A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur 10 in Soft Body Parts of Animals Collected from Deep-Sea Hydrothermal Vent and Methane Seep Fields: Variations in Energy Source and Importance of Subsurface Microbial Processes in the Sediment-Hosted Systems

Toshiro Yamanaka, Sho Shimamura, Hiromi Nagashio, Shosei Yamagami, Yuji Onishi, Ayumi Hyodo, Mami Mampuku, and Chitoshi Mizota

Abstract The stable isotopic signatures of biophilic elements, such as carbon, nitrogen, and sulfur, exhibited in animal soft body parts are excellent indicators for evaluating the pathways of energy and food sources. Thioautotrophic and methanotrophic nutrition prevailed in deep-sea hydrothermal vent and methane seep areas results in sulfide-sulfur and methane- carbon isotopic ratios. In this study, we reevaluated the carbon, nitrogen, and sulfur isotope compositions of animals taken from deep-sea hydrothermal vents and methane seep areas in order to understand the detailed pathways of energy and food sources for the habitants. The results showed that most animals collected from sediment-starved hydrothermal areas rely on thioautotrophic nutrition, using hydrogen sulfide dissolved in venting fluids as the sole primary energy source. On the other hand, animals from sediment-covered hydrothermal vent and cold seep fields show some variations in energy sources, of both hydrothermal and microbial origins. Sediment-covered areas tend to be enriched in biomass and diversity relative to sediment-starved areas. The results suggest that fluid discharged through sediments to the seafloor are strongly affected by subsurface microbial processes and result in increased biomass and diversity of the seafloor animal community.

The online version of this chapter (doi:10.1007/978-4-431-54865-2_10) contains supplementary material, which is available to authorized users. T. Yamanaka (*)  S. Shimamura  H. Nagashio  S. Yamagami  Y. Onishi Graduate School of Natural Science and Technology, Okayama University, 1-1, Naka 3-chome, Kita-ku, Okayama 700-8530, Japan e-mail: [email protected] A. Hyodo Faculty of Science, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan M. Mampuku Graduate School of Social and Cultural Studies, Kyushu University, 4-2-1, Ropponmatsu, Chuo-ku, Fukuoka 810-8560, Japan C. Mizota Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan

J.-i. Ishibashi et al. (eds.), Subseafloor Biosphere Linked to Hydrothermal Systems: TAIGA Concept, 105 DOI 10.1007/978-4-431-54865-2_10, # The Author(s) 2015 106 T. Yamanaka et al.

Keywords Chemosynthesis-based animals  Food ecology  Hydrothermal vent community  Methane seep community  Stable isotopes

10.1 Introduction from À20 to +7 ‰ (e.g., Nelson and Fisher 1995; Mizota and Yamanaka 2003). Such negative δ15N values of marine Since the first discovery of hydrothermal vent communities animal soft tissues have been reported only in communities in 1977, the stable isotopic signatures of chemosynthesis- consisting of chemosynthesis-based animals (e.g., Saino based animal species have been used to evaluate the isola- and Ohta 1989; Fiala-Me´dioni et al. 1993; Mizota and tion of vent communities from the usual marine food web Yamanaka 2003) and cyanobacteria, which have the ability systems that are supported by photoautotrophic primary to fix dinitrogen (À3to+0‰) (Minagawa and Wada 1984; production (e.g., Nelson and Fisher 1995; Mizota and Carpenter et al. 1997). The nitrogen nutrition of symbiotic Yamanaka 2003). Average carbon isotopic composition of bacteria is not well understood. marine photoautotrophic products produced by phytoplankton Stable isotopic signatures are quite useful indicators for have been documented at δ13C ¼ ~ À22 ‰, although they distinguishing chemosynthesis-based animals from the show a wide range (δ13C ¼À16 to À28 ‰), reflecting a phototrophic food web. Their isotopic signatures can possi- variety of carbon fixation pathways together with physico- bly be used to identify the energy source for chemosynthesis. chemical conditions (e.g., Rees et al. 1978). The sulfur Mizota and Yamanaka (2003) reviewed the carbon, nitrogen, isotopic compositions of common marine animals, and sulfur isotopic compositions of chemosynthesis-based supported by the same photoautotrophic production, reflect animals and the associated methane and sulfide data the signature of sulfates dissolved in seawater, which is published prior to 2003, and discussed the flow of chemical uniform throughout the oceans (δ34S ¼ +21 ‰) (Rees energy from emitting fluids to the animal community. In the et al. 1978) and the sole nutrient source of sulfur for primary review, the importance of environmental isotopic data sets of producers. On the other hand, it is reported that the carbon sulfide-, methane-, and nitrogen-issuing species, was isotopic ratios of thioautotrophic microbes that use the emphasized. Nevertheless, environmental isotopic data sets Calvin cycle involving RuBisCO (ribulose 1,5-bisphosphate have not been fully integrated. Some animal clusters have carboxylase/oxygenase) for carbon fixation have a relatively been found far from vents where significant concentrations of narrow range of δ13C values, namely À35 Æ 5 ‰, and it is sulfide and methane have been detected. Furthermore, δ15N known that other types of thioautotrophic microbes have values of nitrate, nitrite, and ammonium from the environ- significantly higher δ13C values (À20 ‰) (e.g., Nelson ment have not been reported. Most reported geochemical and Fisher 1995; Markert et al. 2007). Furthermore, sulfur data from hydrothermal and seep fields are derived from isotopic ratios of thioautotrophic microbes reflect sulfide venting fluids and visible seepages. This implies that the nutrition with a limited kinetic isotope effect (~ À5 ‰) reported values are almost comparable to the end-member through the cell membrane (Fry et al. 1983). In natural (i.e., deep-seated source) values. Therefore, it is difficult to environments, δ34S values of sulfides, which are mainly directly compare the isotopic data to the soft body parts of derived from volcanism and bacterial sulfate reduction, are animals, especially in sediment-hosted systems (i.e., meth- clearly lower than those of sulfate-sulfur dissolved in sea- ane seeps and sediment-covered hydrothermal fields), where water (e.g., Thode 1988; Canfield 2001). In the case of emitting fluids penetrate through thick clastic sediments and methanotrophic microbes, which are another important pri- are subsequently subjected to subsurface microbial transfor- mary producer in the seep food web, carbon isotopic ratios mation. An obvious example is methane seep communities reflect methane nutrition, while sulfur isotopic ratios reflect dominated by thiotrophic animals, which use microbial seawater sulfate-sulfur, similar to photoautotrophs. Some of hydrogen sulfide derived from sulfate reduction with meth- the methane derived from pyrolysis of organic matter have ane as an electron donor (e.g., Mizota and Yamanaka 2003). carbon isotopic ratios similar to those of photoautotrophic In addition, reduced chemical species, such as hydrogen products. Nevertheless, microbial methane, which prevails sulfide and methane discharged from the seafloor, are in anoxic sediments, has significantly lower δ13C values incorporated by chemosynthetic and methanotrophic (À45 ‰), whereas abiotic methane has distinguishably microbes, and the resulting microbial products have been high δ13C values (> À20 ‰) (e.g., Schoell 1988). considered to support not only vent- and seep-endemic ani- However, the nitrogen sources for chemosynthesis-based mal communities but also common benthic and epibenthic animals are not well understood (Kennicutt et al. 1992; animals. In the case of hydrothermal systems, discharge of Fisher et al. 1994). Previously reported δ15N values for soft these chemicals mainly originates from venting chimneys. tissues from thiotrophic and methanotrophic animals range In fact, at hydrothermal fields lacking sediment cover, 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 107

