A Novel Archaeal Lineage in Boiling Hot Springs around Oyasukyo Gorge (Akita, Japan).

A novel deep-branching archaeal lineage was discovered at high-temperature hot springs around Oyasukyo Gorge in Akita Prefecture, Japan. Actively boiling hot spring water contained >1×104 microbes mL-1. The microbial community composition assessed by analyzing 16S rRNA gene amplicons revealed that the dominant bacterial phyla were Proteobacteria and Aquificae (>50% of the microbial composition) in samples collected in 2016 and 2019, respectively. Approximately 10% of the reads obtained in both years were not assigned to any taxonomy. The more detailed phylogenetic positions of the unassigned sequences identified using a clone library and phylogenetic tree showed that they formed a clade that was independent, distantly related to known phyla, and had low similarity (<82%) to all other sequences in available databases. The present results suggest that this novel archaeal phylum-level lineage thrives in boiling hot springs in Japan.

Subseafloor sulphide deposit formed by pumice replacement mineralisation.

Seafloor massive sulphide (SMS) deposits, modern analogues of volcanogenic massive sulphide (VMS) deposits on land, represent future resources of base and precious metals. Studies of VMS deposits have proposed two emplacement mechanisms for SMS deposits: exhalative deposition on the seafloor and mineral and void space replacement beneath the seafloor. The details of the latter mechanism are poorly characterised in detail, despite its potentially significant role in global metal cycling throughout Earth's history, because in-situ studies require costly drilling campaigns to sample SMS deposits. Here, we interpret petrographic, geochemical and geophysical data from drill holes in a modern SMS deposit and demonstrate that it formed via subseafloor replacement of pumice. Samples from the sulphide body and overlying sediment at the Hakurei Site, Izena Hole, middle Okinawa Trough indicate that sulphides initially formed as aggregates of framboidal pyrite and matured into colloform and euhedral pyrite, which were replaced by chalcopyrite, sphalerite and galena. The initial framboidal pyrite is closely associated with altered material derived from pumice, and alternating layers of pumiceous and hemipelagic sediments functioned as a factory of sulphide mineralisation. We infer that anhydrite-rich layers within the hemipelagic sediment forced hydrothermal fluids to flow laterally, controlling precipitation of a sulphide body extending hundreds of meters.

A Miocene impact ejecta layer in the pelagic Pacific Ocean.

Meteorite impacts have caused catastrophic perturbations to the global environment and mass extinctions throughout the Earth's history. Here, we present petrographic and geochemical evidence of a possible impact ejecta layer, dating from about 11 Ma, in deep-sea clayey sediment in the Northwest Pacific. This clay layer has high platinum group element (PGE) concentrations and features a conspicuous negative Os isotope anomaly (187Os/188Os as low as ~0.2), indicating an influx of extraterrestrial material. It also contains abundant spherules that include pseudomorphs suggestive of porphyritic olivine as well as spinel grains with euhedral, dendritic and spherical forms and NiO contents as great as 23.3 wt%, consistent with impact ejecta. Osmium isotope stratigraphy suggests a most plausible depositional age of ~11 Ma (Miocene) for this layer, as determined by fitting with the seawater evolution curve. No large impact crater of this age is known on land, even within the relatively large uncertainty range of the relative Os age. Thus, we suggest that an unrecognised impact event in the middle or late Miocene produced the impact ejecta layer of the Northwest Pacific.

Onboard experiment investigating metal leaching of fresh hydrothermal sulfide cores into seawater.

We observed the initial release rate of metals from four fresh (i.e., without long time exposure to the atmosphere) hydrothermal sulfide cores into artificial seawater. The sulfide samples were collected by seafloor drilling from the Okinawa Trough by D/V Chikyu, powdered under inert gas, and immediately subjected to onboard metal-leaching experiments at different temperatures (5 °C and 20 °C), and under different redox conditions (oxic and anoxic), for 1-30 h. Zinc and Pb were preferentially released from sulfide samples containing various metals (i.e., Mn, Fe, Cu, Zn, Cd, and Pb) into seawater. Under oxic experimental conditions, Zn and Pb dissolution rates from two sulfide samples composed mainly of iron disulfide minerals (pyrite and marcasite) were higher than those from two other sulfide samples with abundant sphalerite, galena, and/or silicate minerals. Scanning electron microscopy confirmed that the high metal-releasing sample contained several galvanic couples of iron disulfide with other sulfide minerals, whereas the low metal-releasing sample contained fewer galvanic couples or were coated by a silicate mineral. The experiments overall confirmed that the galvanic effects with iron disulfide minerals greatly induce the initial release of Zn and Pb from hydrothermal sulfides into seawater, especially under warm oxic conditions.

