http://orcid.org/0000-0002-5614-8464
Figures
Abstract
The Shinkai Seep Field (SSF) in the southern Mariana forearc discovered in 2010 is the deepest (~5,700 m in depth) known serpentinite-hosted ecosystem dominated by a vesicomyid clam, Calyptogena (Abyssogena) mariana. The pioneering study presumed that the animal communities are primary sustained by reducing fluid originated from the serpentinization of mantle peridotite. For understanding the nutrient and energy sources for the SSF community, this study conducted four expeditions to the SSF and collected additional animal samples such as polychaetes and crustaceans as well as sediments, fragments of chimneys developing on fissures of serpentinized peridotite, seeping fluid on the chimneys, and pore water within the chimneys. Geochemical analyses of seeping fluids on the chimneys and pore water of the chimneys revealed significantly high pH (~10) that suggest subseafloor serpentinization controlling fluid chemistry. Stable isotope systematics (carbon, nitrogen, and sulfur) among animals, inorganic molecules, and environmental organic matter suggest that the SSF animal community mostly relies on the chemosynthetic production while some organisms appear to partly benefit from photosynthetic production despite the great depth of SSF.
Citation: Onishi Y, Yamanaka T, Okumura T, Kawagucci S, Watanabe HK, Ohara Y (2018) Evaluation of nutrient and energy sources of the deepest known serpentinite-hosted ecosystem using stable carbon, nitrogen, and sulfur isotopes. PLoS ONE 13(6): e0199000. https://doi.org/10.1371/journal.pone.0199000
Editor: Sébastien Duperron, Museum National d'Histoire Naturelle, FRANCE
Received: September 5, 2017; Accepted: May 30, 2018; Published: June 15, 2018
Copyright: © 2018 Onishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This study was partly supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT; http://www.mext.go.jp/en/) of Japan through Special Coordination Funds ‘TAIGA’ project (20109005; T. Y.), Kiban-S (16H06347; Y. O.) and a grant-in aids for Young Scientists (15H05468; T. O.), and Japan Science and Technology Agency (JST; http://www.jst.go.jp/kisoken/crest/en/index.html) of CREST program (JPMJCR11A2; T. Y.), Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interest exist.
Introduction
Serpentinization of ultramafic rocks such as peridotite produces H2-rich fluid [1] that can yield CH4 by CO2 reduction through biotic (e.g., microbial methanogenesis) and/or abiotic (e.g., Fischer-Tropsch Type reaction) processes [2–5]. H2 and CH4 can be oxidized exothermically, coupled with sulfate reduction by microbes such as sulfate-reducing bacteria (SRB) in the anoxic habitats, leading to H2S production [6]. Consequently, serpentinization-associated fluids can provide relevant energy sources for chemolithoautotrophic primary producers using H2 and H2S, as well as methanotrophs using CH4. Such productivity often becomes the basis for nourishing high-biomass faunal communities (e.g. [7]).
In 2010, an animal community composed mainly of vesicomyid clam, Calyptogena (Abyssogena) mariana [8], was discovered at c. 5500–5700 m depth in a site on the inner trench slope of the southern Mariana forearc named the Shinkai Seep Field (SSF) [9]. Since this clam relies on endosymbiotic chemoautotroph for nutrient, this community was presumed to be chemosynthesis-based [8, 9]. Subsequent expeditions to the SSF until 2015 revealed numerous white chimneys composed of brucite and/or calcium carbonate minerals [10]. Biological surveys in both the clam colony and the chimney sites also revealed a higher diversity of animals, including for example Phyllochaetopterus polychaetes and a gastropod Provanna cingulata [9–11], which possibly feed on chemolithoautotrophic microbes. Although not visible, the presence of chemically unstable brucite-carbonate chimneys [10] and presumably chemosynthetic symbiont-associated animals strongly suggests the existence of alkaline and reducing fluid emission (or discharge). Fluids discharging along fissures of peridotites either exposed to the seafloor or covered by thin sediments is believed to be associated with the subseafloor serpentinization in the SSF, as observed in the Lost City hydrothermal field (discharging fluid temperature ≤91°C [12, 13]) and the South Chamorro Seamount (though shimmering fluid was not observed and temperature anomalies not reported [14, 15]). There has so far been no geochemical data from the SSF, however, to confirm the fluid seepage or the energetic relationship between geological processes and the faunal community there.
Stable isotopic signatures of biophilic elements have been used to elucidate the basal foods and trophic levels for animals around hydrothermal vents and hydrocarbon seeps in the deep-sea (e.g. [16, 17]). Carbon and sulfur isotopic compositions of animal tissues are used to estimate basal foods because there are negligible enrichments of 13C (0 to 1‰; [18]) or depletions of 34S (0 to 5‰; [19]) through trophic levels [20] as well as assimilation of environmental molecules by each primary producer (e.g. [21–23]). Nitrogen isotopic composition is used to assess the trophic level of a species because an increase of +3.4‰ of δ15N is usually considered along with increasing a trophic level [24]. The combined use of these three isotopic signatures for each constituent of an ecosystem can therefore provide an estimation of food-web structure (e.g. [17]).
The purpose of this study is at first to geochemically characterize the SSF seeping fluids that likely support animal communities there and then to elucidate energy and nutrient sources of dominant animals in the SSF. For this purpose, we conducted sampling and chemical analyses of the relevant fluids, as well as carbon, sulfur, and nitrogen isotopic analyses of the animal soft tissues and organic matter in the sediments and the chimneys.
Materials and methods
Study site
The Mariana arc-trench system is a typical non accretionary convergent plate margin [25] where the Pacific Plate is subducting beneath the Philippine Sea Plate. The SSF is located on a deep inner trench slope of the southern Mariana forearc, ~80 km northeast of the Challenger Deep [9] (Fig 1A). The southern Mariana forearc is young and tectonically active, hosting serpentinized peridotite along the deep inner trench slope [9]. In the SSF, eleven clam colony sites mainly composed of C. mariana [8] (CO Sites 1–11 indicated by black numbers in Fig 1B) and four brucite-carbonate chimney structure sites (CH Sites 1–4 indicated by red numbers in Fig 1B) have been identified [10]. These chimney structures were observed to develop from the fissures of the serpentinized peridotite [10].
The black and red numbers beside the yellow and red circles indicate each site number (CO and CH, respectively). The size of yellow circles corresponds to the size of clam colonies (modified from ref. [10]).
