Data_Sheet_1_Fine-Scale Biogeographical Boundary Delineation and Sub-population Resolution in the Symbiodinium thermophilum Coral Symbiont Group From the Persian/Arabian Gulf and Gulf of Oman.DOCX

<p>The adaptation of tropical coral communities to the world's hottest sea, the Persian/Arabian Gulf (PAG), has recently been associated with ecological selection acting on a group of coral-associated algal symbionts, the Symbiodinium thermophilum group. Previous studies have shown that considerable genetic diversity exists within the group and that group members found within the PAG are significantly differentiated from those found externally, in the Gulf of Oman and wider waters. However, little is known about this genetic diversity. As an initial step towards understanding whether this diversity could represent niche adapted, selectable populations within the S. thermophilum group that may act as natural sources of stress tolerant associations to Indo-Pacific reefs, we investigate whether the diversity is structured between populations and where the location of the internal-external genetic partition lies. We use regions of the nuclear ribosomal DNA (ITS1-5.8S-ITS2) and chloroplastic psbA gene (non-coding region) from >100 S. thermophilum group-harbouring Porites spp. (P. lobata, P. lutea, and P. harrisoni) sampled across steep temperature and salinity gradients to conduct analyses of variance and create maximum parsimony networks to assess genetic structure and (dis)similarity within and between populations of S. thermophilum found within the PAG and externally in the Gulf of Oman. Our analyses resolve a sharp genetic boundary between Symbiodinium populations in the western Strait of Hormuz and identify significant genetic structure between populations with as little as 20 km between them demonstrating that differentiation between populations is likely due to factors other than limited connectivity. Further, we hypothesize that genotypes identified outside of the PAG in the Gulf of Oman existing in near-oceanic salinities, yet thermally challenging waters, putatively represent candidates for stress-tolerant symbionts that could act as natural seed populations of stress tolerant genotypes to the wider indo-Pacific.</p

Characterisation of symbiont communities in the southern PAG and Gulf of Oman.

<p>(A) Bar charts indicate the proportion of colonies hosting each ITS2 type or combination of ITS2 types. Vertical separation within the bars indicates colonies where more than one symbiont type was present. (B) Radial phylogenetic tree depicting the relationships between C3 variants found in <i>P</i>. <i>daedalea</i> from PAG reefs, C3 variants from <i>Porites spp</i>. within the region and other C3 variants found elsewhere. The tree was generated by Bayesian inference analysis of the psbA^ncr region using the alignment generated in a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180169#pone.0180169.ref018" target="_blank">18</a>]. The branches containing samples from this study are shown in red with labelled arrows indicating the location of the samples (D: Delma; S: Saadiyat; R: Ras al Khaimah). The accession numbers for samples used to generate the tree are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180169#pone.0180169.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180169#pone.0180169.s004" target="_blank">S4</a> Tables.</p

Genetic structure of <i>Platygyra daedalea</i> in the PAG and Gulf of Oman.

<p><i>Upper panel</i>: Mean monthly averaged maximum SST, 2004–2014. <i>Lower panel</i>: Host haplotype frequencies. Each colour represents a different haplotype and white sectors indicate private haplotypes. SH = Strait of Hormuz.</p

Conspicuous morphological differentiation without speciation in <i>Anemonia viridis</i> (Cnidaria, Actiniaria)

<p><i>Anemonia viridis</i> is a model species for studies of physiological and transcriptomic response to symbiosis and environmental stress (temperature, light, symbiosis breakdown). Five morphs are described in this species, based on morphology, pigment protein content, and major mode of reproduction. Up to now, the taxonomic status of these morphs remains unclear, without clear knowledge of whether the morphological variation amongst the morphs is due to phenotypic plasticity or adaptation. In the present study, we assess the species status of the three most commonly found morphs by coalescent analyses. For this purpose, five markers were designed for genes whose expression is modified under stress (<i>ca2m</i>, <i>duf140</i>, <i>RNAbinding5</i>, <i>tyrK</i>, <i>sym32</i>) and analysed in 34 individuals representing the morphs <i>A. viridis</i> var <i>rufescens</i>, <i>rustica</i> and <i>smaragdina</i> from eight geographic sites (two in the English Channel and six in the Mediterranean Sea). Phylogenetic analyses of individual gene trees showed no clear separation of the morphs. Furthermore, multilocus coalescent analyses using SpedeSTEM and BPP regrouped all the morphs into one species, showing very few genetic differences amongst them. In a further analysis, we checked for clonality amongst 80 individuals of <i>A. viridis</i> var. <i>smaragdina</i> from one geographic site using three microsatellite loci. This morph proved to be as clonal as var. <i>rustica</i>, indicating a similar potential for asexual reproduction.</p

Photoswitching of the chromophore in cerFP505.

