Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity

The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR). They harbour in specialized gill cells two types of endosymbiont (gram—bacteria): sulphide oxidizing bacteria (SOX) and methanotrophic bacteria (MOX). This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike -1700m, Mid-Atlantic Ridge), and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.     

Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.     

Relative abundances of methane- and sulphur-oxidising symbionts in the gills of a cold seep mussel and link to their potential energy sources

Bathymodiolus mussels are key species in many deep-sea chemosynthetic ecosystems. They often harbour two types of endosymbiotic bacteria in their gills, sulphur- and methane oxidisers. These bacteria take up sulphide and methane from the environment and provide energy to their hosts, supporting some of the most prolific ecosystems in the sea. In this study, we tested whether symbiont relative abundances in Bathymodiolus gills reflect variations in the highly spatially dynamic chemical environment of cold seep mussels. Samples of Bathymodiolus aff. boomerang were obtained from two cold seeps of the deep Gulf of Guinea, REGAB (5°47.86S, 9°42.69E, 3170 m depth) and DIAPIR (6°41.58S, 10°20.94E, 2700 m depth). Relative abundances of both symbiont types were measured by means of 3D fluorescence in situ hybridisation and image analysis and compared considering the local sulphide and methane concentrations and fluxes assessed via benthic chamber incubations. Specimens inhabiting areas with highest methane content displayed higher relative abundances of methane oxidisers. The bacterial abundances correlated also with carbon stable isotope signatures in the mussel tissue, suggesting a higher contribution of methane-derived carbon to the biomass of mussels harbouring higher densities of methane-oxidising symbionts. A dynamic adaptation of abundances of methanotrophs and thiotrophs in the gill could be a key factor optimising the energy yield for the symbiotic system and could explain the success of dual symbiotic mussels at many cold seeps and hydrothermal vents of the Atlantic and Gulf of Mexico.     

Shift from widespread symbiont infection of host tissues to specific colonization of gills in juvenile deep-sea mussels

The deep-sea mussel Bathymodiolus harbors chemosynthetic bacteria in its gills that provide it with nutrition. Symbiont colonization is assumed to occur in early life stages by uptake from the environment, but little is known about this process. In this study, we used fluorescence in situ hybridization to examine symbiont distribution and the specificity of the infection process in juvenile B. azoricus and B. puteoserpentis (4–21 mm). In the smallest juveniles, we observed symbionts, but no other bacteria, in a wide range of epithelial tissues. This suggests that despite the widespread distribution of symbionts in many different juvenile organs, the infection process is highly specific and limited to the symbiotic bacteria. Juveniles⩾9 mm only had symbionts in their gills, indicating an ontogenetic shift in symbiont colonization from indiscriminate infection of almost all epithelia in early life stages to spatially restricted colonization of gills in later developmental stages.     

Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, cbbL, and pmoA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of thiotrophic mytilid’s symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Global depression in gene expression as a response to rapid thermal changes in vent mussels

Hydrothermal vent mussels belonging to the genus Bathymodiolus are distributed worldwide and dominate communities at shallow Atlantic hydrothermal sites. While organisms inhabiting coastal ecosystems are subjected to predictable oscillations of physical and chemical variables owing to tidal cycles, the vent mussels sustain pronounced temperature changes over short periods of time, correlated to the alternation of oxic/anoxic phases. In this context, we focused on the short-term adaptive response of mussels to temperature change at a molecular level. The mRNA expression of 23 genes involved in various cell functions of the vent mussel Bathymodiolus azoricus was followed after heat shocks for either 30 or 120 min, at 25 and 30°C over a 48 h recovery period at 5°C. Mussels were genotyped at 10 enzyme loci to explore a relationship between natural genetic variation, gene expression and temperature adaptation. Results indicate that the mussel response to increasing temperature is a depression in gene expression, such a response being genotypically correlated at least for the Pgm-1 locus. This suggests that an increase in temperature could be a signal triggering anaerobiosis for B. azoricus or this latter alternatively behaves more like a ‘cold’ stenotherm species, an attribute more related to its phylogenetic history, a cold seeps/wood fall origin.     

