-6. Samples used in bacterial cloning/sequencing of the ITS2 and psbAncr are labeled on the gel by their psbAncr 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 psbAncr acquired from field samples and isoclonal cultures.
(a) Unrooted phylogenetic analysis of partial (∼500 bases) psbAncr 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 psbAncr 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 psbAncr 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 psbAncr 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 psbAncr 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 psbAncr of common Symbiodinium ITS types characterized from locations throughout the Indo-Pacific (see map).
Despite originating from geographically remote locations, psbAncr 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 psbAncr tree.
Figure 8.
Average pairwise genetic distances compared among intragenomic, inter-individual, and inter-species sequence variation of the psbAncr (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.