A vesicomyid bivalve bed in Site 3 (see Figure 1), with <i>C. regab</i> and <i>L. chuni.</i>

<p>Arrows show <i>L. chuni</i> siphons. Images ROV <i>Victor 6000</i>, WACS cruise – Ifremer. Reprinted from Ifremer WACS cruise under a CC BY license, with permission from Ifremer, original copyright 2011.</p

Physical Proximity May Promote Lateral Acquisition of Bacterial Symbionts in Vesicomyid Clams

<div><p>Vesicomyid clams harbor intracellular sulfur-oxidizing bacteria that are predominantly maternally inherited and co-speciate with their hosts. Genome recombination and the occurrence of non-parental strains were recently demonstrated in symbionts. However, mechanisms favoring such events remain to be identified. In this study, we investigated symbionts in two phylogenetically distant vesicomyid species, <i>Christineconcha regab</i> and <i>Laubiericoncha chuni</i>, which sometimes co-occur at a cold-seep site in the Gulf of Guinea. We showed that each of the two species harbored a single dominant bacterial symbiont strain. However, for both vesicomyid species, the symbiont from the other species was occasionally detected in the gills using fluorescence <i>in situ</i> hybridization and gene sequences analyses based on six symbiont marker genes. Symbiont strains co-occurred within a single host only at sites where both host species were found; whereas one single symbiont strain was detected in <i>C. regab</i> specimens from a site where no <i>L. chuni</i> individuals had been observed. These results suggest that physical proximity favored the acquisition of non-parental symbiont strains in Vesicomyidae. Over evolutionary time, this could potentially lead to genetic exchanges among symbiont species and eventually symbiont displacement. Symbiont densities estimated using 3D fluorescence <i>in situ</i> hybridization varied among host species and sites, suggesting flexibility in the association despite the fact that a similar type of metabolism is expected in all symbionts.</p></div

Unrooted maximum-likelihood phylogenies for six vesicomyid symbiont marker genes.

<p>Boxes indicate clade designations: <i>L. chuni</i>/<i>Vesicomya</i> sp. mtII symB (blue), <i>C. regab</i>/<i>Vesicomya</i> sp. mtI, mtII symA (red). Bootstrap values for 1000 replicates are given in percent above branches and clades (70% only). Scale bars are expressed as number of substitutions per base pair. The evolutionary model tested was GTR+I+G. with the following parameters: proportion of invariable sites = 0.79 (A), 0.56 (B), 0 (C, D, E, F), number of substitution rates (nst) = 6 for all phylogenies, gamma distribution parameter = 0.84 (A), 1.11 (B), 0.11 (C), 0.32 (D),0.15 (E), 0.14 (F). ‘Und. genus’) indicates a temporary genus name. Sp- 10 corresponds to the species numbers given in Audzijonyte <i>et al.</i> 2012.</p

Cross-sections of gills dissected from <i>C. regab</i> (1 and 2) and <i>L. chuni</i> (3) (1:217-V2 and 2:225- V3 and 3:225-V1) and hybridized with Creg821 (Cy5, stained in red) and Lchun821 (Cy3, stained in green) FISH probes, and counterstained with DAPI (in blue).

<p>In addition to the expected symbiont 16S rRNA phylotype, the unexpected phylotype is visible. Arrows indicate bacteria hybridized with both Creg821 (red) and Lchu821 (green). For individuals shown, a single (1 and 2) or two symbionts (3) were detected with 16S rRNA PCR probes. Images from specific probes and DAPI staining(3) do not overlap perfectly, possibly due to low activity in some bacteria. b: bacteriocyte, n: nucleus, os: outer lamellar space, is: intralamellar space.</p

Location of the three study sites on the Regab pockmark:

<p>Site 1 at the north of the pockmark (Guineco Marker 3), Site 2 at the center of the pockmark (Guineco Marker 7) and Site 3 in the southwestern part of the pockmark (Guineco Marker 10). Images ROV <i>Victor 6000</i>, WACS cruise – Ifremer. Reprinted from Ifremer WACS cruise under a CC BY license, with permission from Ifremer, original copyright 2011.</p

FISH probes used in this study, percentage of formamide in hybridization buffer, and target groups.

<p>FISH probes used in this study, percentage of formamide in hybridization buffer, and target groups.</p

Cross-sections of gills dissected from <i>C. regab</i> (217-V4) (1) and <i>L. chuni</i> (225-V6) (2) and hybridized with Creg821 (Cy3, stained in green) and Lchun821 (Cy5, stained in red) FISH probes, and DAPI counterstained (in blue).

<p>For the individuals shown, only a single 16S rRNA symbiont phylotype was identified through PCR. Specific probes and DAPI (2) do not overlap perfectly, possibly due to low activity in some bacteria. b: bacteriocyte, n: nucleus, os: outer lamellar space, is: intralamellar space.</p

Study sites at the Regab pockmark and number of individuals sampled (Cr: <i>Christineconcha regab</i> and Lc: <i>Laubiericoncha chuni</i>).

<p>Study sites at the Regab pockmark and number of individuals sampled (Cr: <i>Christineconcha regab</i> and Lc: <i>Laubiericoncha chuni</i>).</p

Results of direct sequencing (red for sequences from the <i>C. regab</i> symbiont and blue for the <i>L. chuni</i> symbiont), and cloning*.

<p>Percentage of divergence between the two symbiont sequences is given in parentheses in the header after each locus name. Given are details on the percentage (number) of sequences from <i>C. regab</i> and <i>L. chuni</i> symbiont (<b>bold</b>), and the number of clones sequenced in parentheses. ‘x’ indicates sequences obtained without cloning.</p

South Western Pacific map showing the sampling localities of mussels of <i>Idas iwaotakii</i>.

<p>Specimens from Malo and New Caledonia have already been studied by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069680#pone.0069680-Lorion1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069680#pone.0069680-Lorion3" target="_blank">[30]</a>.</p