Sampling procedure and on-board sample processing
Expeditions and dives.
Animals, sediment cores, chimney fragments, and fluid samples were collected using the deep-submergence vehicle (DSV) Shinkai 6500 during expeditions YK13-08, YK14-13, and YK15-11 of the R/V Yokosuka, and using the remotely operated vehicle (ROV) Kaiko (with vehicle Mk-IV) during the expedition KR16-14 of the R/V Kairei (Table 1 and Fig 1B). All four expeditions and sample collections were performed under the permission of the US Fish and Wildlife Service (SUP#12541–12001 for YK13-08, SUP#12541–14001 for YK14-13, SUP#12541–15002 for YK15-11, and SUP#12541–16002 for KR16-14).
Animals.
Animal sampling was conducted using a suction sampler and a scoop operated by the manipulators of the DSV Shinkai 6500 during the dives 6K#1362, 6K#1365, and 6K#1366 in expedition YK13-08 and dive 6K#1404 in expedition YK14-13. Each organism was identified on-board based on their morphology, before being frozen and preserved at −80°C in a deep-freezer for on-shore analyses.
Calyptogena clams were collected from three sites (CO Sites 2, 4, and 7 in Fig 1B) with a scoop, and simultaneously other animal species such as the snail Bayerius cf. arnoldi, polychaetes (unidentified species of Maldanidae, Lumbrineridae, and Eunicidae), crustaceans (unidentified species of amphipods and isopods), nematodes (species unidentified), and anthozoans (species unidentified) were collected in the CO Sites 2 and 4. Bayerius cf. arnoldi and the anthozoans were observed on the seafloor during the dive surveys, while the other animals were not seen on the seafloor due to its smaller size and therefore it is unknown whether they lived on or inside sediment. In CO Site 2, frenulate tubeworms on the sediment were collected with manipulators. Moreover, nautiliniellid polychaetes were found inside the clam shells. In CH Sites 1 and 3, animals on the chimney surface such as B. cf. arnoldi, polychaetes (unidentified species of Phyllochaetopterus, Polynoidae, Lumbrineridae, and Eunicidae), crustaceans (unidentified species of amphipods and leptostracans), nematodes (species unidentified), and anthozoans (species unidentified) were collected by a suction sampler.
POM in seawater.
For suspended particulate organic matter (POM) analyses, seawater samples were collected with a Niskin bottle at the CO Sites 2 and 4 as well as an area approximately 400 km west of the SSF (11°13.58’N, 139°15.18’E) (Table 1). POM in the seawater samples was subsampled on-board by a glass fiber filter (Advantec GC-5, the filter was heated at c. 450°C, 2h before using). The filters were preserved in a −80°C freezer until on-shore analyses. A filtrate at the reference site was used as a reference seawater for fluid chemistry.
Sediment.
Sediment cores were obtained from inside (inside core) and at the edge (edge core) of a clam colony at CO Site 4 using a push corer (30 cm in length and 10 cm in diameter) during expedition YK13-08. Another sediment core was obtained as a reference (reference core) several kilometers away from the SSF (11°40.65’N, 142°59.26’E) (Table 1) during expedition YK14-13. Immediately after on-board recovery, the sediment cores were sliced horizontally at 3–5 cm intervals according to the sedimentary layers with different colors. Pore water was squeezed from about one-quarter of each sediment slice using a plastic syringe with a vice, following the procedures in ref. [26]. Another one-quarter of each sediment slice was provided for sulfide extraction on-board as described later. The remaining sediment was frozen at −80°C and taken back to an on-shore laboratory. An aliquot of the pore water was used for on-board analyses while the rest was filtered using a membrane filter (pore size = 0.45 µm) and stored in a refrigerator until further analyses. For methane analysis, 2 cm3 of sediments were subsampled with a sterilized cut syringe and were placed into 30 mL glass vials with 3 mL water containing 0.1 mL of saturated HgCl2 solution. Each vial was sealed with a Teflon-coated butyl-rubber septum with an aluminum seal and heated to 70°C for ~30 min. After cooling at room temperature, the methane concentration was determined on-board, as described below.
Chimney.
Chimneys of the SSF have been classified into three types based on the mineralogy, the appearance (colors, spiky/tubular textures, and microbial mats), and the animal community inhabiting the surface [10]: (1) the Type I brucite-rich chimney has well developed microbial mats but a lack of densely-colonized animal, likely due to the relatively high flux of the highly alkaline and reduced fluid, (2) the Type II carbonate-rich chimney has obvious microbial mats and densely-colonized animals on it, the relatively lower fluid flux likely allows organisms to settle and flourish, and (3) the Type III brucite-poor chimney is dissolving and alternating after the fluid discharge has ceased.
In this study, eight brucite-carbonate chimneys were collected from the SSF CH Sites (Table 1). Two Type II chimneys were recovered from CH Site 1 (Chim 2) during expedition YK13-08 and CH Site 3 (Chim 4) during expedition YK14-13. Two Type I chimneys were also sampled from CH Site 4 (Chim 5 and 6) during expedition YK14-13. Each of these four chimneys was divided into four vertical sections: the root, lower, middle, and upper sections. Each section of the chimneys was further divided into inner and surfaces when possible. The middle part of the sample Chim 2 was lithified and sliced into round slices. These fragments of chimneys were preserved at −80°C for on-shore analyses. The pore waters of fragile parts of the chimneys (Chim 5 from CH Site 4 during expedition YK14-13, Chim 9 from CH Site 4 and Chim 10 from CH Site 3 during expedition YK15-11, and Chim 16 from CH Site 4 and Chim 18 from CH Site 3 during expedition KR16-14) were also collected by the same methods as for the sediment pore water. For methane analyses, ~5 cm3 of chimney fragments were subsampled from the inner part of the chimney structure with pore water and placed into 140 mL glass vials. When the chimney samples were massive and lithified, an agate mortar and pestle was used to crush the fragments into pebble-sized fragments (<5 mm in diameter) before placed into the vials. Each vial was sealed with a Teflon-coated butyl-rubber septum with an aluminum seal after adding 0.1 mL of saturated HgCl2 solution. The vials were kept at room temperature until on-shore analysis.
Seeping fluids.