<p>A) Absorption spectra after irradiation with 400 nm (on-state) or with 450 nm light (off-state). Deactivation gives rise to a second peak around 390 nm. B) Ground state equilibrium. Irradiation at 400 nm drives the protein to the on-state, 450 nm light turns off the “activatable” fraction of the protein. Bars above the x-axis specify the light treatment, 400 nm light (white bar), 450 nm light (grey bar) or no light (black bar). C) Fluorescence emission of the protein in the on- or off-state was recorded for ≥30 min in the dark after 5 min irradiation with 400 or 450 nm light. Bars above the x-axis specify the light treatment as described above. D) The reversibility of the reaction is shown for eight cycles of activation/deactivation. Bars above the x-axis specify the light treatment as described in (B).</p

Identification of a GFP-like protein from a deep sea ceriantharian.

<p>A) Fluorescence image of a ceriantharian acquired in 530 m depth in the Gulf of Mexico. The depth (ft), temperature (Temp, °C) and the salinity (Salin) measured during image acquisition are displayed in the upper part of the panel. B–C) Microscopic image of two tentacles photographed under white light (B) or blue light excitation using a GFP-filter set (C). D) Absorption, excitation and emission spectra of purified recombinant cerFP505. Fluorescence spectra were recorded using 450 nm light for excitation, whereas the emission was collected at 550 nm. E) Multiple alignment of amino acid sequences from ceriantharian fluorescent proteins and EGFP. The chromophore – forming tripeptide is highlighted in green. Residues interacting directly with, or found in the proximity of, the chromophore in cmFP512 are labeled in blue or red, respectively. Amino acids underlayed in grey or yellow are involved in A/B or A/C interface interactions in cmFP512, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003766#pone.0003766-Nienhaus1" target="_blank">[59]</a>. The numberings for cerFP505 and EGFP are given above and below the sequences, respectively. Regions forming ß-sheets in EGFP are underlined.</p

Photophysical properties of ceriantharian fluorescent proteins.

a<p>values calculated from the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003766#pone-0003766-g003" target="_blank">Figure 3C</a>.</p>b<p>data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003766#pone.0003766-Wiedenmann5" target="_blank">[43]</a>.</p>c<p>data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003766#pone.0003766-Ip2" target="_blank">[63]</a>.</p>d<p>data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003766#pone.0003766-Patterson2" target="_blank">[81]</a>.</p

Expression of cerFP505 in mammalian cells.

<p>A) cerFP505, cmFP512 and EGFP were expressed in HEK293 cells. Photographs were taken 12 h after transfection. B) The upper panels show the expression and maturation of cerFP505 <i>in vivo</i> in HEK293 cells. Images were excised from a time-lapse movie. The time-points of image acquisition are indicated in the panels as minutes after transfection. The increase in fluorescence intensity of cells in the green channel was analyzed for each image of the time-lapse movie. The graph displays the mean of the individual measurement normalized to 1.0, error bars denote standard deviations. C) Photobleaching of cerFP505, cmFP512 and EGFP in HEK293 cells under continuous irradiation with blue light under the fluorescence microscope. The upper panels are images from a time-lapse movie showing the photobleaching of cells expressing cerFP505. Imaging time-points are given and correspond to minutes of irradiation. The graph displays the mean values of the green fluorescence emission of blue-light irradiated cells expressing cerFP505, cmFP512 and EGFP. Error bars denote standard deviations. <i>Inset</i>: Photobleaching measured <i>in vitro</i> using purified recombinant protein samples.</p

Fluorescence of <i>S. pistillata</i> and <i>A. squarrosa</i> from depths ≥60 m.

<p>Images, taken after the specimens were collected, show the daylight appearance (left column) and the blue-light-induced fluorescence of the upper and underside of the branches (middle/right column).</p

Changes in fluorescence of colour morphs of <i>Euphyllia paradivisa</i> from mesophotic depths in response to altered light environments.

<p>The daylight appearance and blue-light- induced fluorescence of the green (a), red (b) and yellow (c) colour morphs are imaged after one year of culture under low intensity daylight (upper row) and in complete darkness (lower row). Fluorescence emission spectra in the range of GFP-like proteins (450–650 nm) and chlorophyll (650–800 nm), measured by excitation with ~450 nm, are shown for the displayed individuals. Peak positions are indicated by arrows.</p