Evaluation of the toxicity of leaches from hydrothermal sulfide deposits by means of a delayed fluorescence-based bioassay with the marine cyanobacterium Cyanobium sp. NIES-981.

The commercial use of metals such as copper, lead, and zinc has markedly increased in recent years, resulting in increased interest in deep-sea mining of seafloor hydrothermal sulfide deposits. However, the full extent of the impact of deep-sea mining at hydrothermal field deposits on the environment remains unclear. In addition to impacting the deep sea, the leaching of heavy metals from extracted sulfide mineral may also affect the upper ocean zones as the sulfide rock is retrieved from the seafloor. Here, we used a delayed fluorescence-based bioassay using the marine cyanobacterium Cyanobium sp. NIES-981 to evaluate the toxicity of three sulfide core samples obtained from three drill holes at the Izena Hole, middle Okinawa Trough, East China Sea. Leaches from two of the cores contained high concentrations of zinc and lead, and they markedly inhibited delayed fluorescence in Cyanobium sp. NIES-981 compared with control. By examining the toxicity of artificial mixed-metal solutions with metal compositions similar to those of the leaches, we confirmed that this inhibition was a result of high zinc and lead concentrations into the leaches. In addition, we conclude that this delayed fluorescence-based bioassay is a viable method for use by deep-sea mining operations because it is quicker and requires less laboratory space and equipment than the standard assay.

Population history of deep-sea vent and seep Provanna snails (Mollusca: Abyssochrysoidea) in the northwestern Pacific.

BACKGROUND: Gastropods of the genus Provanna are abundant and widely distributed in deep-sea chemosynthetic environments with seven extant species described in the northwestern Pacific. METHODS: We investigated the population history and connectivity of five Provanna species in the northwestern Pacific through population genetic analyses using partial sequences of the cytochrome c oxidase subunit I gene. RESULTS: We found that P. subglabra, the most abundant and genetically diverse species, is genetically segregated by depth. Among the five species, the three comparatively shallower species (P. lucida, P. kuroshimensis, P. glabra) had a more constant demographic history compared to the deeper species (P. subglabra, P.  clathrata). DISCUSSION: Environmental differences, especially depth, appears to have a role in the segregation of Provanna snails. The population of P. clathrata in the Irabu Knoll appears to have expanded after P. subglabra population. The remaining three species, P. lucida, P. kuroshimensis, and P. glabra, are only known from a single site each, all of which were shallower than 1,000 m. These data indicate that Provanna gastropods are vertically segregated, and that their population characteristics likely depend on hydrothermal activities.

Horizontal sliding of kilometre-scale hot spring area during the 2016 Kumamoto earthquake.

We report horizontal sliding of the kilometre-scale geologic block under the Aso hot springs (Uchinomaki area) caused by vibrations from the 2016 Kumamoto earthquake (Mw 7.0). Direct borehole observations demonstrate the sliding along the horizontal geological formation at ~50 m depth, which is where the shallowest hydrothermal reservoir developed. Owing to >1 m northwest movement of the geologic block, as shown by differential interferometric synthetic aperture radar (DInSAR), extensional open fissures were generated at the southeastern edge of the horizontal sliding block, and compressional deformation and spontaneous fluid emission from wells were observed at the northwestern edge of the block. The temporal and spatial variation of the hot spring supply during the earthquake can be explained by the horizontal sliding and borehole failures. Because there was no strain accumulation around the hot spring area prior to the earthquake and gravitational instability could be ignored, the horizontal sliding along the low-frictional formation was likely caused by seismic forces from the remote earthquake. The insights derived from our field-scale observations may assist further research into geologic block sliding in horizontal geological formations.

Leaching of Metals and Metalloids from Hydrothermal Ore Particulates and Their Effects on Marine Phytoplankton.