For on-site fluid sampling, we used a ‘D-WHATS’ sampler, a version of the WHATS-series gas tight fluid samplers [27, 28], and an all titanium fluid inlet with a temperature probe. D-WHATS is designed to resist pressure up to 100 MPa and has four 50 mL bottles made by high tensile titanium alloy. We attempted to collect discharging geofluids holding up the fluid inlet of the sampler just above the colonies (CO Sites 2 and 4) and the chimneys (CH Sites 1, 3, and 4) during the expeditions YK13-08, YK14-13, YK15-11, and KR16-14. Although no visual signature of fluid discharge was seen, a chemical signature suggesting the existence of seepage was detected (see Results). As a reference seawater sample, we used the filtrate after removal of the reference POM.
Since the fluid seepage was evidently very slow, we developed a seafloor-deployed fluid accumulator, named the proto-GATS (Geofluid Accumulating Trap System) marker (hereinafter, referred to as GATS marker), in order to accumulate the invisible but presumably discharging fluids on board in haste. The concept of the GATS marker is the displacement of pre-filled seawater by presumably buoyant (lower-density) deep-sourced seeping fluid. The GATS marker consists of a 40 cm-long transparent polycarbonate tube (generally used for push corer), a vinyl chloride plate for sealing one end of the tube fixed with stainless-steel screw nails and resin type adhesive, a buoyant marker attached to the outside of the plate for keeping the GATS marker vertical, and four stainless-steel chains at the open-ended side serving as ballasts. The expected sample volume of the GATS marker (~3,300 mL) is sufficient to fill the bottles (50 mL) and fluid path tubes of the D-WHATS (~100 mL). Two GATS markers (named GATS-blue and -red) were deployed on the base of Type II chimneys at CH Site 3 after the elongated chimneys were broken during the dive 6K#1433 in expedition YK15-11. During the dive KIV#709 in expedition KR16-14 (471 days after deployment), we identified whitish precipitations on the inner wall and edges of GATS-blue, whereas black precipitations were identified on the inner wall of GATS-red. For each GATS marker, approximately 1 L of the ~3 L fluid accumulated was carefully sampled by D-WHATS.
Analytical methods
Analytical methods used in this study are listed in Table 2. Animal soft tissue samples for carbon and nitrogen isotope measurements were treated with 1 N HCl, followed by treatment with chloroform:methanol mixture (3:1 by volume) to remove carbonates [29] and lipids [30]. Then, they were lyophilized and pulverized. For sulfur isotope measurements, animal soft tissues were dialyzed repeatedly using 1M LiCl solution to remove excess seawater sulfates [31, 32]. All organic sulfur species in the dialyzed soft tissue were converted to sulfate using a Parr Bomb 1108 apparatus and then recovered as a BaSO4 precipitate in accordance with a previously reported methodology [31, 32].
Fluid pH, alkalinity, and concentrations of ammonium and silica of all fluid samples were measured on-board using pH electrode and colorimetric analyses as described by ref. [33]. Major cation (Na+, Mg2+, K+, and Ca2+) and anion (Cl−, Br−, and SO42−) in the pore water and fluid samples were quantified with inductivity coupled plasma optical emission spectrometer (ICP-OES) and ion chromatography, respectively. The accuracies and precisions of ICP-OES and ion chromatography analyses were verified using IAPSO seawater. Nitrate concentrations were measured by HPLC [34] and calibrated using the CSK (The International Coordination Group for Cooperative Study of the Kuroshio and Adjacent Region) standard distributed by Wako Pure Chemical Industries, Ltd.
H2S concentrations of the fluid samples were quantified by the weight of HgS, which was precipitated by the addition of a HgCl2 solution. Analytical precisions of the H2S concentration were estimated to be within 5%. The precipitated HgS was converted to BaSO4 by adding inverse aqua regia (nitric acid:hydrochloric acid = 3:1, by volume), and subsequently bromine for dissolution and oxidation, following the method in ref. [35]. Sulfate ions dissolved in the fluids were also recovered as BaSO4 precipitates.
For total organic carbon (TOC) and total nitrogen (TN) measurements, frozen POM, sediment, and chimney samples were lyophilized. A 1 N HCl solution was added to remove carbonate before being dried in vacuo using NaOH pellets as a desiccant. To measure sulfide concentrations in sediment samples, a portion of the sediment was used for the extraction of acid volatile sulfide-sulfur (AVS) on-board, following the referenced procedures in ref. [36]. The AVS extracted as CdS precipitates was oxidized using a few drops of c. 30% hydrogen peroxide solution and converted into BaSO4 precipitate by the addition of a BaCl2 solution. The resulting BaSO4 precipitates and dried sediment fractions used for AVS extractions were precisely weighed, and sulfide-sulfur concentrations were calculated in mmol/kg dry sediment. The quantitative analytical error associated with the overall process of TOC, TN, and AVS determinations was less than 5%.
TOC and TN levels in the POM, sediment, and chimney samples and all carbon, nitrogen, and sulfur isotopic compositions of TOC, TN, the BaSO4 precipitates, glass fiber filters containing POM and animal samples were measured using EA/irMS. All the isotopic values are expressed using δ notation in permil deviation (‰) from international reference materials (VPDB: Vienna Pee Dee Belemnite for δ13C, atmospheric N2 for δ15N, and CDT: Cañon Diablo troilite for δ34S). Analytical errors associated with the overall process of these determinations were less than 5% for the quantitative analyses and less than ±0.2, ±0.3, and ±0.3‰ for δ13C, δ15N and δ34S isotopic compositions, respectively. Stable carbon and oxygen isotopic compositions of carbonate minerals of the chimneys were determined using an irMS with an automated carbonate reaction system. Analytical precision for carbon and oxygen isotope analyses was better than 0.06‰.
Methane concentrations of sediments and chimneys were determined using an in-house standard comprising 100 ppm CH4 over a He matrix (purity is 99.9999%: Iwatani Gasnetwork Corporation, Osaka, Japan). The measurement was performed on-board for sediment samples, and on-shore for chimney samples. The overall error rate for the analysis was expected to be within 10%. Carbon isotopic compositions were determined by continuous-flow isotope ratio mass spectrometry (CF-irMS) with an on-line gas preparation and introduction system connected with a mass spectrometer (for further details, see ref. [37]). The concentration was presented in mole abundance per cm cubic sediment/chimney volume (mol/cm3). As listed above, isotopic values are expressed using δ13C in permil deviation (VPDB‰). The analytical precision was estimated to be 0.3‰.
The TOC, TN, TOC/TN atomic ratio, and isotopic values of sediment, chimney, and animal samples were examined using mean comparison t-tests or one-way ANOVA in order to evaluate the statistical significance of the differences observed.