Fig. 10.1 Maps showing the sample locations of this study. Open circles indicate hydrothermal fields. Open squares indicate methane seep fields. The Wakamiko site in Kagoshima Bay and the Shinkai seep field of Southern Mariana are categorized as methane seeps hydrothermal discharge is mostly confined to venting animals and environments located near hydrothermal vents chimneys and underlying hydrothermal mounds. Hydrother- and methane seeps of the extensive areas indicated in mal fields covered by thick clastic sediments have a few Fig. 10.1. We discuss the variations in energy and food sources additional pathways for hydrothermal fluid discharge, e.g., of the environments from the view point of “TAIGA” (sub- diffusion into aquifers in overlying sediments. The fluids seafloor fluid flow system, Urabe, Chap. 1), together with the diffused within the sediments provide reduced chemical spe- importance of sub-seafloor microbial processes. cies and other nutrients to subsurface microbes. Some of the microbes may be grazed upon, thereby supporting benthic animals, and the others may generate reduced chemical spe- 10.2 Materials and Methods cies once more. Such secondary chemicals are also thought to support chemosynthesis-based animals that inhabit hydro- 10.2.1 Geological Background of the Sample thermal field and methane seep communities mentioned Materials above. Since 2003, reconnaissance surveying and sampling of 10.2.1.1 Okinawa Trough chemosynthesis-based animals using submersibles from The Okinawa Trough (Fig. 10.1) is a typical back-arc rifting Japan and other countries is ongoing, and the following isoto- basin between the east margin of the East China Sea and the pic analyses have been made. In this Chapter, we have com- Ryukyu Islands (e.g., Letouzey and Kimura 1986). The trough piled the latest isotopic data from soft tissues of diverse is filled with thick clastic sediments derived from surrounding 108 T. Yamanaka et al. continents and islands, together with volcaniclastic sediments et al. 2009; de Ronde et al. 2011; Gamo et al. 2001), due to associated with arc and back-arc volcanism in this area minimal contribution of sedimentary organic matter to the (Tsugaru et al. 1991; Ishibashi et al. Chap. 29). Vigorous hydrothermal fluids. Bio-diversity of the animal hydrothermal activities associated with volcanism have communities observed at the Snail and Yamanaka sites in been observed in the trough, and the chemistry of emitting the South Mariana Trough was very low (Kojima and

