The discovery of new deep-sea hydrothermal vent communities in the southern ocean and implications for biogeography

Since the first discovery of deep-sea hydrothermal vents along the Galápagos Rift in 1977, numerous vent sites and endemic faunal assemblages have been found along mid-ocean ridges and back-arc basins at low to mid latitudes. These discoveries have suggested the existence of separate biogeographic provinces in the Atlantic and the North West Pacific, the existence of a province including the South West Pacific and Indian Ocean, and a separation of the North East Pacific, North East Pacific Rise, and South East Pacific Rise. The Southern Ocean is known to be a region of high deep-sea species diversity and centre of origin for the global deep-sea fauna. It has also been proposed as a gateway connecting hydrothermal vents in different oceans but is little explored because of extreme conditions. Since 2009 we have explored two segments of the East Scotia Ridge (ESR) in the Southern Ocean using a remotely operated vehicle. In each segment we located deep-sea hydrothermal vents hosting high-temperature black smokers up to 382.8°C and diffuse venting. The chemosynthetic ecosystems hosted by these vents are dominated by a new yeti crab (Kiwa n. sp.), stalked barnacles, limpets, peltospiroid gastropods, anemones, and a predatory sea star. Taxa abundant in vent ecosystems in other oceans, including polychaete worms (Siboglinidae), bathymodiolid mussels, and alvinocaridid shrimps, are absent from the ESR vents. These groups, except the Siboglinidae, possess planktotrophic larvae, rare in Antarctic marine invertebrates, suggesting that the environmental conditions of the Southern Ocean may act as a dispersal filter for vent taxa. Evidence from the distinctive fauna, the unique community structure, and multivariate analyses suggest that the Antarctic vent ecosystems represent a new vent biogeographic province. However, multivariate analyses of species present at the ESR and at other deep-sea hydrothermal vents globally indicate that vent biogeography is more complex than previously recognised.     

Antarctic marine biodiversity and deep-sea hydrothermal vents

The diversity of many marine benthic groups is unlike that of most other taxa. Rather than declining from the tropics to the poles, much of the benthos shows high diversity in the Southern Ocean. Moreover, many species are unique to the Antarctic region. Recent work has shown that this is also true of the communities of Antarctic deep-sea hydrothermal vents. Vent ecosystems have been documented from many sites across the globe, associated with the thermally and chemically variable habitats found around these, typically high temperature, streams that are rich in reduced compounds and polymetallic sulphides. The animal communities of the East Scotia Ridge vent ecosystems are very different to those elsewhere, though the microbiota, which form the basis of vent food webs, show less differentiation. Much of the biological significance of deep-sea hydrothermal vents lies in their biodiversity, the diverse biochemistry of their bacteria, the remarkable symbioses among many of the marine animals and these bacteria, and the prospects that investigations of these systems hold for understanding the conditions that may have led to the first appearance of life. The discovery of diverse and unusual Antarctic hydrothermal vent ecosystems provides opportunities for new understanding in these fields. Moreover, the Antarctic vents south of 60°S benefit from automatic conservation under the Convention on the Conservation of Antarctic Marine Living Resources and the Antarctic Treaty. Other deep-sea hydrothermal vents located in international waters are not protected and may be threatened by growing interests in deep-sea mining.     

Hidden Historical Habitat-Linked Population Divergence and Contemporary Gene Flow of a Deep-Sea Patellogastropod Limpet

Hydrothermal vents and hydrocarbon seeps in the deep ocean are rare oases fueled by chemosynthesis. Biological communities inhabiting these ecosystems are often distributed in widely separated habitats, raising intriguing questions on how these organisms achieve connectivity and whether habitat types facilitate intraspecific divergence. The deep-sea patellogastropod limpet Bathyacmaea nipponica that colonizes both vents and seeps across ∼2,400 km in the Northwest Pacific is a feasible model to answer these questions. We analyzed 123 individuals from four vents and three seeps using a comprehensive method incorporating population genomics and physical ocean modeling. Genome survey sequencing and genotyping-by-sequencing resulted in 9,838 single-nucleotide polymorphisms for population genomic analyses. Genetic divergence and demographic analyses revealed four habitat-linked (i.e., three seep and one vent) genetic groups, with the vent genetic group established via the opportunistic invasion of a few limpet larvae from a nearby seep genetic group. TreeMix analysis uncovered three historical seep-to-vent migration events. ADMIXTURE and divMigrate analyses elucidated weak contemporary gene flow from a seep genetic group to the vent genetic group. Physical ocean modeling underlined the potential roles of seafloor topography and ocean currents in shaping the genetic connectivity, contemporary migration, and local hybridization of these deep-sea limpets. Our study highlighted the power of integrating genomic and oceanographic approaches in deciphering the demography and diversification of deep-sea organisms. Given the increasing anthropogenic activities (e.g., mining and gas hydrate extraction) affecting the deep ocean, our results have implications for the conservation of deep-sea biodiversity and establishment of marine protected areas.     