Isotopic signatures of supposed basal food sources and evaluation of the proportion of each food source
Four basal food sources were supposed as the nutrient and energy sources of the animals in the SSF. Three of the four sources are chemosynthetic products expected from abundant sulfide and methane in environment, including the following types: 1. Thioautotrophic microbes using the Calvin-Benson-Bassham cycle as a carbon fixation pathway (hereinafter, referred to as thiotroph-CBB), 2. Thioautotrophic microbes using the reductive tricarboxylic acid cycle (thiotroph-rTCA), and 3. Methanotrophic microbes. Methanotrophs are generally regarded as heterotrophs [7], but in the present study we regard them as chemosynthetic microbes (unless otherwise noted). Fourth basal food source is photosynthetic products by marine phytoplankton near the sea surface.
The relative contributions of four basal food sources for the SSF animals can be evaluated by carbon and sulfur isotopes assuming isotope mass balance models [38] as followings:
Eq. 1
Eq. 2
Eq. 3
where, f, δ13C, and δ34S, are respectively the proportions and carbon and sulfur isotope ratios of each animal and sources represented by subscripts (x: animal examined; tc: thiotroph-CBB; tr: thiotroph-rTCA; mt: methanotroph; pp: photosynthetic production).
The δ13C value of the thiotroph-CBB which and -rTCA are known to be typically range between −40 to −30‰ [16, 39] and −15 to −10‰ [40, 41], respectively. The δ13Ctc and δ13Ctr values are assumed to be −35‰ and −12.5‰, respectively (Table 3). The δ13Cmt value generally reflects that of the methane assimilated [42]. The δ13C values of the methane at CO and CH Sites were determined by porewater analyses at −48‰ and −10‰ in CO and CH sites, respectively (see Results). The δ13Cpp values has been reported in a range from c. −25 to c. −20‰ [38, 43−45]. The δ13C value of seawater POM at the reference site was in fact −24.6‰ (see Results). The δ34S value s of thiotrophs (both δ34Stc and δ34Str) are assumed to be identical to that of hydrogen sulfide utilized because of the known little isotope effect during assimilation [19]. The δ34S values of methanotrophs and photosynthetic products (δ34Smt and δ34Spp) typically range from c. +15 to c. +21‰ (c.f. [17]) as well as seawater sulfate (+21‰ [46]). The sulfur isotope mass balance (Eq. 2) can be approximated as a following:
The δ34Ssulfide at CO and CH sites were respectively assumed to be −10‰ (identical with AVS in shallow sediment) and +24‰ (identical with H2S in fluid) (see Results). The δ34Ssulfate was assumed to be +20‰ [46]. Because there are four variables in these three equations (Eqs. 1, 3, and 4), the proportions (f) cannot be constrained in general. In the case of animals which have δ34S values identical to δ34Ssulfide or δ34Ssulfate, the sulfur sources can be constrained to be sulfide (ftc + ftr = 1) or sulfate (fmt + fpp = 1), respectively, from Eqs. 3 and 4.
The δ15N value of the photosynthetic product has been reported in a range from c. +3 to c. +10‰ [47, 48], and the seawater POM at the reference site had δ15N = +7.5‰ (see Results). On the other hand, the δ15N values of chemoautotrophs and methanotrophs, probably controlled by δ15N values of their nitrogen sources and isotope effects on assimilation, are still poorly constrained [49, 50]. Nonetheless, δ15N values of organic matter including animal tissues are not higher than +7.5‰, photosynthetic product value, indicating significant contribution of chemosynthetically-produced nitrogen nutrient as basal food source because δ15N value increases along with increasing trophic level.
Results
Fluids and pore waters
The chemical compositions of fluid samples collected just above the seafloor at the CO and CH Sites by the D-WHATS sampler were almost identical to those of the reference seawater sample (Table 4). On the other hand, the two fluid samples collected from the GATS markers (GATS-red and -blue fluid) showed distinct fluid chemistry from the ambient seawater. The GATS-blue fluid exhibited a significantly high pH (9.89), and low sulfate (7.6 mM) and Mg2+ (7.4 mM) concentrations. The sulfate and Mg2+ depletions were also significant in the GATS-red fluid but the magnitude of depletion was smaller. In addition, the pH value of the GATS-red fluid sample was lower than that of the reference seawater.
δ34S values of sulfate (+20.3 and +20.4‰) in the GATS markers were almost identical to those of the ambient seawater (c. +21‰ [46]). The alkaline GATS-blue fluid contained a significant amount of H2S (1.3 mM) with higher δ34S value (+24.2‰) than the seawater sulfate δ34S value.
Fig 2 shows plots of major ions versus sulfate concentrations in all the fluid and pore water samples. Concentrations of some major ions, such as Na+, K+, Ca2+, and Cl−, were nearly constant against the changes in sulfate concentrations. On the other hand, Mg2+ and Si concentrations in some fluid samples were significantly lower than those in the reference seawater.
Filled circles represent samples collected from around the clam colonies and near the chimneys in the SSF. Filled black squares represent the results of the fluid samples, excluding the GATS fluids. Filled red and blue squares represent the results of GATS-red and -blue fluids, respectively. Open circles and squares represent the pore water samples squeezed from sediments beneath the clam colony and the brucite-carbonate chimneys, respectively. Dashed lines represent a plausible mixing line between fluid samples containing GATS fluids and the ambient seawater.
Chemical compositions of pore water obtained from the inside and the edge core in CO Site 4 were almost identical to the reference core with exceptions of sulfate and methane (Table 4). Sulfate concentrations in the pore water acquired from the deep layer (>10 cm below sea floor (cmbsf)) of the inside core were slightly lower (26.7–26.8 mM) than those from the edge core (28.6–28.8 mM) and also the reference core (27.7–28.8 mM). Methane concentrations of the clam colony sediment were approximately ten times higher inside the colony (5.9–19.7 μmol/cm3 sediment) than on the edge region (1.0–2.0 μmol/cm3 sediment; Table 4). Methane-enriched deeper sediment layers of the inside core (7.5, 12.5 and 16 cmbsf) showed δ13C values between −48.3 to −47.4‰. On the other hand, methane-depleted chimney pore waters (<0.5 μmol/cm3 chimney fragment) showed higher δ13C values ranging from −21.5 to −8.6‰ (Table 4).