Seafloor massive sulfide deposits have attracted much interest as mineral resources. Therefore, the potential environmental impacts of full-scale mining should be considered. In this study, we focused on metal and metalloid contamination that could be triggered by accidental leakage and dispersion of hydrothermal ore particulates from mining vessels into surface seawater. We determined the leaching potential of metals and metalloids from four hydrothermal ores collected from the Okinawa Trough into aerobic seawater and then evaluated the toxic effects of ore leachates on a phytoplankton species, Skeletonema marinoi-dohrnii complex, which is present ubiquitously in the ocean. Large amounts of metals and metalloids were released from the ground hydrothermal ores into seawater within 5 min under aerobic conditions. The main components of leachates were Zn + Pb, As + Sb, and Zn + Cu, which were obtained from the Fe-Zn-Pb-rich and Zn-Pb-rich zero-age, Ba-rich, and Fe-rich ores, respectively. The leachates had different chemical compositions from those of the ore. The rapid release and difference in chemical compositions between the leachates and the ores indicated that substances were not directly dissolved from the sulfide-binding mineral phase but from labile phases mainly on the adsorption-desorption interface of the ores under these conditions. All ore leachates inhibited the growth of S. marinoi-dohrnii complex but with different magnitudes of toxic effects. These results indicate that the fine particulate matter of hydrothermal ores is a potential source of toxic contamination that may damage primary production in the ocean. Therefore, we insist on the necessity for the prior evaluation of toxic element leachability from mineral ores into seawater to minimize mining impacts on the surface environment.

Deepest and hottest hydrothermal activity in the Okinawa Trough: the Yokosuka site at Yaeyama Knoll.

Since the initial discovery of hydrothermal vents in 1977, these 'extreme' chemosynthetic systems have been a focus of interdisciplinary research. The Okinawa Trough (OT), located in the semi-enclosed East China Sea between the Eurasian continent and the Ryukyu arc, hosts more than 20 known vent sites but all within a relatively narrow depth range (600-1880 m). Depth is a significant factor in determining fluid temperature and chemistry, as well as biological composition. However, due to the narrow depth range of known sites, the actual influence of depth here has been poorly resolved. Here, the Yokosuka site (2190 m), the first OT vent exceeding 2000 m depth is reported. A highly active hydrothermal vent site centred around four active vent chimneys reaching 364°C in temperature, it is the hottest in the OT. Notable Cl depletion (130 mM) and both high H2 and CH4 concentrations (approx. 10 mM) probably result from subcritical phase separation and thermal decomposition of sedimentary organic matter. Microbiota and fauna were generally similar to other sites in the OT, although with some different characteristics. In terms of microbiota, the H2-rich vent fluids in Neuschwanstein chimney resulted in the dominance of hydrogenotrophic chemolithoautotrophs such as Thioreductor and Desulfobacterium. For fauna, the dominance of the deep-sea mussel Bathymodiolus aduloides is surprising given other nearby vent sites are usually dominated by B. platifrons and/or B. japonicus, and a sponge field in the periphery dominated by Poecilosclerida is unusual for OT vents. Our insights from the Yokosuka site implies that although the distribution of animal species may be linked to depth, the constraint is perhaps not water pressure and resulting chemical properties of the vent fluid but instead physical properties of the surrounding seawater. The potential significance of these preliminary results and prospect for future research on this unique site are discussed.

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor.

Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the Mid-Okinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ13C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to ~45 °C. However, in the past, higher temperatures of 106-198 °C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

Helium and methane sources and fluxes of shallow submarine hydrothermal plumes near the Tokara Islands, Southern Japan.

Shallow submarine volcanoes have been newly discovered near the Tokara Islands, which are situated at the volcanic front of the northern Ryukyu Arc in southern Japan. Here, we report for the first time the volatile geochemistry of shallow hydrothermal plumes, which were sampled using a CTD-RMS system after analyzing water column images collected by multi-beam echo sounder surveys. These surveys were performed during the research cruise KS-14-10 of the R/V Shinsei Maru in a region stretching from the Wakamiko Crater to the Tokara Islands. The 3He flux and methane flux in the investigated area are estimated to be (0.99-2.6) × 104 atoms/cm2/sec and 6-60 t/yr, respectively. The methane in the region of the Tokara Islands is a mix between abiotic methane similar to that found in the East Pacific Rise and thermogenic one. Methane at the Wakamiko Crater is of abiotic origin but affected by isotopic fractionation through rapid microbial oxidation. The helium isotopes suggest the presence of subduction-type mantle helium at the Wakamiko Crater, while a larger crustal component is found close to the Tokara Islands. This suggests that the Tokara Islands submarine volcanoes are a key feature of the transition zone between the volcanic front and the spreading back-arc basin.

Rapid growth of mineral deposits at artificial seafloor hydrothermal vents.