fluids has been characterized by high volatiles (i.e., CO2 Watanabe, Chap. 26). Dense Alviniconcha snails (Wheat and methane) and ammonium concentrations, suggesting et al. 2003), crabs (Gandalfus sp.) and sea anemones the significant interaction of magma with sedimentary (Marianactis sp.) have been identified. High bio-diversity, organic matter (e.g., Ishibashi and Urabe 1995; Kawagucci, including dense long-necked barnacles (Volcanolepas Chap. 35). Therefore, many of the hydrothermal activities in osheai), shrimp (Alvinocaris sp.), and vestimentiferan the Okinawa Trough are interpreted as a typical sediment- tubeworms (Lamellibrachia sp.), has been observed in the hosted system. Chemosynthesis-based animal communities animal community at the Brother Volcano site (Kermadec have been found around the hydrothermal venting area, Arc), while many types of animals, such as Rimicaris and they have been characterized by the presence of dense shrimp, Bathimodiolus mussels, Alviniconcha snails (de Bathymodiolus mussels, vestimentiferan tubeworms, and Ronde et al. 2011), and Brachyuran crabs, have been galatheid crab communities (e.g., Fujikura et al. 2012; observed at the Kairei hydothermal field on Hakuho Knoll Watanabe and Kojima, Chap. 40). (Central Indian Ridge) (Hashimoto et al. 2001; Watanabe Animal samples were obtained from the following and Beedessee, Chap. 16). hydrothermal fields: Minami-Ensei knoll, Izena Hole, Iheya Ridge, Iheya North knoll, Tarama knoll, Daiyon- 10.2.1.4 Sagami and Kagoshima Bays and Yonaguni knoll, and Hatoma knoll (Fig. 10.1). Kuroshima Knoll Sagami and Kagoshima Bays and Kuroshima Knoll 10.2.1.2 Izu-Ogasawara Arc (Fig. 10.1) are areas with significantly high methane flux The Izu-Ogasawara Islands (Fig. 10.1) are a volcanic arc from the seafloor (Masuzawa et al. 1992; Yamanaka et al. associated with subduction of the Pacific Plate beneath the 2013). Sagami Bay and Kuroshima Knoll are located on the Philippine Plate. Deep-sea hydrothermal activities have fore-arc regions, which are related to subduction of the been identified in the calderas and craters of the submerged Philippine Plate, where compression stress associated with volcanoes (e.g., Glasby et al. 2000). The hydrothermal areas subduction is a possible driving force for methane seepage. lack clastic sediments except for a small amount of In Kagoshima Bay, methane rich gas is emitted from volcaniclastic sediment that fills in the craters and calderas fumaroles, together with volcanic gas associated with the (e.g., Iizasa 1993; Takano et al. 2004). Relative to the fluids volcanic activity of the Wakamiko volcano. Sedimentary emitting from the Okinawa Trough, the resulting chemistry organic matter has been considered a primary source for of the hydrothermal fluids is characterized by significantly methane as a result of microbial metabolism or pyrolysis. lower methane and ammonium concentrations, i.e., a Chemosynthesis-based animal communities have been sediment-starved system (e.g., Ishibashi and Toki 2012). found around the seepages and have been characterized by Chemosynthesis-based animal communities have been the occurrence of dense Bathymodiolus mussel, Calyptogena found around the fluid venting areas and have been clam, and vestimentiferan tubeworm communities (e.g., characterized by occurrence of dense Bathymodiolus Fujikura et al. 2012). mussels (e.g., Fujikura et al. 2012). Animal samples were collected from the following 10.2.1.5 Additional Methane Seep Fields hydrothermal fields: Myojin Knoll, Sumisu caldera, Suiyo Methane seepages from the seafloor have been reported seamount, and Kaikata seamount (Fig. 10.1). from South Chamorro Seamount (Northern Mariana Fore-arc) (Yamanaka et al. 2003b), Shinkai Seep Field 10.2.1.3 Additional Hydrothermal Fields (Southern Mariana Fore-arc) (Ohara et al. 2012), and the Animal samples were also collected from the following western edge of Sunda Trough (off southwest Java) (Okutani hydrothermal fields and used for isotopic analyses: three and Soh 2005) (Fig. 10.1), and characteristic animal sites at the southern most portion of the Mariana back-arc communities have also been reported around the seepages. spreading center in the South Mariana Trough (the Snail, Calyptogena clams have been observed at all of these sites, Archaean, and Yamanaka sites), Brothers Volcano in the while Bathymodiolus mussels are primarily observed on the Kermadec Arc (New Zealand), and Hakuho Knoll at the South Chamorro Seamount. The South Chamorro Seamount Rodriguez Triple Junction (Central Indian Ridge) is a large serpentine mud volcano, while the Shinkai Seep (Fig. 10.1). The lack of clastic sediments in the hydrothermal Field is an outcrop of serpentinized peridotite (Ohara et al. fields reflects ammonium concentrations < 20 μM/kg (Kato 2012). This suggests that methane emitting from these sites 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 109 may be an abiotic product of the reaction between carbon 10.2.3 Analytical Procedures dioxide and dihydrogen, which are expected to occur during serpentinization (Berndt et al. 1996). Serpentinization of the Soft tissues of bivalve and gastropoda samples were dis- seafloor may be the cause of hydrothermal activity at sites sected into gill, foot, mantle, adductor and viscera sections, such as the Rainbow and Lost City hydrothermal fields and subjected to isotopic analyses. For vestimentiferan (Kelley et al. 2005). A detailed geochemical study has tubeworm samples, the soft body parts were dissected into recently been conducted on the Shinkai Seep Field (Ohara gill, vestimentifera, and trophosome sections, and the et al. 2012). Therefore, in this study we have categorized trophosome sections were used for the analyses. Muscle this system as a methane seep. samples were separated from the crustacean species. Other Methane seepage at the Sunda Trough is located in a fore- animals, such as limpets, sponges, polychaetes, etc., were arc basin covered with thick clastic sediments, suggesting used as whole-body samples due to small size or complex that the tectonic background is comparable with the cold anatomy. The tissues dissected for analyses were treated seepages of Sagami Bay. This means that the primary source with 1 M hydrochloric acid to remove carbonates, then of methane is expected to be organic matter of sedimentary freeze-dried prior to grinding. Preparation for sulfur isotopic origin. measurements in the soft tissues was described previously (Mizota et al. 1999; Yamanaka et al. 2000a, b). Briefly, to remove excess seawater sulfate, the dissected soft tissues were dialyzed repeatedly in cellulose bags against 1 M lith- 10.2.2 Animal, Sediment, and Fluid Sampling ium chloride solution at 5 C. Dialyzed samples were then Procedures freeze-dried and pulverized. Samples of large size (1 g dry weight) were placed in a Parr bomb 1108 Oxygen Combus- The animal samples used in this study are listed in Table 10.1. tion Vessel (a stainless steel vessel filled with oxygen gas In addition to common marine benthic animals, such as under high-pressure (30 kg/cm2) and a few milliliters of sponges, sea cucumbers, starfishes, and fishes, well known distilled water). After combustion, the dried samples were chemosynthesis-based animals, including three bivalves completely converted into gas, and all sulfur compounds (Calyptogena, Bathymodiolus, Acharax), two shrimps were trapped as sulfates in the distilled water within the

(Alvinocaris, Limicaris), two tubeworms (Lamellibrachia, vessel. The resulting sulfates were precipitated as BaSO4. Alaysia), one gastropoda (Alviniconcha), one galatheid The dried soft tissue samples were measured for carbon, crab (Shinkaia), among others, were analyzed for the carbon, nitrogen, and sulfur isotopic ratios using an EA/irMS nitrogen, and sulfur isotopic ratios in their soft tissues. (IsoPrime coupled with Euro Vector EA3000, GV The sampling areas of the animals used in this study are Instruments, UK, and Delta Plus coupled with CE Instru- also indicated in Table 10.1. The deep-sea hydrothermal fields ment NA2500, Thermo Quest, USA). Small-sized samples of Okinawa Trough, Izu-Ogasawara Arc, Mariana Trough in for sulfur isotope analysis were wrapped within tin capsules the North Pacific Ocean, Kermadec Arc in the South Pacific and directly subjected to EA/irMS. Ocean, and Rodriguez Triple Junction in the Indian Ocean, Sulfide-sulfur were recovered from the substrate sediments and the methane seeps (cold seeps) of Kagoshima and by treatment with warm (80 C) 30 % hydrogen peroxide Sagami Bays, Ryukyu and Mariana fore-arcs in the Pacific solution to generate sulfates, which were finally converted into

Ocean, and Sunda Trough in the Indian Ocean, were BaSO4. Dissolved sulfide in the hydrothermal fluids was fixed subjected to sample collection using submersibles (ROVs/ on board in order to precipitate zinc sulfide, and then it was

