The contrasted evolutionary fates of deep‐sea chemosynthetic mussels (Bivalvia,Bathymodiolinae)

Bathymodiolinae are giant mussels that were discovered at hydrothermal vents and harboring chemosynthetic symbionts. Due to their close phylogenetic relationship with seep species and tiny mussels from organic substrates, it was hypothesized that they gradually evolved from shallow to deeper environments, and specialized in decaying organic remains, then in seeps, and finally colonized deep‐sea vents. Here, we present a multigene phylogeny that reveals that most of the genera are polyphyletic and/or paraphyletic. The robustness of the phylogeny allows us to revise the genus‐level classification. Organic remains are robustly supported as the ancestral habitat for Bathymodiolinae. However, rather than a single step toward colonization of vents and seeps, recurrent habitat shifts from organic substrates to vents and seeps occurred during evolution, and never the reverse. This new phylogenetic framework challenges the gradualist scenarios “from shallow to deep.” Mussels from organic remains tolerate a large range of ecological conditions and display a spectacular species diversity contrary to vent mussels, although such habitats are yet underexplored compared to vents and seeps. Overall, our data suggest that for deep‐sea mussels, the high specialization to vent habitats provides ecological success in this harsh habitat but also brings the lineage to a kind of evolutionary dead end.     

Methane-based symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan

Abstract. Bathymodiolus platifrons , a chemosynthetic mussel from cold seeps off Japan, relies for its nutrition on the productivity of methylotrophic or methanotrophic endosymbionts. High densities of bacterial symbionts appearing to be type I methanotrophs were observed in transmission electron micrographs of gill tissues. Methanol dehydrogenase activity in gill tissue from a single individual was positive compared to non-methanotrophic control samples, indicating a high potential for methanotrophy. Stable isotopic ratios of carbon in symbiont-containing gill tissue, as well as host tissues, were extremely depleted in ¹³C, and similar to values reported for other methanotrophic species. TEMs of gill tissue showing symbionts in various stages of digestion support the hypothesis that carbon transfer from symbionts to B. platifrons occurs through intracellular digestion of the symbionts. Discovery of methane- or methanolbased symbioses in B. platifrons from cold seeps in Sagami Bay extends the range of such symbioses to include cold seeps and hydrothermal vents, and supports the idea that environmental methane levels control the distribution of these symbioses.     

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.     

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.     

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.     

Siboglinid evolution shaped by habitat preference and sulfide tolerance

Siboglinids are tube-dwelling annelids that inhabit marine reducing habitats such as anoxic mud bottoms, seeps and hydrothermal vents. As adults, they lack a functional digestive system and rely on chemoautotrophic microbial endosymbionts. Based on morphological analyses, Siboglinidae form a clade with the Sabellariidae, Serpulidae and Sabellidae within the Annelida. The sister group to this clade is the Oweniidae. Three subgroups constitute the Siboglinidae: Frenulata typically inhabit anoxic sediments, Sclerolinium (a.k.a., Monilifera) live on decaying organic matter or reduced sediments and Vestimentifera are mostly found at hydrocarbon seeps and hydrothermal vents. Recent studies suggest that Sclerolinum is the sister group to the Vestimentifera. Within the Vestimentifera, the species inhabiting bare-rock hydrothermal vents represent a derived clade. The seep-inhabiting genus Lamellibrachia forms a basal branch within the Vestimentifera. Trends in siboglinid evolution are most notable with regard to the level of sulfide tolerance and type of substrate. Basal groups inhabit soft substrate with only slightly elevated sulfide levels, whereas more derived species colonize hard substrate and tolerate elevated temperatures and high levels of sulfide. The type of substrate correlates with tube morphology and the function of the opisthosome. The role of the symbionts in habitat selection needs further investigation.     