Seafloor massive sulphide deposits are potential resources for base and precious metals (Cu-Pb-Zn ± Ag ± Au), but difficulties in estimating precise reserves and assessing environmental impacts hinder exploration and commercial mining. Here, we report petrological and geochemical properties of sulphide chimneys less than 2 years old that formed where scientific boreholes vented hydrothermal fluids in the Iheya-North field, Okinawa Trough, in East China Sea. One of these infant chimneys, dominated by Cu-Pb-Zn-rich sulphide minerals, grew a height of 15 m within 25 months. Portions of infant chimneys are dominated by sulphate minerals. Some infant chimneys are sulphide-rich similar to high-grade Cu-Pb-Zn bodies on land, albeit with relatively low As and Sb concentrations. The high growth rate reaching the 15 m height within 25 months is attributed to the large hydrothermal vent more than 50 cm in diameter created by the borehole, which induced slow mixing with the ambient seawater and enhanced efficiency of sulphide deposition. These observations suggest the possibility of cultivating seafloor sulphide deposits and even controlling their growth and grades through manipulations of how to mix and quench hydrothermal fluids with the ambient seawater.

ESR dating of barite in sulphide deposits formed by the sea-floor hydrothermal activities.

Barite is a mineral newly found to be practically useful for electron spin resonance (ESR) dating of sulphide deposits formed by the sea-floor hydrothermal activities. The recent studies for the properties of the ESR dating signal in barite are summarised in the present paper as well as the formulas for corrections for accurate dose-rate estimation are developed including the dose-rate conversion factors, shape correction for gamma-ray dose and decay of (226)Ra. Although development of the techniques for ESR dating of barite has been completed, further comparative studies with other dating techniques such as U-Th and (226)Ra-(210)Pb dating are necessary for the technique to be widely used.

Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs.

Two thermophilic, strictly anaerobic, Gram-negative bacteria, designated strains AZM34c06(T) and AZM44c09(T), were isolated from terrestrial hot springs in Japan. The optimum growth conditions for strain AZM34c06(T) were 60 °C, pH 7.4 and 0% additional NaCl, and those for strain AZM44c09(T) were 70 °C, pH 7.4 and 0% additional NaCl. Complete genome sequencing was performed for both strains, revealing genome sizes of 2.19 Mbp (AZM34c06(T)) and 2.01 Mbp (AZM44c09(T)). Phylogenetic analyses based on 16S rRNA gene sequences and the concatenated predicted amino acid sequences of 33 ribosomal proteins showed that both strains belonged to the genus Thermotoga. The closest relatives of strains AZM34c06(T) and AZM44c09(T) were the type strains of Thermotoga lettingae (96.0% similarity based on the 16S rRNA gene and 84.1% similarity based on ribosomal proteins) and Thermotoga hypogea (98.6 and 92.7% similarity), respectively. Using blast, the average nucleotide identity was 70.4-70.5% when comparing strain AZM34c06(T) and T. lettingae TMO(T) and 76.6% when comparing strain AZM44c09(T) and T. hypogea NBRC 106472(T). Both values are far below the 95% threshold value for species delineation. In view of these data, we propose the inclusion of the two isolates in the genus Thermotoga within two novel species, Thermotoga profunda sp. nov. (type strain AZM34c06(T) = NBRC 106115(T) = DSM 23275(T)) and Thermotoga caldifontis sp. nov. (type strain AZM44c09(T) = NBRC 106116(T) = DSM 23272(T)).

Growth temperatures of archaeal communities can be estimated from the guanine-plus-cytosine contents of 16S rRNA gene fragments.

Prokaryote growth temperatures in environmental samples are difficult to measure because it is hard to culture viable prokaryotes in natural environments. We comprehensively surveyed growth temperatures and 16S rRNA sequences of prokaryotes to estimate their growth temperatures based on guanine-plus-cytosine contents (P(GC)) of their 16S rRNA sequences. We focused on archaea because of the wide range of growth temperatures within this group. Their minimum (Tmin), optimum (Topt) and maximum (Tmax) growth temperatures correlated strongly with PGC of their 16S rRNA genes. Linear regression equations were established to approximate Tmin, Topt and Tmax from P(GC). We also established a linear regression equation for calculating P(GC) of 16S rRNA genes based on the melting temperatures (Tm) of PCR fragments, without using a clone library or sequencing. Environmental samples were obtained from a wide variety of microbial natural habitats. Tm of archaeal 16S rRNA genes amplified by real-time PCR were determined by melting curve analysis. Based on those values, P(GC) of 16S rRNA genes and mean Tmin, Topt and Tmax were calculated using the linear regression equations. These temperatures correlated strongly with the in situ temperatures. Tmax agreed particularly well with these temperatures, suggesting many archaea live at their maximum growth temperatures.