Experimental ecology at deep-sea hydrothermal vents: a perspective

In situ and laboratory experiments conducted over the past quarter of a century have greatly increased our understanding of the ecology of deep-sea hydrothermal systems. Early experiments suggested that chemosynthetic primary production constituted the principal source of organic matter for biological communities associated with vents, although subsequent studies have revealed many complexities associated with interactions between microbes and higher organisms inhabiting these ecosystems. A diversity of host-microbial symbiont relationships has been identified and experimental studies have revealed the exquisite physiological adaptations within the giant tubeworm, Riftia pachyptila, for the uptake, fixation, and assimilation of carbon. In vitro experiments demonstrated the unusual sulfide binding properties of tubeworm hemoglobin that prevent inhibition of the cytochrome-c oxidase enzyme system during transport of sulfide to symbiont-bearing tissues. Studies of respiration and growth of several species of vent organisms conducted over the past two decades transformed earlier views that low metabolism and slow growth are characteristics of all organisms inhabiting all deep-sea environments. Results of recent experiments suggest that metabolic rates correlate with the degree of mobility of the organisms rather than with any specific attribute of the deep-sea environment itself, and growth rates of certain vent organisms (e.g., R. pachyptila) were found to be among the highest in any marine environments. While extreme thermal tolerance has been suggested as characteristic of certain vent fauna (e.g., alvinellid polychaetes and alvinocarid shrimp), the majority of vent metazoans live at temperatures below 20 °C and additional experiments are necessary to reconcile field experiments documenting thermal tolerance in situ, thermal tolerance in vivo, and thermal sensitivity of biochemical constituents of vent organisms. Transplantation and clearance experiments, as well as in situ characterization of vent fluid chemistry, have greatly increased our understanding of organism–environment interactions. Early analyses of metazoan egg size and larval morphology, coupled with in vivo larval culture experiments, available physical oceanographic data, and genetic studies of gene flow, have contributed greatly to our understanding of mechanisms of dispersal between widely separated vent sites. The documentation of invertebrate colonization and succession of new vents following a volcanic eruption, and a series of manipulative field experiments, provide considerable insights into the relative roles of abiotic conditions and biotic interactions in structuring vent communities. Recent and emerging technological developments, such as in situ chemical analyzers, observatory approaches, and laboratory-based pressure culture systems, should provide invaluable new experimental tools for tackling many remaining questions concerning the ecology of deep-sea hydrothermal systems.     

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.     

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.     

Characterization of Bacterial Communities in Deep-Sea Hydrothermal Vents from Three Oceanic Regions

Deep-sea hydrothermal vents are considered to be one of the most spectacular ecosystems on Earth. Microorganisms form the basis of the food chain in vents controlling the vent communities. However, the diversity of bacterial communities in deep-sea hydrothermal vents from different oceans remains largely unknown. In this study, the pyrosequencing of 16S rRNA gene was used to characterize the bacterial communities of the venting sulfide, seawater, and tubeworm trophosome from East Pacific Rise, South Atlantic Ridge, and Southwest Indian Ridge, respectively. A total of 23,767 operational taxonomic units (OTUs) were assigned into 42 different phyla. Although Proteobacteria, Actinobacteria, and Bacteroidetes were the predominant phyla in all vents, differences of bacterial diversity were observed among different vents from three oceanic regions. The sulfides of East Pacific Rise possessed the most diverse bacterial communities. The bacterial diversities of venting seawater were much lower than those of vent sulfides. The symbiotic bacteria of tubeworm Ridgeia piscesae were included in the bacterial community of vent sulfides, suggesting their significant ecological functions as the primary producers in the deep-sea hydrothermal vent ecosystems. Therefore, our study presented a comprehensive view of bacterial communities in deep-sea hydrothermal vents from different oceans.     

The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO₂, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.     