Gill-symbiosis in mytilidae associated with wood fall environments

Bivalves belonging to the genera Idas and Adipicola were collected from wood fall environments in the west Pacific (Vanuatu islands) between 300 and 890 m depths in 2004. Bacterial symbionts were checked by three complementary techniques: histological and DAPI staining, in situ hybridization (FISH), and TEM. No bacteria were detected inside the gills of the two species, rejecting the endosymbiosis hypothesis. However, results from our study demonstrated the existence of ectosymbionts colonizing microvilli differentiated at the apical surface of the cells constituting the lateral zone of gill filaments. These ectosymbionts are γ-Proteobacteria due to their strong hybridization with the specific probe GAM42; in contrast no hybridization was obtained from either gills or other host tissues by using the oligonucleotide probes specific to α- β- and δ-Proteobacteria. Based on TEM observations, these Gram-negative bacterial symbionts are not methanotrophic due to the lack of concentric stacking of intracellular membranes in their cytoplasm. Such ectosymbionts may represent thioautotrophic bacteria as already described in various Mytilidae from hydrothermal vents and cold seeps. Unfortunately, no phylogenetic analysis could be done in this study to compare their DNA sequence to that of other marine invertebrate symbionts described to date.     

Comparative analysis of symbiont ratios and gene expression in natural populations of two Bathymodiolus mussel species

Bathymodiolus mussels associated with deep-sea hydrothermal vents and cold seeps harbor chemosynthetic endosymbiotic bacteria in bacteriocytes located in the gill epithelium. Two distinct morphotypes of γ-proteobacteria, sulfur- and methane-oxidizing, have been identified and form a dual symbiosis in B. azoricus mussels from the Mid-Atlantic Ridge and in B. aff. boomerang from cold seeps in the Gulf of Guinea. Thiotrophic bacteria (SOX) are capable of fixing CO2 in the presence of sulfide or thiosulfate and methanotrophic bacteria (MOX) use methane both as a carbon and an energy source. In this study we used quantitative real-time PCR to test whether symbiont abundance and gene expression varied between the two mussel species. Results showed that B. azoricus from two hydrothermal sites had similar ratios and gene expression pattern for both symbiont types. In B. aff. boomerang, SOX ratio and ATP sulfurylase gene expression show differences between specimens collected on the different sites. Analysis of symbiont ratios in both species indicated a clear dominance of MOX symbionts in B. aff. boomerang and SOX symbionts in B. azoricus. We also evidenced that the species from the deeper sites (B. aff. boomerang) and mussels collected from sulfur and methane rich habitats showed higher symbiont ratio suggesting that environmental parameters may have significant impacts on the symbiont ratios in Bathymodiolus mussels.     

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-water chemosynthetic ecosystem research during the census of marine life decade and beyond: a proposed deep-ocean road map

The ChEss project of the Census of Marine Life (2002-2010) helped foster internationally-coordinated studies worldwide focusing on exploration for, and characterization of new deep-sea chemosynthetic ecosystem sites. This work has advanced our understanding of the nature and factors controlling the biogeography and biodiversity of these ecosystems in four geographic locations: the Atlantic Equatorial Belt (AEB), the New Zealand region, the Arctic and Antarctic and the SE Pacific off Chile. In the AEB, major discoveries include hydrothermal seeps on the Costa Rica margin, deepest vents found on the Mid-Cayman Rise and the hottest vents found on the Southern Mid-Atlantic Ridge. It was also shown that the major fracture zones on the MAR do not create barriers for the dispersal but may act as trans-Atlantic conduits for larvae. In New Zealand, investigations of a newly found large cold-seep area suggest that this region may be a new biogeographic province. In the Arctic, the newly discovered sites on the Mohns Ridge (71 °N) showed extensive mats of sulfur-oxidisng bacteria, but only one gastropod potentially bears chemosynthetic symbionts, while cold seeps on the Haakon Mossby Mud Volcano (72 °N) are dominated by siboglinid worms. In the Antarctic region, the first hydrothermal vents south of the Polar Front were located and biological results indicate that they may represent a new biogeographic province. The recent exploration of the South Pacific region has provided evidence for a sediment hosted hydrothermal source near a methane-rich cold-seep area. Based on our 8 years of investigations of deep-water chemosynthetic ecosystems worldwide, we suggest highest priorities for future research: (i) continued exploration of the deep-ocean ridge-crest; (ii) increased focus on anthropogenic impacts; (iii) concerted effort to coordinate a major investigation of the deep South Pacific Ocean - the largest contiguous habitat for life within Earth's biosphere, but also the world's least investigated deep-ocean basin.     