HyperDolphin and Kaiko, DSV/Shinkai 6500 belonging to converted to sulfate for recovery as BaSO4 precipitate. The JAMSTEC and RV/Ropos belonging to Canadian Scientific BaSO4 precipitate was converted into SO2 gas, as described by Submersible Facility). Yanagisawa and Sakai (Yanagisawa and Sakai 1983). During the dive studies, animal samples were collected The concentration and carbon isotope composition of the with a suction sampler or manipulator equipped with the dissolved methane in the water samples were determined submersible. Fluid and sediments used for geochemical following the methods of Tsunogai et al. (2000), using characterization were collected using all-titanium ALVIN- an isotope-ratio-monitoring-GC/MS (MAT 252, Thermo type fluid samplers (Von Damm et al. 1985), gas-tight Quest, USA). The methane content in the samples was calcu- 44 WHATS fluid samplers (Tsunogai et al. 2003), multi- lated by comparing the CO2 output with that of a working cylinder type ROCS fluid samplers (Yamanaka et al. standard gas containing c. 500 ppm methane in nitrogen. 2013), and NISKIN bottles equipped with the submersibles. All of the isotopic values were expressed using δ notation The fluid and sediment samples were collected as close as as a per mill deviation (‰) from international reference possible to the animal habitat in order to evaluate the rela- materials (VPDB for δ13C, CDT for δ34S and atmospheric 15 tionship between the animal soft bodies and the energy N2 for δ N, respectively). Analytical errors associated with sources for the chemosynthesis-based animal communities, the overall process of these determinations were 0.2, 0.3, and using isotopic signatures. 0.3‰, respectively. Table 10.1 Animal samples discussed in this paper 110 Organism Sampling Info Site (marks Genus (Order or Submarsible Dive Year/ Type Area Field in figures) Longitude Latitude Depth (m) Category Family) Species Food ecologya type # Month Note Hydrothermal Okinawa Minami- (MEn) 12738.3920E2823.4760N 701–709 Sea sponge (Demospongiae) ? Heterotroph (F) HyperDolphin 1327, 2011/9 system Trough Ensei knoll 1328 Izena Hole JADE (IzJ) 12704.8840E2716.2960N 1,305 Crab Shinkaia crosnieri Heterotroph HyperDolphin 1184 2010/9 and ectosymbiosis 12704.5000E2715.9360N 1,536 Crab Paralomis verrilli Heterotroph (C) HyperDolphin 1193 2010/9 12704.5000E2715.9360N 1,536 Crab Paralomis ? Heterotroph (C) HyperDolphin 1193 2010/9 12704.8840E2716.2960N 1,305 Shrimp Alvinocaris longirostris Heterotroph HyperDolphin 1184 2010/9 and ectosymbiosis 12704.8770E2716.2990N 1,536 Shrimp Opaepele loihi Heterotroph HyperDolphin 1192 2010/9 and exosymbiosis 12704.4940E2715.9420N 1,520 Bivalve Bathymodiolus platifrons Endosymbiosis HyperDolphin 1188 2010/9 (M) 12704.8770E, 2716.2990N, 1,536 Gastropoda Provanna ? Heterotroph HyperDolphin 1192, 2010/9 12704.5000E 2715.9360N (G) 1193 12704.8840E2716.2960N 1,300–1,535 Limpet Lepetodrilus nux Heterotroph HyperDolphin 1184, 2010/9 (G) 1192, 1193 12704.8770E, 2716.2990N, 1,536 Limpet Bathyacmaea secunda Heterotroph HyperDolphin 1192, 2010/9 12704.5000E 2715.9360N (G) 1193 12704.8780E2716.3020N 1,306 Fish (Zoarcidae) ? Heterotroph (C) HyperDolphin 1192 2010/9 12704.5000E2715.9360N 1,536 Starfish (Goniasteridae) ? Heterotroph HyperDolphin 1193 2010/9 (D) 12704.5000E2715.9360N 1,536 Starfish (Asteroidea) ? Heterotroph HyperDolphin 1193 2010/9 (D) 12704.8840E2716.2960N 1,305 Sea sponge (Demospongiae) ? Heterotroph (F) HyperDolphin 1184 2010/9 12704.5000E2715.9360N 1,536 Sea sponge (Hexachtinellida) ? Heterotroph (F) HyperDolphin 1193 2010/9 12704.5000E2715.9360N 1,536 Sea sponge (Demospongiae) ? Heterotroph (F) HyperDolphin 1193 2010/9 12704.4940E2715.9420N 1,520 Polychaeta Branchipolynoe pettiboneae Heterotroph (P) HyperDolphin 1188 2010/9 Parasite on Bathymodiolus mussels 12704.7990E2716.2260N 1,335 Polychaeta Paradialychone ? Endosymbiosis HyperDolphin 1193 2010/9 (T) Hakurei 12704.1340E, 2714.9400N, 1,593 Crab Shinkaia crosnieri Heterotroph HyperDolphin 1191, 2010/9, (IzH) 12704.1410E 2714.9440N and 1311 2011/8 ectosymbiosis 12704.0890E2714.8150N 1,617 Bivalve Bathymodiolus japonicus Endosymbiosis HyperDolphin 1329 2011/10 (M) Ihaya Ridge (IhR) 12658.1880E2733.0180N 1,399 Crab Shinkaia crosnieri Heterotroph HyperDolphin 1183 2010/9 and al. et Yamanaka T. ectosymbiosis 12658.1880E2733.0180N 1,399 Shrimp Alvinocaris longirostris Heterotroph HyperDolphin 1183 2010/9 and ectosymbiosis 12658.1880E2733.0180N 1,399 Bivalve Calyptogena okutanii Endosymbiosis HyperDolphin 1183 2010/9 (T) 0ACmiaino h tbeIooi opstoso abn irgn n Sulfur and Nitrogen, Carbon, of Compositions Isotopic Stable the of Compilation A 10