Comparative Population Structure of Two Deep-Sea Hydrothermal-Vent-Associated Decapods (Chorocaris sp. 2 and Munidopsis lauensis) from Southwestern Pacific Back-Arc Basins

Studies of genetic connectivity and population structure in deep-sea chemosynthetic ecosystems often focus on endosymbiont-hosting species that are directly dependent on chemical energy extracted from vent effluent for survival. Relatively little attention has been paid to vent-associated species that are not exclusively dependent on chemosynthetic ecosystems. Here we assess connectivity and population structure of two vent-associated invertebrates—the shrimp Chorocaris sp. 2 and the squat lobster Munidopsis lauensis—that are common at deep-sea hydrothermal vents in the western Pacific. While Chorocaris sp. 2 has only been observed at hydrothermal vent sites, M. lauensis can be found throughout the deep sea but occurs in higher abundance around the periphery of active vents We sequenced mitochondrial COI genes and deployed nuclear microsatellite markers for both species at three sites in Manus Basin and either North Fiji Basin (Chorocaris sp. 2) or Lau Basin (Munidopsis lauensis). We assessed genetic differentiation across a range of spatial scales, from approximately 2.5 km to more than 3000 km. Population structure for Chorocaris sp. 2 was comparable to that of the vent-associated snail Ifremeria nautilei, with a single seemingly well-mixed population within Manus Basin that is genetically differentiated from conspecifics in North Fiji Basin. Population structure for Munidopsis lauensis was more complex, with two genetically differentiated populations in Manus Basin and a third well-differentiated population in Lau Basin. The unexpectedly high level of genetic differentiation between M. lauensis populations in Manus Basin deserves further study since it has implications for conservation and management of diversity in deep-sea hydrothermal vent ecosystems.     

Intense but variable autotrophic activity in a rapidly flushed shallow‐water hydrothermal plume (Kueishantao Islet,Taiwan)

Shallow‐water hydrothermal plumes concomitantly host both photosynthetic and chemoautotrophic organisms in a single biotope. Yet, rate measurements to quantify the contributions of different autotrophic activity types are scarce. Herein, we measured the light and dark dissolved inorganic carbon (DIC) uptake rates in the plume water of the Kueishantao hydrothermal field using the ¹³C‐labeling approach. Seventy percent of the plume‐water samples had chemoautotrophy as the dominant mode of carbon fixation, with the dark DIC uptake rates (up to 18.6 mg C/m³/h) within the range of the primary production in productive inner‐shelf waters. When considered alongside the geochemical and microbiological observations, the rate data reveal the distribution of different trophic activities in the hydrothermal plume. The autotrophic activity at the initial phase of plume dispersal is low. This is explained by the short response time the chemoautotrophs have to the stimulation from vent‐fluid discharge, and the harmful effects of hydrothermal substances on phytoplankton. As plume dispersal and mixing continue, chemoautotrophic activities begin to rise and peak in waters that have low‐to‐moderate Si(OH)₄ content. Toward the plume margin, chemoautotrophy declines to background levels, whereas photosynthesis by phytoplankton regains importance. Our results also provide preliminary indication to the loci of enhanced heterotrophy in the plume. Results of artificial mixing experiments suggest that previously formed plume water is the primary source of microbial inoculum for new plume water. This self‐inoculation mechanism, in combination with the intense DIC uptake, helps to sustain a distinct planktonic autotrophic community in this rapidly flushed hydrothermal plume.     

Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian Ocean

Here we describe novel forms of structural integration between endo- and episymbiotic microbes and an unusual new species of snail from hydrothermal vents in the Indian Ocean. The snail houses a dense population of gamma-proteobacteria within the cells of its greatly enlarged esophageal gland. This tissue setting differs from that of all other vent mollusks, which harbor sulfur-oxidizing endosymbionts in their gills. The significantly reduced digestive tract, the isotopic signatures of the snail tissues, and the presence of internal bacteria suggest a dependence on chemoautotrophy for nutrition. Most notably, this snail is unique in having a dense coat of mineralized scales covering the sides of its foot, a feature seen in no other living metazoan. The scales are coated with iron sulfides (pyrite and greigite) and heavily colonized by epsilon- and delta-proteobacteria, likely participating in mineralization of the sclerites. This novel metazoan-microbial collaboration illustrates the great potential of organismal adaptation in chemically and physically challenging deep-sea environments.     

Archaeal diversity and community development in deep-sea hydrothermal vents

Over the past 35 years, researchers have explored deep-sea hydrothermal vent environments around the globe and studied a number of archaea, their unique metabolic and physiological properties, and their vast phylogenetic diversity. Although the pace of discovery of new archaeal taxa, phylotypes and phenotypes in deep-sea hydrothermal vents has slowed recently, bioinformatics and interdisciplinary geochemistry-microbiology approaches are providing new information on the diversity and community composition of archaea living in deep-sea vents. Recent investigations have revealed that archaea could have originated and dispersed from ancestral communities endemic to hydrothermal vents into other biomes on Earth, and the community structure and productivity of chemolithotrophic archaea are controlled primarily by variations in the geochemical composition of hydrothermal fluids.     