Life in Sulfidic Environments: Historical Perspective and Current Research Trends

SYNOPSIS. The discovery of the deep-sea hydrothermal vents andtheir associated fauna in 1977 was a watershed for the recentinterest in the biology of sulfidic environments. From the initialquestions concerning how organisms survived at the high sulfideconcentrations around the vents, research quickly focused onthe previously unrecognized sulfuroxidizing chemoautotrophicinvertebrate/bacterial symbioses whose nature was discoveredin 1980. There followed a successful and frenzied search forother reducing habitats harboring non-vent chemoautotrophicsymbioses. The interest in the biology of species living insulfidic habitats has since expanded to include the non-symbioticspecies, species with sulfuroxidizing symbionts and specieswith methanotrophic symbionts, all of which must be adaptedto tolerate sulfide and many of which are adapted to utilizesulfide in various ways.     

Symbioses between deep-sea mussels (Mytilidae: Bathymodiolinae) and chemosynthetic bacteria: diversity,function and evolution

Mussels of the subfamily Bathymodiolinae thrive around chimneys emitting hot fluids at deep sea hydrothermal vents, as well as at cold seeps and on sunken organic debris (sunken wood, whale falls). Despite the absence of light-driven primary production in these deep-sea ecosystems, mussels succeed reaching high biomasses in these harsh conditions thanks to chemosynthetic, carbon-fixing bacterial symbionts located in their gill tissue. Since the discovery of mussel symbioses about three decades ago our knowledge has increased, yet new findings are published regularly regarding their diversity, role and evolution. This article attempts to summarize current knowledge about symbiosis in Bathymodiolinae, focusing on mussel species for which information is available regarding both hosts and symbionts. Moreover, new data obtained from small mussels inhabiting sunken woods around the Philippines are provided. Indeed, mussel species from organic falls remain poorly studied compared to their vent and seep relatives despite their importance for the understanding of the evolution of symbiosis in the subfamily Bathymodiolinae. To cite this article: S. Duperron et al., C. R. Biologies 332 (2009).     

Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean

Specimens of Lamellibrachia ( Annelida: Siboglinidae ) were recently discovered at cold seeps in the eastern Mediterranean. In this study, we have investigated the phylogeny and function of intracellular bacterial symbionts inhabiting the trophosome of specimens of Lamellibrachia sp. from the Amon mud volcano, as well as the bacterial assemblages associated with their tube. The dominant intracellular symbiont of Lamellibrachia sp. is a gammaproteobacterium closely related to other sulfide-oxidizing tubeworm symbionts. In vivo uptake experiments show that the tubeworm relies on sulfide for its metabolism, and does not utilize methane. Bacterial communities associated with the tube form biofilms and occur from the anterior to the posterior end of the tube. The diversity of 16S rRNA gene phylotypes includes representatives from the same divisions previously identified from the tube of the vent species Riftia pachyptila , and others commonly found at seeps and vents.     