12658.1880E2733.0180N 1,399 Bivalve Acharax ? Endosymbiosis HyperDolphin 1183 2010/9 (T) 12658.1880E2733.0180N 1,399 Tube worm Alaysia ? Endosymbiosis HyperDolphin 1183 2010/9 (T) Iheya North (IhN) 12704.4940E2715.9420N 1,520 Crab Shinkaia crosnieri Heterotroph HyperDolphin 1188 2010/9 Knoll and ectosymbiosis 12704.4940E2715.9420N 1,520 Bivalve Calyptogena soyoae Endosymbiosis HyperDolphin 1188 2010/9 (T) 12704.4940E2715.9420N 1,520 Bivalve Bathymodiolus platifrons Endosymbiosis HyperDolphin 1188 2010/9 (M) 12653.9930E2747.2860N 993 Tube worm Lamellibrachia ? Endosymbiosis HyperDolphin 222 2003/9 (T) 12653.9930E2747.2860N 993 Barnacle Ashinkailepas seepiophilia heterotroph (F) HyperDolphin 222 2003/9 Tarama (Tr) 12432.1650E2505.4690N 1,732 Shrimp Alvinocaris? ? Heterotroph HyperDolphin 1322 2011/9 Found in the knoll and gut of Liparis ectosymbiosis 12432.3490E2505.5760N 1,556 Fish Liparis ? Heterotroph (C) HyperDolphin 1034 2009/7 12432.1650E2505.4690N 1,732 Fish Liparis ? Heterotroph (C) HyperDolphin 1322 2011/9 12432.3100E2505.5540N 1,588 Starfish (Asteroidea) ? Heterotroph HyperDolphin 1034 2009/7 Preyed on (D) sponge 12432.3100E2505.5540N 1,588 Sea sponge (Hexachtinellida) ? Heterotroph (F) HyperDolphin 1034 2009/7 12432.1360E2505.3260N 1,850 Soft coral (Alcyonacea) ? Heterotroph (F) HyperDolphin 1108 2010/4 12432.1240E2505.3030N 1,862 Sea Enypniaster eximia Heterotroph HyperDolphin 1108 2010/4 cucumber (D) 12432.3640E2505.6310N 1,500 Octopus Benthoctopus ? Heterotroph (C) HyperDolphin 1033 2009/7 Daiyon- (Yo) 12241.9990E2450.9310N 1,336 Crab Shinkaia crosnieri Heterotroph Shinkai2000 1273 2001/5 Yonaguni and knoll ectosymbiosis 12241.9990E2450.9310N 1,336 Bivalve Bathymodiolus platifrons Endosymbiosis Shinkai2000 1273 2001/5 (M) Hatoma (Ht) 12350.3690E2451.4510N 1,523 Crab Shinkaia crosnieri Heterotroph Shinkai2000 1352 2002/5 knoll and ... ectosymbiosis 12350.3690E2451.4510N 1,523 Shrimp Alvinocaris longirostris Heterotroph Shinkai2000 1352 2002/5 and ectosymbiosis 12350.3690E2451.4510N 1,523 Shrimp Lebbeus washingtonianus Heterotroph Shinkai2000 1352 2001/5 (D) 12350.3690E2451.4510N 1,523 Crab Munidopsis ryukyuensis Heterotroph Shinkai2000 1352 2002/5 (D) 12350.3650E2451.5580N 1,484 Crab Paralomis ? Heterotroph (C) Shinkai2000 1361 2002/6 12350.3690E2451.4510N 1,523 bivalve Bathymodiolus platifrons Endosymbiosis Shinkai2000 1270 2001/5 (M) Izu- Myojin (My) 139520E32060N 1,274 Crab Gandalfus yunohana Heterotroph HyperDolphin 185 2003/6 Ogasawara knoll (D) arc 13952.0810E3206.2780N 1,303 Bivalve Bathymodiolus septemdierim Endosymbiosis HyperDolphin 1284 2011/6 (T) (continued) 111 Table 10.1 (continued) 112 Organism Sampling Info Site (marks Genus (Order or Submarsible Dive Year/ Type Area Field in figures) Longitude Latitude Depth (m) Category Family) Species Food ecologya type # Month Note Sumisu (Sm) 14004.2580E3128.1790N 686 Bivalve Bathymodiolus septemdierim Endosymbiosis HyperDolphin 84 2002/3 caldera (T) 14004.2580E3128.1790N 686 Sea sponge Characella ? Endosymbiosis HyperDolphin 84 2002/3 Provided from (T) Dr. Lindsay 14004.2580E3128.1790N 686 Tube worm Lamellibrachia ? Endosymbiosis HyperDolphin 84 2002/3 (T) Suiyo (Sy) 14038.6680E2834.2680N 1,381 Bivalve Bathymodiolus septemdierim Endosymbiosis HyperDolphin 1285 2011/6 Seamount (T) Kaikata (Kt) 14104.30E2642.50N 508 Crab Gandalfus yunohana Heterotroph HyperDolphin 186 2003/6 Provided from Seamount (D) Dr. Tsuchida 14104.2500E2642.5000N 448 Crab Leptodius exaratus Heterotroph Shinkai2000 1234 2000/11 Provided from (D) Dr. Tsuchida 14104.2500E2642.5000N 448 Fish Symphurus orientalis Heterotroph (C) Shinkai2000 1234 2000/11 Provided from Dr. Tsuchida South Mariana Snail site 14337.1950E1257.1670N 2,860 Crab Gandalfus yunohana Heterotroph Shinkai6500 793 2003/10 Mariana backarc (Sn) (D) Trough spreading 14337.1670E1257.1850N 2,861 Gastropoda Alviniconca hessri Endosymbiosis ROPOS 776 2004/3 center (T) Archaean 14337.9010E1256.3700E 2,990 Gastropoda Phymorhynchus ? Heterotroph ROPOS 781 2004/3 site (Ar) (D) Yamanaka 14336.7890E1256.6600N 2,827 Bernacle Neoverruca brachylepadoformis Heterotroph (F) ROPOS 779 2004/3 site (Ym) 14336.7960E1253.6380N 2,828 Sea Marianactis ? Heterotroph ditto 774 2004/3 anemone (D) Kermadec Brothers Lower 17904.3020E3452.7220S 1,336 Tube worm Lamellibrachia sp.1 Endosymbiosis Shinkai6500 854 2004/11 Arc Volcano Cone site (T) (Br) 17904.3020E3452.7220S 1,336 Tube worm Lamellibrachia sp.2 Endosymbiosis ditto 854 2004/11 (T) 17904.3020E3452.7220S 1,336 Barnacle Vulcanolepas osheai Heterotroph (F) ditto 854 2004/11 Rodriguez Hakuho Kaiko field 7002.400E2519.160S 2,432, 2,442 Bivalve Bathymodiolus marisindicus Endosymbiosis Kaiko 168, 2000/8 Triple Knoll (RtK) (T) 169 Junction, Central Indian Ridge Methane seep Ryukyu arc Kuroshima (Kr) 12411.5470E, 2407.7980N, 638, 624 Bivalve Bathymodiolus hirtus Endosymbiosis Shinkai2000 1355, 2002/5, knoll 12411.5350E 2407.8050N (M) 1364 6 (cold seep) 12411.5470E, 2407.7980N, 638, 624 Bivalve Bathymodiolus securiformis Endosymbiosis Shinkai2000 1355, 2002/5, 12411.5350E 2407.8050N (M) 1364 6 12411.5470E, 2407.7980N, 638, 624 Bivalve Calyptogena kawamurai Endosymbiosis Shinkai2000 1355, 2002/5, 12411.5350E 2407.8050N (T) 1364 6

Kyushu Kagoshima Wakamiko 13048.0520E3139.7420N 102 Tube worm Lamellibrachia satsuma Endosymbiosis HyperDolphin 686 2007/6 al. et Yamanaka T. Island Bay (KgW) (T) Sagami off (SbH) 13913.3960E3500.9260N 928 Shrimp Alvinocaris longirostris Heterotroph HyperDolphin 1293 2011/6 Trough Hatsushima and ectosymbiosis (Sagami Bay) 13913.3800E3500.9400N 910 Bivalve Bathymodiolus platifrons Endosymbiosis HyperDolphin 1291 2011/6 (M) 0ACmiaino h tbeIooi opstoso abn irgn n Sulfur and Nitrogen, Carbon, of Compositions Isotopic Stable the of Compilation A 10