Characteristics of microbial communities in crustal fluids in a deep-sea hydrothermal field of the suiyo seamount.

To directly access the sub-seafloor microbial communities, seafloor drilling has been done in a deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific. In the present study, crustal fluids were collected from the boreholes, and the bacterial and archaeal communities in the fluids were investigated by culture-independent molecular analysis based on 16S rRNA gene sequences. Bottom seawater, sands, rocks, sulfide mound, and chimneys were also collected around the boreholes and analyzed for comparisons. Comprehensive analysis revealed the characteristics of the microbial community composition in the crustal fluids. Phylotypes closely related to cultured species, e.g., Alteromonas, Halomonas, Marinobacter, were relatively abundant in some crustal fluid samples, whereas the phylotypes related to Pelagibacter and the SUP05-group were relatively abundant in the seawater samples. Phylotypes related to other uncultured environmental clones in Alphaproteobacteria and Gammaproteobacteria were relatively abundant in the sand, rock, sulfide mound, and chimney samples. Furthermore, comparative analysis with previous studies of the Suiyo Seamount crustal fluids indicates the change in the microbial community composition for 3 years. Our results provide novel insights into the characteristics of the microbial communities in crustal fluids beneath a deep-sea hydrothermal field.

Iron-based microbial ecosystem on and below the seafloor: a case study of hydrothermal fields of the southern mariana trough.

Microbial community structures in deep-sea hydrothermal vents fields are constrained by available energy yields provided by inorganic redox reactions, which are in turn controlled by chemical composition of hydrothermal fluids. In the past two decades, geochemical and microbiological studies have been conducted in deep-sea hydrothermal vents at three geographically different areas of the Southern Mariana Trough (SMT). A variety of geochemical data of hydrothermal fluids and an unparalleled microbiological dataset of various samples (i.e., sulfide structures of active vents, iron-rich mats, borehole fluids, and ambient seawater) are available for comparative analyses. Here, we summarize the geochemical and microbiological characteristics in the SMT and assess the relationship between the microbial community structures and the fluid geochemistry in the SMT by thermodynamic modeling. In the high temperature vent fluids, aerobic sulfide-oxidation has the potential to yield large amounts of bioavailable energy in the vent fluids, which is consistent with the detection of species related to sulfide-oxidizing bacteria (such as Thiomicrospira in the Gammaproteobacteria and Sulfurimonas in the Epsilonproteobacteria). Conversely, the bioavailable energy yield from aerobic iron-oxidation reactions in the low-temperature fluids collected from man-made boreholes and several natural vents were comparable to or higher than those from sulfide-oxidation. This is also consistent with the detection of species related to iron-oxidizing bacteria (Mariprofundus in the Zetaproteobacteria) in such low-temperature samples. The results of combination of microbiological, geochemical, and thermodynamic analyses in the SMT provide novel insights into the presence and significance of iron-based microbial ecosystems in deep-sea hydrothermal fields.

Meiothermus hypogaeus sp. nov., a moderately thermophilic bacterium isolated from a hot spring.

A novel moderately thermophilic, red-pigmented bacterium, designated strain AZM34c11(T), was isolated from the bottom of a 1000 m-deep drilled well located in a Japanese hot spring. Cells were Gram-negative and grew optimally at 50 °C, at pH 7.6 and with 0-0.3% (w/v) NaCl. Analysis of the 16S rRNA gene sequence revealed that the isolate belonged to the genus Meiothermus. Levels of 16S rRNA gene sequence similarity between strain AZM34c11(T) and the type strains of recognized Meiothermus species were 88.2-94.8%. Strain AZM34c11(T) was distinguished from recognized Meiothermus species by its cellular fatty acid profile: iso-C(16:0) was one of the predominant components and hydroxy fatty acids were present only in trace amounts. The strain could also be differentiated based on its growth substrate preferences and characteristic enzyme reactions. On the basis of these results, strain AZM34c11(T) is considered to represent a novel species of the genus Meiothermus, for which the name Meiothermus hypogaeus sp. nov. is proposed. The type strain is AZM34c11(T) (=NBRC 106114(T) =DSM 23238(T)).

Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

Archaeal diversity and distribution along thermal and geochemical gradients in hydrothermal sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa trough.

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the ecophysiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.