Comparative composition, diversity and trophic ecology of sediment macrofauna at vents, seeps and organic falls

Sediments associated with hydrothermal venting, methane seepage and large organic falls such as whale, wood and plant detritus create deep-sea networks of soft-sediment habitats fueled, at least in part, by the oxidation of reduced chemicals. Biological studies at deep-sea vents, seeps and organic falls have looked at macrofaunal taxa, but there has yet to be a systematic comparison of the community-level attributes of sediment macrobenthos in various reducing ecosystems. Here we review key similarities and differences in the sediment-dwelling assemblages of each system with the goals of (1) generating a predictive framework for the exploration and study of newly identified reducing habitats, and (2) identifying taxa and communities that overlap across ecosystems. We show that deep-sea seep, vent and organic-fall sediments are highly heterogeneous. They sustain different geochemical and microbial processes that are reflected in a complex mosaic of habitats inhabited by a mixture of specialist (heterotrophic and symbiont-associated) and background fauna. Community-level comparisons reveal that vent, seep and organic-fall macrofauna are very distinct in terms of composition at the family level, although they share many dominant taxa among these highly sulphidic habitats. Stress gradients are good predictors of macrofaunal diversity at some sites, but habitat heterogeneity and facilitation often modify community structure. The biogeochemical differences across ecosystems and within habitats result in wide differences in organic utilization (i.e., food sources) and in the prevalence of chemosynthesis-derived nutrition. In the Pacific, vents, seeps and organic-falls exhibit distinct macrofaunal assemblages at broad-scales contributing to ß diversity. This has important implications for the conservation of reducing ecosystems, which face growing threats from human activities.     

Diversity in mussel beds at deep-sea hydrothermal vents and cold seeps

Remarkably little is known about fundamental distinctions (or similarities) between the faunas of deep‐sea hydrothermal vents and seeps. Low species richness at vents has been attributed to the transient nature of vent habitats and to toxic effects of hydrogen sulphide and heavy metals in vent effluents. Seeps are arguably more stable and more chemically benign than vents. They have also been regarded as more diverse, but until now there has not been a rigorous test of this hypothesis. We evaluated diversity indices for invertebrates associated with mussel beds at six vents and two seeps and found that invertebrate diversity was significantly higher at seeps than vents, although some vent mussel beds supported nearly the same diversity as seep mussel beds. Lower diversity at vents may be a consequence of a greater physiological barrier to invasion at vents than at seeps. Diversity was lowest where spacing between vents was greatest, suggesting that risks of extinction as a result of dispersal‐related processes may contribute to the pattern of diversity observed at vents.     

Symbiotic diversity in marine animals: the art of harnessing chemosynthesis

Chemosynthetic symbioses between bacteria and marine invertebrates were discovered 30 years ago at hydrothermal vents on the Galapagos Rift. Remarkably, it took the discovery of these symbioses in the deep sea for scientists to realize that chemosynthetic symbioses occur worldwide in a wide range of habitats, including cold seeps, whale and wood falls, shallow-water coastal sediments and continental margins. The evolutionary success of these symbioses is evident from the wide range of animal groups that have established associations with chemosynthetic bacteria; at least seven animal phyla are known to host these symbionts. The diversity of the bacterial symbionts is equally high, and phylogenetic analyses have shown that these associations have evolved on multiple occasions by convergent evolution. This Review focuses on the diversity of chemosynthetic symbionts and their hosts, and examines the traits that have resulted in their evolutionary success.     

Ecology and Biogeography of Free-Living Nematodes Associated with Chemosynthetic Environments in the Deep Sea: A Review

Background

Here, insight is provided into the present knowledge on free-living nematodes associated with chemosynthetic environments in the deep sea. It was investigated if the same trends of high standing stock, low diversity, and the dominance of a specialized fauna, as observed for macro-invertebrates, are also present in the nematodes in both vents and seeps.

Methodology

This review is based on existing literature, in combination with integrated analysis of datasets, obtained through the Census of Marine Life program on Biogeography of Deep-Water Chemosynthetic Ecosystems (ChEss).

Findings

Nematodes are often thriving in the sulphidic sediments of deep cold seeps, with standing stock values ocassionaly exceeding largely the numbers at background sites. Vents seem not characterized by elevated densities. Both chemosynthetic driven ecosystems are showing low nematode diversity, and high dominance of single species. Genera richness seems inversely correlated to vent and seep fluid emissions, associated with distinct habitat types. Deep-sea cold seeps and hydrothermal vents are, however, highly dissimilar in terms of community composition and dominant taxa. There is no unique affinity of particular nematode taxa with seeps or vents.