Although chemical compositions of pore waters retrieved from porous chimneys were variable, they were generally characterized by high pH and low concentrations of silica and sulfate when compared to the reference seawater level. Mg2+ and Ca2+ concentrations in the chimney pore waters exhibited a wide variation (0.3–55.2 mM and 6.98–20.23 mM, respectively).
POM, sediments, and chimneys
The TOC (50.071–0.404 mg-C/L) and TN (0.007–0.069 mg-N/L) levels of POM in seawater just above the clam colonies were higher than those of the reference site (0.031 mg-C/L and 0.002 mg-N/L). δ13C (−29.4 to −27.3‰) and δ15N values (+5.2 to +5.7‰) of the colony-water POM were both lower than those of the reference POM (δ13C = −24.6‰ and δ15N = +7.5‰). The δ13C and δ15N value of the reference POM fell into the range of typical oceanic phytoplankton (δ13C = c. −25 to c. −20‰ and δ15N = c. +3 to c. +10‰ [38, 43−45, 47, 48]) (Table 5). Sulfur isotope ratio of the POM samples cannot be determined due to low sulfur content.
The TOC and TN levels of the inside and edge cores at CO Site 4 were higher at shallower part in each core and obviously higher than those of the reference core through the depth (t-test, p < 0.05 in TOC and TN of both cores and reference core). Notably, the TOC level in the shallow layer (<5 cmbsf) of the inside core was greater than 1 wt%. δ13C values of TOC in both cores were lowest in the surface TOC-enriched layer (−31.8‰) and increased with depth to −27.0‰. In turn, δ13C values of TOC in the reference core were −25.0 to −23.8‰ and identical to that of the reference seawater POM (Table 6 and Fig 3). AVS levels were 5.1−19.1 mM-S/kg in the inside core while not detectable (<0.3 mM-S/kg) in the other cores. δ34S values of AVS of the inside core ranged from −18.6 to −9.2‰. As benthic biological community likely relies on nutrients in the sediment surface (<10 cmbsf) rather than the deep subseafloor (>10 cmbsf), δ34Ssulfate in Eq. 2 for CO Site is assumed to be −10‰, an averaged δ34S value at the sediment surface (0–10 cmbsf).
Upper plots show levels of TOC (a), TN (b), and AVS (c) in the sediments. The lower plots (d, e, and f) show each isotopic composition corresponding to the upper plots. Solid circles and squares represent samples taken from the inside and edge of the clam colony, respectively. Open squares represent the reference core sample.
The TOC and TN levels of a Type I chimney sample from CH Site 4 (Chim 6) were higher at the chimney surface than the inner part (t-test, p < 0.05 for TOC and TN in surface and inner) (Table 7). δ13C values of the inner part TOC showed a narrow range from −27.3 to −26.0‰, whereas those of the surfaces TOC varied widely from −30.3 to −22.8‰. δ15N values of TN in the inner part (−1.6 to +11.4‰) were similar to or higher than those of the surface (−3.6 to −1.4‰) (t-test, p < 0.01). Chimney surface of another Type I chimney sample from CH Site 4 (Chim 5) had similar TOC, TN, δ13C, and δ15N values of the surface part of the sample Chim 6 (Table 7).
Each of TOC, TN, δ13C and δ15N values of the porous Type II chimney sample from CH Site 3 (Chim 4) was comparable between surface and inner parts (Table 7). TOC and TN levels of the Type II chimney (both inner and surface) were 0.019−0.471 wt% and 0.002−0.100 wt%, respectively, and were higher (up to ten times) than Type I chimneys (Table 7). δ13C values of the chimney varied widely from −31.5 to −13.5‰, whereas δ15N values showed a narrow range from −3.6 to −1.4‰. A lithified Type II chimney sample from CH Site 1 (Chim 2) had lower TOC and TN levels and lower δ13C and δ15N values at the inner parts were lower than the surfaces.
Carbon and oxygen isotopic compositions of carbonate minerals were obtained from the four chimney samples (Chim 2, 4, 5, and 6) (Table 7). δ18O values (+3.5 ± 1.2‰) were uniform among all chimney carbonates, while δ13C values were highly varied from −8.8 to +23.6‰.
Animals in CO Sites
Stable isotopic compositions of gill tissues of C. mariana from the CO Sites 2, 4 and 7 were not significantly different (one-way ANOVA, p > 0.05 for δ13C, δ15N and δ34S values). The isotope ratios obtained in this study (δ13C = −35.0 ± 1.1‰, δ15N = +3.3 ± 1.0‰, and δ34S = −10.7 ± 3.0‰; mean ± SD; n = 9; Table 8) were almost identical to the values from the CO Sites 1 and 2 reported previously (δ13C = −34.6 ± 0.5‰, δ15N = +4.2 ± 1.4‰; n = 3, and δ34S values were not reported [9]). Among tissues of the clams, the δ15N values of gill tissues were c. 3‰ lower those of the other tissues (Table 8). This difference of δ15N values between the gill and the other tissues of the clam has been observed also in other chemosynthetic communities (e.g. [51–54]), and interpreted as TN of gill tissue is occupied by symbiotic microbial cells and not representative to the host clams body. The clams and frenulate tubeworms had δ34S values of −10.7 ± 3.0‰ and −10.5‰, respectively, that were similar to those of AVS values (−10‰) in the associated shallow sediments. δ13C values of the frenulate tubeworms (−22.2 and −20.6‰) were much higher than those of the clams (t-test, p < 0.01).
The nautiliniellid polychaetes inhabiting inside the clam shells had δ13C of −33.4 ± 0.8‰ and δ34S of −9.0 ± 1.1‰ that were comparable to those of the host clams (−33.5 ± 1.4‰ and −7.4 ± 2.6‰, respectively) (t-test, p > 0.05 for δ13C and δ34S values). In turn, the nautiliniellid polychaetes had ~4‰ higher δ15N values (+11.7 ± 0.7‰) than those of the host clam tissues (t-test, p < 0.05) (Table 8 and Figs 4 and 5).
In (a) and (b), yellow, orange, blue, and green shadows represent the expected ranges of the isotopic compositions of thiotroph-CBB, thiotroph-rTCA, methanotroph in CH Sites, photosynthetic organic matter (Table 3). The ranges of methanotroph in CO Sites are not shown. In (c), green shadow represents the expected ranges of the δ34S values of photosynthetic organic matter and methanotroph (δ34Ssulfate) and yellow shadows with red and blue lines, respectively, represent the δ34S values of thiotrophs (δ34Ssulfide) in CO and CH Sites.