13913.4940E, 3500.2220N, 1,180, 910 Bivalve Bathymodiolus japonicus Endosymbiosis HyperDolphin 524, 2006/3, 13913.3800E 3500.9400N (M) 1291 2011/6 13913.3220E3500.9550N 855 Bivalve Calyptogena okutanii Endosymbiosis HyperDolphin 1291 2011/6 (T) 13913.3220E3500.9550N 855 Bivalve Calyptogena soyoae Endosymbiosis HyperDolphin 1291 2011/6 (T) 13913.4820E3500.2220N 1,172 Bivalve Acharax johnsoni Endosymbiosis HyperDolphin 528 2006/3 (T) 13913.4820E3500.2220N 1,172 Bivalve (Thyasiridae) ? Endosymbiosis HyperDolphin 528 2006/3 (T) 13913.4820E3500.2220N 1,172 Bivalve Conchocela bisecta Endosymbiosis HyperDolphin 528 2006/3 (T) 13913.4580E3500.1680N 1,173 Gastropoda Provanna glabra Heterotroph (C) HyperDolphin 525 2006/3 13913.4580E, 3500.1680N, 1,173, 1,172 Gastropoda Margarites shinkai Heterotroph HyperDolphin 525, 2006/3 13913.4820E 3500.2220N (D) 528 13913.4940E3500.2220N 1,180 Gastropoda Phymorhynchus buccinoides Heterotroph HyperDolphin 524 2006/3 (D) 13913.4580E3500.1680N 1,173 Gastropoda Bathyacmaea nipponica Heterotroph HyperDolphin 525 2006/3 (D) 13913.4940E3500.2220N 1,180 Gastropoda Oenopota sagamiana Heterotroph HyperDolphin 524 2006/3 (D) 13913.4580E3500.1680N 1,173 Starfish Ophiuroidea ? Heterotroph HyperDolphin 525 2006/3 (D) 13913.5600E3500.0540N 1,184 Sea Actiniaria ? Heterotroph HyperDolphin 524 2006/3 anemone (D) 13913.4820E3500.2220N 1,172 Tube worm Lamellibrachia ? Endosymbiosis HyperDolphin 528 2006/3 (T) 13913.4820E3500.2220N 1,172 Tube worm Alaysia ? Endosymbiosis HyperDolphin 528 2006/3 (T) 13913.4580E3500.1680N 1,173 Polychaeta Nicomache ohtai Heterotroph HyperDolphin 525 2006/3 (D) Northern South Chamorro 14600.2100E1346.9990N 2,900 Bivalve Bathymodiolus ? Endosymbiosis Kaiko 165 2000/6 Mariana arc seamount (SCh) (D) ... Southern Shinkai (SmS) 14302.940E1139.090N 5,620 Bivalve Calyptogena mariana Endosymbiosis Shinkai6500 1234 2010/9 Provided from Mariana arc Seep Field (T) Dr. Ohara Sunda off southwest corner 10547.2020E, 724.6000S, 2,390, 2,100 Bivalve Calyptogena garuda Endosymbiosis Shinkai6500 716, 2002/10 Provided from Trough of Java (Jv) 10546.9980E 724.6000S (T) 727 Dr. Soh aReported food ecology in previous literature: heterotroph (C): Carniver, Scavenger and/or Grazer, (D): Detritus and sediment feeder, (F): Filter feeder, (G): Grazer, (P) Parasite, ectosymbiosis (T) and endosymbiosis (T): harboring only thioautotrophic endosymbiont, endosymbiosis (M): harboring only methanotrophic endosymbiont, endosymbiosis (d): harboring both thiotrophic and methanotrophic endosymbionts 113 114 T. Yamanaka et al.