Conclusions

It seems that shallow water relatives, rather than typical deep-sea taxa, have successfully colonized the reduced sediments of seeps at large water depth. For vents, the taxonomic similarity with adjacent regular sediments is much higher, supporting rather the importance of local adaptation, than that of long distance distribution. Likely the ephemeral nature of vents, its long distance offshore and the absence of pelagic transport mechanisms, have prevented so far the establishment of a successful and typical vent nematode fauna. Some future perspectives in meiofauna research are provided in order to get a more integrated picture of vent and seep biological processes, including all components of the marine ecosystem.     

Do Larval Supply and Recruitment Vary among Chemosynthetic Environments of the Deep Sea?

Background

The biological communities that inhabit chemosynthetic environments exist in an ephemeral and patchily distributed habitat with unique physicochemical properties that lead to high endemicity. Consequently, the maintenance and recovery from perturbation of the populations in these habitats is, arguably, mainly regulated by larval supply and recruitment.

Methodology/Principal Findings

We use data from the published scientific literature to: (1) compare the magnitudes of and variability in larval supply and settlement and recruitment at hydrothermal vents, seeps, and whale, wood and kelp falls; (2) explore factors that affect these life history processes, when information is available; and (3) explore taxonomic affinities in the recruit assemblages of the different chemosynthetic habitats, using multivariate statistical techniques. Larval supply at vents can vary across segments by several orders of magnitude for gastropods; for bivalves, supply is similar at vents on different segments, and at cold seeps. The limited information on larval development suggests that dispersal potential may be highest for molluscs from cold seeps, intermediate for siboglinids at vents and lowest for the whale-bone siboglinid Osedax. Settlement is poorly studied and only at vents and seeps, but tends to be highest near an active source of emanating fluid in both habitats. Rate of recruitment at vents is more variable among studies within a segment than among segments. Across different chemosynthetic habitats, recruitment rate of bivalves is much more variable than that of gastropods and polychaetes. Total recruitment rate ranges only between 0.1 and 1 ind dm⁻² d⁻¹ across all chemosynthetic habitats, falling above rates in the non-reducing deep sea. The recruit assemblages at vents, seeps and kelp falls have lower taxonomic breadth, and include more families and genera that have many species more closely related to each other than those at whale and wood falls. Vents also have the most uneven taxonomic structure, with fewer recruits represented by higher taxonomic levels (phyla, orders, classes) compared to seeps and wood and kelp falls, whereas the opposite is true at whale falls.

Conclusions/Significance

Based on our evaluation of the literature, the patterns and regulatory factors of the early history processes in chemosynthetic environments in the deep sea remain poorly understood. More research focused on these early life history stages will allow us to make inferences about the ecological and biogeographic linkages among the reducing habitats in the deep sea.     

Physiological Ecology of Sulfide Metabolism in Hydrothermal Vent and Cold Seep Vesicomyid Clams and Vestimentiferan Tube Worms

SYNOPSIS. The primary ecosystem-structuring organisms at manyhydrothermal vents and cold seeps are phylogenetically relatedand quite similar physiologically and anatomically. Vestimentiferantube worms and Vesicomyid clams in particular all rely on chemoautotrophicsulfur-oxidizing symbionts and have blood which binds sulfidewith high affinity and capacity. However, there are significantdifferences between cold seep and hydrothermal vent environments,including large differences in flow rate of the emitted fluidand the chemistry of that fluid. Here we review extant dataon the hydrothermal vent species, present new data on the physiologicallyrelevant chemical microhabitat of cold seep vestimentiferansand vesicomyids, and compare the physiological ecology of theseep species to their hydrothermal vent relatives     

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.