Yellow, orange, blue, and green shadows represent the expected ranges of the isotopic compositions of thiotroph-CBB, thiotroph-rTCA, methanotroph, and photosynthetically-derived organic matter, respectively. In CO Sites, the ranges of isotopic signatures of methanotroph were not shown due to the low δ13C value (c. −48‰).
The amphipods and lumbrinerid polychaetes in CO Site 2, and the eunicid polychaetes in CO Site 4 had δ13C values of −24.6 ± 1.0‰, −26.1‰, and −31.7‰, respectively (Table 8). These animals had high δ34S values (+16.6 ± 1.5‰, +19.8‰, and +17.2‰), that close to those of seawater sulfate (+21‰ [46]). The δ15N values of the lumbrinerid polychaetes (+4.9‰) were clearly lower than that of the reference POM (+7.5‰). While the amphipods had variable δ15N values (+7.7 ± 3.0‰), the eunicid polychaetes had the highest δ15N value of +12.4‰ in the SSF.
The isopods, B. cf. arnoldi, maldanid polychaetes, and amphipods in CO Site 4 had similar δ13C and δ34S values (one-way ANOVA, p > 0.05) of c. −32‰ and c. +2‰, respectively (Table 8). The δ13C and δ34S values of these animals are lower than those of photosynthetically-derived organic matter and seawater sulfate, respectively (Fig 4). These animals had significantly different δ15N values (one-way ANOVA, p < 0.05) from +7.1 ± 0.1‰ (isopods) to +10.8 ± 0.1‰ (maldanid polychaetes).
The anthozoans were also characterized by a relatively low δ13C (−29.2 ± 0.9‰) and δ34S (−3.9 ± 1.2‰) values. The anthozoans had relatively high δ15N value of +9.2 ± 0.5‰. The nematode from CO Site 2 had a δ13C value (−25.3‰) comparable to the photosynthetic signature and the highest δ15N value (+10.4‰) among the animals in the CO Sites.
Animals in CH Sites
The δ13C and δ34S values of all the animals in CH Sites (Phyllochaetopterus polychaetes, anthozoans, amphipods, lumbrinerid polychaetes, polynoid polychaetes, eunicid polychaetes, leptostracans, B. cf. arnoldi, and nematodes) ranged between −28.4 and −22.3‰ and between +14.3 and +21.6‰, respectively (Fig 4). The δ13C and δ34S values of the CH Sites animals are comparable to those of the photosynthetic organic matter and seawater sulfate, respectively. In addition, the δ13C and δ34S values of the CH Sites animals are generally higher than those of the CO Sites animals (Fig 4). Moreover, most of the δ15N values of animals in CH Sites were lower than that of the reference POM (+7.5‰) while the nematodes had δ15N values of +8.0‰ (Fig 4).
Discussion
Evidence for active shimmering of serpentinization-associated fluid from the SSF
The highly alkaline fluids occupying the GATS-blue (pH = 9.89) and chimney pore waters (pH = 10.0) demonstrate serpentinization-involved geofluid actively emits from the SSF seafloor. The pH value of the fluids are in fact as high as those observed in other serpentinization-associated seafloor geofluid systems (9–9.8 for Lost City hydrothermal field [12]; 12.5 for South Chamorro Seamount [55]). The highly alkaline fluids rich in reducing chemical species, such as methane and hydrogen sulfide, are confirmed and can provide available energy and nutrient sources for chemosynthetic community. It is noted that the distinct chemical compositions between the two GATS fluid samples, especially in pH, suggest that the original fluid chemistry of fluids emitting around each GATS may be different. However, it remains unclear whether the differences reflect the original fluid chemistry or not due to a possible alteration of fluid chemistry within the GATS chamber until the sampling. A consistency of fluid chemistry between GATS-blue and chimney porewaters (Fig 3) nevertheless implies appropriate sampling at least for the GATS-blue.
The δ34S values of sulfate in the GATS fluids, comparable to the ambient seawater sulfate-sulfur, suggest that sulfate in the GATS fluids was derived from the ambient seawater. The sulfate concentrations of the GATS fluids are thus hypothesized as an indicator for a mixing between two end-member fluids, the sulfate-rich seawater ([SO42−] = 28 mM) and the sulfate-free seeping fluid ([SO42−] = 0 mM). The mixing model based on the sulfate concentration (Fig 3) can provide estimated fluid chemistry of the seeping end-member. The estimated “negative” Mg2+ and Si concentrations in the SSF seeping fluid (Fig 3) are consistent with those obtained from the Lost City hydrothermal vent fluids [12] and pore water fluids of the Mariana serpentinite mud volcanoes [55] as well as the trend of experimental study in the serpentinization of peridotite [56]. They can be explained by Mg precipitation from a mixed fluid between the Mg-enriched seawater and the Mg-free seeping fluid. The Mg depletion in the seeping fluid seems reasonable because water/rock interaction during the exothermic serpentinization removes Mg from the liquid phase [12] while the precipitation of brucite from the mixed fluid due to high pH condition further removes seawater-derived Mg. The precipitation of Mg minerals from the mixing fluid is suggested by the brucite-like whitish precipitation observed in the GATS-blue marker.
The 34S-enriched H2S in the GATS fluid can be produced from more 34S-enriched sulfate through microbial sulfate reduction (MSR) activity [23]. As 32S-preferential sulfate consumption by subseafloor SRB populations and subsequent subseafloor deposition of sulfide minerals such as pyrite remove 32S from the ascending fluid, and the remnant 34S-enriched sulfate at the later stage of MSR occurring on the seafloor could be a source of 34S-enriched H2S in the GATS fluid. The 34S-enriched H2S may also reflect complex sulfur cycles carried out by the microbial population [57] in the GATS marker during approximately one year until sampling. However, the GATS-blue fluid contained very high levels of H2S relative to venting fluid at the Lost City (<64 μM [12]), while pore water fluids of serpentinite mud volcanoes contain a similar level of H2S (0.2–1.3 mM [55]). Although sulfate are plausibly lacking in fluids from the SSF and the Lost City field [12], the sulfate concentrations in pore fluids of the serpentinite mud volcanoes are as high as ambient seawater [55]. Those results suggest that the subsurface microbial activity, especially MSR, is sufficiently active to deprive the SSF fluid of sulfate.