collections off Hatsushima, in Sagami Bay. Nearly all data 10.3 Analytical Results for Isotopic were beyond the range of common marine organisms. Composition Calyptogena clams, as shown by open circles, were located in the typical thioautotrophic range, as were the specimens 10.3.1 Isotopic Compositions of Animal obtained from hydrothermal fields, with one exception: Samples from Hydrothermal Fields Calyptogena soyoae off Hatsushima (δ34S ¼ +16.8 ‰). Except for two groups of samples from Kuroshima knoll The analytical results for the isotopic compositions of ani- (δ34S ¼ +10.6, +7.2 for gill tissues), methanotrophic mal soft body parts and issuing fluids are summarized in Bathymodiolus mussels had high δ34S values, which are Tables 10.2 and 10.3. Diagrams of the relationships among comparable to those of common marine organisms. Some nitrogen and sulfur isotopic ratios vs. carbon isotopic ratios of the bivalves (Bathymodiolus, Calyptogena, and Acharax) are shown in Figs. 10.2 and 10.3. Abbreviations in the showed significantly low δ15N values < À5 ‰. figures indicate the locations of the samples (red-colored Bathymodiolus mussels also showed significantly low δ15N abbreviations indicate sediment-starved hydrothermal fields, values as well as the lowest δ13C values. It appeared that the see Table 10.1), and the asterisks to the right of the abbre- δ13C and δ15N values of these samples are positively viations indicate the species that harbor methanotrophic correlated (R2 ¼ 0.85). endosymbionts. The diagrams also indicate the approximate ranges of common marine organisms that rely on phototrophic products (insert enclosed by green dotted box). The δ13C vs. δ15N plot in Fig. 10.2 shows that many 10.3.3 Stable Isotopic Composition of the animals, especially crustacean species, were among the range Issuing Fluids Associated with Animal of common marine organisms, while the δ13Cvs.δ34Splot Communities (Fig. 10.3) indicates that most samples had lower δ34Svalues (< +15 ‰), relative to the common marine organisms. 10.3.3.1 Hydrogen Sulfide Calyptogena clams, indicated by open circles, had a typical The δ34S values of hydrogen sulfide dissolved in the hydro- thioautotrophic range of δ13C ¼À35 Æ 5 ‰ and thermal fluids ranged from nearly 0 to +12 ‰ (Table 10.2), δ34S  +15 ‰,whileBathymodiolus mussels, which harbor whereas those from methane-rich seeps were less than thioautotrophic and/or methanotrophic endosymbionts in their À20 ‰, except for the fumarolic gas emitting from gill tissues, showed wider δ13C values ranging from À50 to Wakamiko submarine volcano (Table 10.3). Thiotrophic À25 ‰ and significantly low δ15Nvalues(<0 ‰,Fig.10.2). animals, which are known to harbor only thioautotrophic Nearly all methanotrophic mussels had high δ34S values, close endsymbionts or feed on thioautotrophic products, are to +15 ‰, while the thiotrophic mussels had significantly expected to have δ34S values close to that of the associated lower δ34S values, relative to the other mussels. Vestimentif- hydrogen sulfide. Therefore, the apparent sulfur isotopic eran tubeworms, indicated by open cross symbols, also had a fractionation between the soft tissue of thiotrophic animals wide range of δ13Cvalues(À35 to À12 ‰), indicating that and the associated hydrogen sulfide collected from the they harbor thioautotrophic endosymbionts with various hydrothermal systems should be zero or nearly zero. As types of carbon fixation pathways in addition to the Calvin shown in Fig. 10.6, most δ34S values of the animals were cycle. Certain animal samples (Alvinocaris shrimp, Alaysia lower than those of the hydrogen sulfides, while some tubeworms at Iheya Ridge field, Ashinkailepas barnacles at animals showed values slightly higher than those of the Iheya North knoll, Bathymodiolus mussels at Sumisu hydrogen sulfides. On the other hand, methanotrophic spe- caldera, and Lamellibrachia tubeworms at Brother seamount) cies, which are known to harbor methanotrophic had significantly lower δ34Svalues(< À10 ‰), relative endsymbionts, and heterotrophs relying on phototrophic to the hydrogen sulfide issuing from the associated hydrother- products are expected to have high δ34S values, ranging mal vents. from +15 to +21 ‰, since they assimilate seawater sulfates with δ34S values of ~+21 ‰. Figures 10.7 and 10.8 show the apparent differences between the δ34S values of animal soft 10.3.2 Isotopic Compositions of Animal tissues and seawater-sulfate for samples from hydrothermal Samples from Methane Seep Fields systems and methane seeps, respectively. The diagrams show that, with some exceptions, especially bivalves Analytical results for animal soft tissues and issuing fluids inhabiting methane seeps (Bathymodiolus japonicas, B. are summarized in Table 10.3. Figures 10.4 and 10.5 show platifrons, B. hirtus, and B. securifornis in Fig. 10.8), δ34S relationships comparable to those shown for the hydrother- values for all animal tissues were significantly lower than mal fields. Samples without abbreviations represent that of seawater-sulfate. Table 10.2 Carbon, nitrogen, and sulfur isotope compositions of benthic animal bodies sampled around hydrothermal fields and associated environmental geochemical data Sulfur and Nitrogen, Carbon, of Compositions Isotopic Stable the of Compilation A 10 Soft body parts Issuing fluids Hydrogen Carbon Nitrogen Sulfur Methane Ammonium sulfide 13 15 34 13 34 Genus (Order or δ CVPDB δ NAir δ SCDT δ CVPDB δ SCDT 15 Area Field Site Family) Species Tissues (‰)SD(‰)SD(‰)SDNumber (‰) δ NAir (‰) (‰) References Okinawa Minami- (Demospongiae) ? Bulk À26.6 À0.6 +6.9 10.6 1 À26.0 to nr nr *Kawagucci et al. (2013) Trough Ensei knoll À24.7* (Demospongiae) ? Bulk À29.7 À0.2 +11.9 1 (Demospongiae) ? Bulk À26.0 +2.7 +13.6 1 Izena Hole JADE Shinkaia crosnieri Muscle À23.1 0.4 +5.2 0.3 À3.8 3 À41.0 to nr +3.6 to *Ishibashi et al. (1995), À36.0* +7.7** **Kim et al. (1989), Sakai Paralomis verrilli Muscle À24.2 +5.6 À5.4 1 et al. (1990), Gamo et al. (2001) Paralomis ? Muscle À24.8 1.1 +6.8 1.2 +7.5 2 Alvinocaris longirostris Muscle À21.0 1.6 +4.3 1.1 À0.7, 4 +2.7 Egg À19.7 3.5 +0.5 2.2 +1.1, 4 +1.6 Opaepele loihi Muscle À17.6 0.7 À0.6 0.4 nd 3 Bathymodiolus platifrons Foot À30.4 0.9 À0.4 1.1 +7.5, 4 +7.7 Gill À30.1 0.9 0.0 0.8 +8.5, 3 +9.1 Gill À31.5 0.6 nd +10.6 2 Mantle À31.5 1.1 À0.2 +11.4 2 Provanna ? Bulk À25.3 0.9 +3.0 2.5 +5.7 4 Lepetodrilus nux Bulk À20.8 2.3 +2.3 0.4 +3.5 5 Bathyacmaea secunda Bulk À26.6 1.3 +0.9 1.7 +8.6 4 (Zoarcidae) ? Bulk À25.7 +7.7 À4.1 1 (Goniasteridae) ? Bulk À42.3 +8.3 nd 1 À

(Asteroidea) ? Bulk 34.4 +8.3 +14.6 1 ... (Demospongiae) ? Bulk À21.2 +1.8 +5.6 1 (Hexachtinellida) ? Bulk À29.6 +7.3 +10.9 7.1 1 (Hexachtinellida) ? Bulk À30.6 +8.0 +6.9 1 (Demospongiae) ? Bulk À29.5 +7.9 nd 1 (Demospongiae) ? Bulk À38.5 À6.6 nd 1 Branchipolynoe pettiboneae Bulk À29.7 0.6 +6.4 0.5 +8.9 4 Paradialychone ? Bulk À33.4 1.1 +2.7 0.3 +3.6 3 Hakurei Shinkaia crosnieri Muscle À22.1 1.4 +4.7 1.3 +0.1 6 À32.0* nr +5.5 to *Kawagucci et al. (2010) +7.8 Bathymodiolus japonicus Gill À31.3 1.3 À3.3 0.1 À8.0 3 Iheya Ridge Shinkaia crosnieri Muscle À23.7 0.7 +4.4 0.6 À1.1 3 À41.2* nr À0.3 to *Ishibashi et al. (1995), +3.0** **Kim et al. (1990), Gamo Alvinocaris longirostris Muscle À26.1 2.0 +6.9 1.1 À21.0 3 et al. (1991) Egg À34.7 0.3 +3.3 0.8 À21.0 3 Calyptogena okutanii Gill À35.2 0.2 +3.1 5.1 +7.7 2 Foot À33.9 1.0 +4.0 2.9 +2.0 3