Unexpected co-occurrence of six bacterial symbionts in the gills of the cold seep mussel Idas sp. (Bivalvia: Mytilidae)

Bathymodioline mussels occur in chemosynthesis-based ecosystems such as cold seeps, hydrothermal vents and organic debris worldwide. Their key adaptation to these environments is their association with bacterial endosymbionts which ensure a chemosynthetic primary production based on the oxidation of reduced compounds such as methane and sulfide. We herein report a multiple symbiosis involving six distinct bacterial 16S rRNA phylotypes, including two belonging to groups not yet reported as symbionts in mytilids, in a small Idas mussel found on carbonate crusts in a cold seep area located north to the Nile deep-sea fan (Eastern Mediterranean). Symbionts co-occur within hosts bacteriocytes based on fluorescence in situ hybridizations, and sequencing of functional genes suggests they have the potential to perform autotrophy, and sulfide and methane oxidation. Previous studies indicated the presence of only one or two symbiont 16S rRNA phylotypes in bathymodioline mussels. Together with the recent discovery of four bacterial symbionts in the large seep species Bathymodiolus heckerae , this study shows that symbiont diversity has probably been underestimated, and questions whether the common ancestor of bathymodioline mussels was associated with multiple bacteria.     

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2–F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels.Subject terms: Metagenomics, Population genetics, Symbiosis, Microbiome     

Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon continental margin (Southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 microM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Species Distribution and Population Connectivity of Deep-Sea Mussels at Hydrocarbon Seeps in the Gulf of Mexico

Hydrocarbon seepage is widespread and patchy in the Gulf of Mexico, and six species of symbiont containing bathymodiolin mussels are found on active seeps over wide and overlapping depth and geographic ranges. We use mitochondrial genes to discriminate among the previously known and a newly discovered species and to assess the connectivity among populations of the same species in the northern Gulf of Mexico (GoM). Our results generally validate the morphologically based distribution of the three previously known GoM species of Bathymodiolus, although we found that approximately 10% of the morphologically based identifications were incorrect and this resulted in some inaccuracies with respect to their previously assigned depth and geographical distribution patterns. These data allowed us to confirm that sympatry of two species of Bathymodiolus within a single patch of mussels is common. A new species of bathymodiolin, Bathymodiolus sp. nov., closely related to B. heckerae was also discovered. The two species live at the same depths but have not been found in sympatry and both have small effective population sizes. We found evidence for genetic structure within populations of the three species of Bathymodiolinae for which we had samples from multiple sites and suggest limited connectivity for populations at some sites. Despite relatively small sample sizes, genetic diversity indices suggest the largest population sizes for B. childressi and Tamu fisheri and the smallest for B. heckerae and B. sp. nov. among the GoM bathymodiolins. Moreover, we detected an excess of rare variants indicating recent demographic changes and population expansions for the four species of bathymodiolins from the Gulf of Mexico.     

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days.Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane.The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

Extracellular and Mixotrophic Symbiosis in the Whale-Fall Mussel Adipicola pacifica: A Trend in Evolution from Extra- to Intracellular Symbiosis

Background

Deep-sea mussels harboring chemoautotrophic symbionts from hydrothermal vents and seeps are assumed to have evolved from shallow-water asymbiotic relatives by way of biogenic reducing environments such as sunken wood and whale falls. Such symbiotic associations have been well characterized in mussels collected from vents, seeps and sunken wood but in only a few from whale falls.