Major ion concentrations of the estimated SSF end-member fluid, comparable to those of the seawater, suggest that water source of the SSF fluid may be seawater penetrating the crust around the SSF rather than the slab-derived fluid. If dehydration of the subducting slab occupies a majority of the fluid source discharging at the SSF, like in forearc serpentinite seamounts, major ion concentrations of the estimated end-member fluid would be different from those of the seawater due to deep subseafloor processes through geological time spans [58]. However, such trends could not be found in the estimated SSF fluids chemistry (see Fig 2). In turn, fresh basaltic glass associated with igneous activity of the backarc basin was recognized near the SSF area [59], and the igneous activity might be as a possible driving force for fluid circulation of the SSF geofluid system like seafloor hydrothermal system.
Chemosynthetic primary production in the sediments beneath clam colonies and in the chimneys in the SSF
In the SSF, chemosynthetic primary production in the sediments beneath the clam colony and in the chimneys likely accounts for the main food sources for the animals as the great biomass of local benthic community cannot be sustained only by phytoplankton-derived sinking organic matter. The large variation in δ13C values of sedimentary TOC beneath the clam colony suggests that there are more than two primary productions such as thiotroph-CBB, thiotroph-rTCA, and/or methanotrophs. These chemosynthetic activities are plausibly supported not only by methane and sulfide derived from deep-sourced ascending fluid but also by biogenic sulfide through MSR within sediments. The MSR in the sediment is fueled by the ascending fluid, because the AVS levels increases with depth. Therefore, the fluid is expected to contain some electron donors for SRB such as organic compounds, methane and/or hydrogen (e.g. [6, 23]).
The chimneys are thought to form by precipitation minerals originating in the ascending fluid associated with serpentinization of mantle peridotite and have been classified into three types [10] as mentioned above: (1) the Type I brucite-rich chimney has microbial mats but a lack of colonized animal under higher fluid flux condition of the highly alkaline and reduced fluid, (2) the Type II carbonate-rich chimney has obvious microbial mats and densely-colonized animals on it under lower fluid flux condition, (3) the Type III brucite-poor chimney is dissolving and alternating after the fluid discharge has ceased.
The Type I chimney had low TOC and TN levels in the inner part of the chimney that is consistent with the visual observation of low biological accumulation [10]. As marine microbial organic matter generally has a TOC/TN atomic ratio of 4 to 6 [60, 61], the high TOC/TN atomic ratios together with low TOC and TN levels of the Type I chimney suggest the occurrence of in situ organic matter decomposition that increases TOC/TN atomic ratio. The high δ15N value (up to +11.4‰) of the inner part of the chimney is not conflict with this interpretation [62]. Thus, chemosynthetic primary production is considered to be active on the surface of Type I chimneys and all sections of porous Type II chimneys, which have low δ15N values and TOC/TN atomic ratios.
The variation of δ13C values of the TOC on the surface of Type I chimneys and all sections of porous Type II chimneys possibly reflect the compositional and functional diversity of microbes in the fluid and/or chimney structures (Table 5). For example, thiotroph-CBB, which has a low δ13C value (−35‰), could be dominant in the microbial community in the surface of lower and middle sections of the chimneys (Table 5). On the other hand, thiotroph-rTCA and/or methanotrophs, which have a high δ13C value (−15‰ and −22 to −9‰, respectively), were expected to be dominant in the root and upper sections of the chimney surfaces. Another possible explanation for this wide range of the δ13C values of the TOC may be due to differences in carbon source availability from seeping fluids, as observed in the Lost City hydrothermal field, where remarkable 13C-enriched organic matters were reported from carbonate chimneys and fissures associated with an extremely carbon-limited environment (−27.7 to −2.8‰; [13, 63, 64]). The wide range of δ13C values in the carbonate may support the notion that the differences of dissolved inorganic carbon (DIC) availability or the δ13C of the source DIC affect the isotopic signature of producers. Further studies, such as microbial community analyses, biomarker compositions and compound-specific isotope analysis, may reveal the sources of the organic matter in more detail.
On the other hand, on the surface of the largely lithified Type II chimney sample (Chim 2, Table 5), relatively high δ15N values and low TOC/TN atomic ratios suggest that organic matter with high TOC and TN levels may reflect incorporation of photosynthetic organic matter as the ultimate source. This suggests that the remains of organisms feeding on suspended organic matter are incorporated in the chimney surface. The δ13C values of carbonates in the chimney surfaces were similar to that of DIC in ambient seawater (c. 0‰), indicating that the source of DIC forming surfaces of the carbonate chimney was provided mainly from ambient seawater. This suggests that the supply of reduced chemical species from the ascending fluid is limited to the lithified Type II chimney surface.
Evaluation of basal foods and food-web structure of the animal community
Clam colony sites.
The Calyptogena clams and the frenulate tubeworms are expected to completely rely on symbionts for their energy and nutrients. Their symbionts are typically occupied by thiotrophic microbes (e.g. [7, 65, 66]), and the δ34S values of these animals are indeed identical to those of AVS (−10‰) in surface sediments. These biological and isotopic characteristics strongly suggest that the proportions of methanotrophic and photosynthetic products to their nutrient sources are negligible (fmt = fpp ≈ 0). It allows their basal carbon sources calculated from the δ13C values based on Eq 1. The calculation yields that CBB-derived carbon occupies nearly 100% for the clams and approximately half (40%) for the tubeworms (Table 9 and Fig 5).
As majority of the organic matter in Calyptogena gill is typically occupied by endosymbiont’s cells [54], δ15N values of the clam gill (c. +3‰; Table 8) can be regarded as a representative value of the primary producer for the 15N-based trophic level estimation. The higher δ15N values of the other tissues than those of gill are reasonable as a higher trophic level from the symbiont. In addition, as the δ15N value of the thioautotrophs estimated here (c. +3‰) is lower than that of the photosynthetic product (+7.5‰), the δ15N value of animals not higher than c. +7.5‰ is a likely indication for significant contribution of the chemosynthetic products in the CO Sites.
The nautiliniellid polychaetes, sampled from the mantle cavities of the Calyptogena clam, are expected to obtain energy and nutrients from the host clam. Indeed, the carbon and sulfur isotopic compositions of the nautiliniellid polychaetes suggest the thiotroph-CBB as a majority carbon and sulfur sources (95%) as well as the host clam (Table 9 and Fig 5). The δ15N values (c. +12‰) of the nautiliniellid polychaetes, which is c. 4‰ higher than that of the clam body (c. ≤+8‰) and corresponds to an increase of the trophic level, are consistently explained by the life style.