深海热液口Epsilon-变形菌的物种多样性与环境适应机理

Epsilon-变形菌是近年来宏基因组调查发现的深海极端环境如热液喷口富集的重要微生物类群,在海洋碳、氮、氢、硫循环中发挥重要作用。目前对这个纲的研究较少,主要来自于16S rRNA的分类鉴定以及深度测序拼接的基因组序列分析。本文总结了目前对Epsilon-变形菌纲的生态分布及多样性调查研究结果,并对深海热液喷口的Epsilon-变形菌的多种能量代谢方式、强大的趋化运动系统以及与底栖生物的共生关系进行了阐述。这些结果初步揭示了Epsilon-变形菌对深海极端环境的适应机制,并推动对这个极端环境富集的细菌分支的生物学特征认知与资源利用。     

Growth and phylogenetic properties of novel bacteria belonging to the epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Artificial construction of the biocoenosis of deep-sea ecosystem via seeping methane

Deep-sea ecosystems, such as cold seeps and hydrothermal vents, have high biomass, even though they are located in the benthic zone, where no sunlight is present to provide energy for organism proliferation. Based on the coexistence of the reduced gases and chemoautotrophic microbes, it is inferred that the energy from the reduced gases supports the biocoenosis of deep-sea ecosystems. However, there is no direct evidence to support this deduction. Here, we developed and placed a biocoenosis generator, a device that continuously seeped methane, on the 1000-m deep-sea floor of the South China Sea to artificially construct a deep-sea ecosystem biocoenosis. The results showed that microorganisms, including bacteria and archaea, appeared in the biocoenosis generator first, followed by jellyfish and Gammaridea arthropods, indicating that a biocoenosis had been successfully constructed in the deep sea. Anaerobic methane-oxidizing archaea, which shared characteristics with the archaea of natural deep-sea cold seeps, acted as the first electron acceptors of the emitted methane; then, the energy in the electrons was transferred to downstream symbiotic archaea and bacteria and finally to animals. Nitrate-reducing bacteria served as partners to complete anaerobic oxidation of methane process. Further analysis revealed that viruses coexisted with these organisms during the origin of the deep-sea biocoenosis. Therefore, our study mimics a natural deep-sea ecosystem and provides the direct evidence to show that the chemical energy of reduced organic molecules, such as methane, supports the biocoenosis of deep-sea ecosystems.     

Insights into the functional role of fungi in deep-sea hydrothermal vents through the analysis of stable isotopes of carbon,nitrogen, and sulfur

The functional diversity of fungi remains poorly explored in the deep-sea, particularly in hydrothermal vents. Here, we approached this gap through the analysis of stable isotopes of carbon (δ¹³C), nitrogen (δ¹⁵N), and sulfur (δ³⁴S) of fourteen isolates obtained from three deep-sea vent systems of the southern Gulf of California. The δ¹³C results indicated that 60% of the isolates relied on mixed carbon sources fixed by the Calvin-Benson-Bassham and the reductive Tricarboxylic Acid (rTCA) cycles, whereas 40% relied exclusively on rTCA carbon. The δ¹⁵N and δ³⁴S values suggested a dependence on local and external nitrogen sources and the assimilation of chemosynthetic and photosynthetic inputs. Fungal δ¹³C and δ¹⁵N overlapped with those of primary and secondary vent macroconsumers, implying the assimilation of bacterial and invertebrate necromass and their ecological role as parasites. These findings provide insights into the unexplored trophic versatility of fungi in chemosynthetic ecosystems, highlighting their importance in deep-sea trophic dynamics.     

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.     

Gamma- and epsilonproteobacterial ectosymbionts of a shallow-water marine worm are related to deep-sea hydrothermal vent ectosymbionts

The marine oligochaete worm Tubificoides benedii is often found in high numbers in eutrophic coastal sediments with low oxygen and high sulfide concentrations. A dense biofilm of filamentous bacteria on the worm's tail end were morphologically described over 20 years ago, but no further studies of these epibiotic associations were done. In this study, we used fluorescence in situ hybridization and comparative sequence analysis of 16S rRNA and protein-coding genes to characterize the microbial community of the worm's tail ends. The presence of genes involved in chemoautotrophy (cbbL and cbbM) and sulfur metabolism (aprA) indicated the potential of the T. benedii microbial community for chemosynthesis. Two filamentous ectosymbionts were specific to the worm's tail ends: one belonged to the Leucothrix mucor clade within the Gammaproteobacteria and the other to the Thiovulgaceae within the Epsilonproteobacteria. Both T. benedii ectosymbionts belonged to clades that consisted almost exclusively of bacteria associated with invertebrates from deep-sea hydrothermal vents. Such close relationships between symbionts from shallow-water and deep-sea hosts that are not closely related to each other are unusual, and indicate that biogeography and host affiliation did not play a role in these associations. Instead, similarities between the dynamic environments of vents and organic-rich mudflats with their strong fluctuations in reductants and oxidants may have been the driving force behind the establishment and evolution of these symbioses.