Methodology/Principal Finding

Here we report symbioses in the gill tissues of two mussels, Adipicola crypta and Adipicola pacifica, collected from whale-falls on the continental shelf in the northwestern Pacific. The molecular, morphological and stable isotopic characteristics of bacterial symbionts were analyzed. A single phylotype of thioautotrophic bacteria was found in A. crypta gill tissue and two distinct phylotypes of bacteria (referred to as Symbiont A and Symbiont C) in A. pacifica. Symbiont A and the A. crypta symbiont were affiliated with thioautotrophic symbionts of bathymodiolin mussels from deep-sea reducing environments, while Symbiont C was closely related to free-living heterotrophic bacteria. The symbionts in A. crypta were intracellular within epithelial cells of the apical region of the gills and were extracellular in A. pacifica. No spatial partitioning was observed between the two phylotypes in A. pacifica in fluorescence in situ hybridization experiments. Stable isotopic analyses of carbon and sulfur indicated the chemoautotrophic nature of A. crypta and mixotrophic nature of A. pacifica. Molecular phylogenetic analyses of the host mussels showed that A. crypta constituted a monophyletic clade with other intracellular symbiotic (endosymbiotic) mussels and that A. pacifica was the sister group of all endosymbiotic mussels.

Conclusions/Significance

These results strongly suggest that the symbiosis in A. pacifica is at an earlier stage in evolution than other endosymbiotic mussels. Whale falls and other modern biogenic reducing environments may act as refugia for primal chemoautotrophic symbioses between eukaryotes and prokaryotes since the extinction of ancient large marine vertebrates.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.     

Annual spawning of the hydrothermal vent mussel, Bathymodiolus azoricus, under controlled aquarium, conditions at atmospheric pressure

Dense macrofaunal communities of modioliform mussels are a major component of many hydrothermal vent and cold seep ecosystems. The hydrothermal vent mussel Bathymodiolus azoricus, that dominates hydrothermal vent communities near the Azores Triple Junction, can be maintained in aquaria at atmospheric pressure. Cages containing these mussels were placed over diffuse vent outlets and recovered at different times. Cages recovered in January 2003 contained mussels with ripe gonads while those recovered in July, August and November 2001 and in April 2003 did not. Mussels collected post-spawning in April 2003 spawned in the aquaria in January 2004. Young mussels recruited to the cages in April 2003. The data support a main single period of spawning and of juvenile recruitment for B. azoricus.     

Diversity, relative abundance and metabolic potential of bacterial endosymbionts in three Bathymodiolus mussel species from cold seeps in the Gulf of Mexico

Cold seeps in the Gulf of Mexico are often dominated by mussels of the genus Bathymodiolus that harbour symbiotic bacteria in their gills. In this study, we analysed symbiont diversity, abundance and metabolic potential in three mussel species from the northern Gulf of Mexico: Bathymodiolus heckerae from the West Florida Escarpment, Bathymodiolus brooksi from Atwater Valley and Alaminos Canyon, and 'Bathymodiolus' childressi, which co-occurs with B. brooksi in Alaminos Canyon. Comparative 16S rRNA sequence analysis confirmed a single methanotroph-related symbiont in 'B.' childressi and a dual symbiosis with a methanotroph- and thiotroph-related symbiont in B. brooksi. A previously unknown diversity of four co-occurring symbionts was discovered in B. heckerae: a methanotroph, two phylogenetically distinct thiotrophs and a methylotroph-related phylotype not previously described from any marine invertebrate symbiosis. A gene characteristic of methane-oxidzing bacteria, pmoA, was identified in all three mussel species confirming the methanotrophic potential of their symbionts. Stable isotope analyses of lipids and whole tissue also confirmed the importance of methanotrophy in the carbon nutrition of all of the mussels. Analyses of absolute and relative symbiont abundance in B. heckerae and B. brooksi using fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization indicated a clear dominance of methanotrophic over thiotrophic symbionts in their gill tissues. A site-dependent variability in total symbiont abundance was observed in B. brooksi, with specimens from Alaminos Canyon harbouring much lower densities than those from Atwater Valley. This shows that symbiont abundance is not species-specific but can vary considerably between populations.     

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

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, Christineconcha regab and Laubiericoncha chuni, 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 in situ 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 C. regab specimens from a site where no L. chuni 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 in situ 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.     

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.