The amphipods and the lumbrinerid polychaetes in CO Site 2 had the δ34S values as high as c. +20‰ that indicate little contributions of thiotrophic products for their basal sulfur sources (ftc = ftr ≈ 0) based on Eq. 4. The δ13C values and Eq. 1 with an assumption of both ftc and ftr of 0 yield that the photosynthetic-derived carbon likely occupies nearly 100% for their basal carbon sources (Table 9 and Fig 5). The δ15N values of these animals were highly diverse even within the CO Site 2 that did not allow us to evaluate their trophic level. Although possible δ15N heterogeneity within an individual [67] may cause the variation, reasons remain unidentified at this moment.
The eunicid polychaetes in CO Site 4 also had the high δ34S values (>+15‰) that suggest slight contribution of thiotrophic products as food sources (ftc = ftr ≈ 0). Their δ13C values and Eq. 1 yield that the eunicid polychaetes rely a large part (c. 70%) of the basal carbon source on the photosynthesis-derived organic matter. The high δ15N value (+12.4‰) is consistent with the digestion of the photosynthesis-derived organic matter. However, its potential preys could not be found among the animals in the present analyses (Fig 4). This suggests that other animals in the site that do not rely on thioautotrophic products and are preyed upon by the eunicid polychaetes, but we did not sample them.
The isopods, the snail B. cf. arnoldi, the maldanid polychaetes, and the amphipods in CO Site 4 had the intermediate δ34S values between the δ34Ssulfide and δ34Ssulfate (Fig 4) that prevents us from estimating the proportions among each four basal source. In turn, the intermediate δ34S values and Eq. 4 yield that approximately half of their basal food sources are likely derived from the chemosynthetic (thiotrophic) products (Table 9). The low δ15N values of isopods (c. +7‰), comparable to those of the syntrophic Calyptogena body, imply that the isopods directly uptake chemosynthetic microbes. The high δ15N values (>+9‰) of the snails, maldanid polychaetes, and the amphipods imply that trophic levels of these animals were likely higher than those of the isopods. This isotopic suggestion seems consistent with behaviors of B. cf. arnoldi who behaves as carnivore in various communities [68]. Both δ13C and δ34S values are similar between the snails and isopods (Fig 5), implying their similar basal foods. It is also the case for the maldanid polychaetes (Fig 5) while the maldanid polychaetes in marine sediments behave deposits feeder or detritus feeders [69].
The anthozoans, known as suspension feeder, in the CO Site 4 had ~3‰ higher δ15N values than the suspended POM around their habitat (Figs 4 and 5), supporting the suspended POM feeding. It is also supported by similar δ13C values between the anthozoans and POM. In turn, the δ34S values (c. −4‰) of anthozoans indicate that majority of their nutrient sources were derived from the thiotrophic microbes (ftc + ftr ≈ 0.8). Because the suspended POM had the intermediated δ13C and δ15N values between those of sediment beneath the clam colony and the photosynthetic product (δ34S was not obtained), the thiotrophy-derived sulfur would be supplied by re-suspension of the sediment. The basal food source of the nematodes in CO Site 2 cannot be constrained due to lack of δ34S measurement, the intermediate δ13C value among the sources, and the δ15N value higher than +9‰.
Chimney sites
The δ15N values of the animals in the CH Sites that were lower than +7.5 strongly suggest significant contributions of chemosynthetic-derived nitrogen as the nitrogen nutrient source. It is however difficult to strictly constrain proportions of each chemosynthetic metabolism due to the δ13C value of methane (−21.5 to −8.6‰) comparable to those of photosynthesis (−25‰) and thiotroph-rTCA (−12.5‰) as well as the δ34Ssulfide value (+24.2‰) comparable to δ34Ssulfate (+24‰) (Table 4).
The Phyllochaetopterus polychaetes and the anthozoans, colonized on/in the chimneys and attached to chimney walls, respectively, had the lowest δ15N values (c. +0‰) in the CH Sites animals (Fig 4 and Table 8). As the Phyllochaetopterus polychaetes and the anthozoans are known to suspension feeders [70], they likely relied on the chemosynthetic products attached on the chimney and from the suspended POM. The δ15N values of the other animals in CH Sites are ≥3.5‰ higher than those of the Phyllochaetopterus polychaetes and the anthozoans, suggesting that the others were secondary (or higher) consumers. The lumbrinerid and the polynoid polychaetes had the δ13C values lower than the reference POM. It suggests a certain amount of thiotroph-CBB-derived carbon contributing to their basal food source although actual foods digested remain unknown.
Conclusion
In this study, we collected fluid, sediment, chimney, and animal samples from the deepest known serpentinite-hosted animal communities named SSF. Seeping fluids recovered by trapping in the GATS markers had distinct chemical compositions from the ambient seawater. Particularly, high pH of the accumulated GATS fluid is the first direct evidence for on-going seepage of serpentinization-associated geofluid in this area. The highly alkaline fluids rich in hydrogen sulfide and methane can serve as an energy and nutrient sources to sustain chemosynthetic productions and associated animal communities. Carbon, nitrogen, and sulfur isotope systematics of various components in the SSF suggest that most of the animals living there rely on chemosynthetic production. On the other hand, some animals use not only chemosynthetic products but also photosynthetic products. It means that sinking particles from the euphotic zone are also important nutritional sources for animals inhabiting the SSF, despite being at a depth of ~5,700 m.
Acknowledgments
We are grateful to the Captains and crews of R/V Yokosuka and Kairei and the operation teams of the DSV Shinkai 6500 and ROV Kaiko for their skillful operations. The facilities for isotopic ratio measurement of carbonate were provided from the Center for Advanced Marine Core Research, Kochi University under the direction of Dr. Minoru Ikehara. We would like to thank Dr. Uta Konno and Dr. Ken Takai for their effort to sampling and methane analysis. Methane isotope analysis was also supported by Mr. Yohei Matsui, Dr. Eiji Tasumi, and Mrs. Keiko Tanaka. We also thank Dr. Ken Takai for helpful comments on the manuscript. We also thank Prof. Hitoshi Chiba and Dr. Chong Chen for correcting English on the manuscript. This article is part of a doctoral thesis of the first author (Y. O.). We appreciate the valuable comments given by two anonymous reviewers and editorial handling by Dr. Sébastien Duperron, Academic Editor of this journal.
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