Acharax ? Bulk À31.7 0.5 +1.6 2.3 À6.5 3 115 (continued) Table 10.2 (continued) 116 Soft body parts Issuing fluids Hydrogen Carbon Nitrogen Sulfur Methane Ammonium sulfide 13 15 34 13 34 Genus (Order or δ CVPDB δ NAir δ SCDT δ CVPDB δ SCDT 15 Area Field Site Family) Species Tissues (‰)SD(‰)SD(‰)SDNumber (‰) δ NAir (‰) (‰) References Alaysia ? Bulk À17.5 0.5 +3.5 0.2 À22.0 3 Iheya North Shinkaia crosnieri Muscle À32.4 +4.1 +6.9 1 À54.0 to nr +8.0 to *Kawagucci et al. (2011), Knoll À52.8* +12.2** **Yamanaka et al. (2000b) Calyptogena soyoae Gill À37.5* +2.0 +1.8* 1 *Mae et al. (2007) Mantle À36.3* +4.5 +0.4*1 ditto Bathymodiolus platifrons Gill À45.2 0.9 À4.4 0.2 +13.3 3 Bathymodiolus platifrons Gill À49.2* 1.6* À4.2* 0.3* +13.4* 3 *Mae et al. (2007) Mantle À49.0* 2.9* À3.7* 0.5* +18.3*3 ditto Lamellibrachia ? Trophosome À22.3 1.3 +2.9 0.9 nd 6 Ashinkailepas seepiophilia Bulk À22.8 0.4 +7.2 0.7 À11.0 5 Tarama Alvinocaris? ? Muscle À15.1 À0.6 +6.8 1 À38.4* nr *Inoue and Ueno, personal knoll communication Liparis ? Muscle À22.3 +9.2 À7.3 1 Liparis ? Muscle À25.7 +13.6 +7.5 1 (Asteroidea) ? Bulk À32.4 +15.1 +8.7 1 (Hexachtinellida) ? Bulk À34.3 +10.6 +5.0 1 ditto Bulk À35.1 +9.9 +4.8 1 ditto Bulk À32.1 +7.7 +2.5 1 (Alcyonacea) ? Bulk À26.3 +11.6 +13.8 1 Enypniaster eximia Bulk À20.7 +12.8 +14.6 1 Benthoctopus ? Muscle À23.1 +14.0 +11.5 1 Yonaguni Shinkaia crosnieri Muscle À22.9 0.0 +3.1 0.1 +5.4 3 À27.3 to nr +13.0 *Konno et al. (2006) knoll À24.8* Muscle À17.3 +1.0 +4.6 1 Bathymodiolus platifrons Gill À25.1 0.1 À7.5 1.4 +13.5 2 Mantle À26.2 0.2 À5.9 1.0 +13.2 2 Bathymodiolus platifrons Gill À24.5* À6.8* +11.1* 1 *Naraoka et al. 2008 Hatoma Shinkaia crosnieri Muscle À24.6 1.6 +5.1 0.7 +8.3 6 À51.3 to nr +8.0 to *Naraoka et al. (2008) knoll À44.3* +12.0 Alvinocaris longirostris Muscle À32.2 5.8 +5.2 1.2 +11.1 3.9 6 Lebbeus washingtonianus Muscle À30.8 0.1 +5.5 0.0 nd 2 Munidopsis ryukyuensis Muscle À28.3 0.8 +3.8 0.7 nd 4 Paralomis ? Muscle À26.7 +9.0 nd 1 Bathymodiolus platifrons Gill À44.8* À4.4* +13.9* 1 *Naraoka et al. (2008) Izu- Myojin Gandalfus yunohana Muscle À15.9 2.2 +8.1 0.4 nd 5 À16.3* nr +4.9 to *Tsunogai et al. (2000), Ogasawara knoll +5.6** **Yamanaka et al. (2000a,

arc 2000b) al. et Yamanaka T. Bathymodiolus septemdierim Gill À34.4 0.7 +0.8 0.8 +2.9, 4 +3.1 Sumisu Bathymodiolus septemdierim Gill À34.5 À4.2 À24.4 1 nr nr nr caldera 0ACmiaino h tbeIooi opstoso abn irgn n Sulfur and Nitrogen, Carbon, of Compositions Isotopic Stable the of Compilation A 10

Characella ? Bulk À34.8 +11.1 À5.9 1 Harboring thioautotrophic endosymbiont (Nishijima et al. (2010) Lamellibrachia ? Trophosome À12.9 +6.1 À18.5 1 Suiyo Bathymodiolus septemdierim Gill À35.3 À2.4 À0.3 1 À7.4 to nr +3.1** *Tsunogai et al. (2005), Seamount À5.3* **Yamanaka et al. (2000a, 2000b) mantle À32.8 À1.9 +2.1 1 Gill À36.0* À5.1* +4.3* 1 *Naraoka et al. (2008) Gill À34.1 1.1 À1.9 1.7 À0.1 2 Kaikata Gandalfus yunohana Muscle À20.2 2.3 +9.9 0.7 nd 12 nr nr nr Seamount Leptodius exaratus Muscle À21.9 0.4 +8.4 0.6 nd 2 Symphurus orientalis Muscle À17.4 +10.1 nd 1 South Mariana Snail site Gandalfus yunohana Muscle À21.2 1.8 +9.7 1.2 +7.5 0.6 4 nr nr +7.0 - *Kakegawa et al. (2008) Mariana backarc +8.7* (Sulfide minerals) Trough spreading Alviniconca hessri Muscle À31.3 0.4 +3.6 0.8 +9.8 0.6 4 center Archaean Phymorhynchus ? foot À30.8 +4.8 +4.4 1 nr nr À1.8 to *Kakegawa et al. (2008) site +1.7* (Sulfide minerals) Yamanaka Neoverruca brachylepadoformis Bulk À21.8 0.7 +10.4 0.8 nd 5 nr nr +5.1 to *Kakegawa et al. (2008) site +6.3* (Sulfide minerals) Marianactis ? Bulk À17.7 +9.7 +4.6 1 Kermadic Brothers Lower Lamellibrachia ? Trophosome À7.5 1 +1.9 0.4 nd 3 À37.0 to nr À8.0 to *de Ronde et al. (2011) Arc volcano Cone site À33.9* À4.8* Lamellibrachia ? Trophosome À12.9 0.3 +3.7 0.0 nd 3 Vulcanolepas osheai Bulk À13.0 1.7 +7.8 1.1 nd 4 Central Hakuho Kaiko field Bathymodiolus marisindicus Gill À31.3* 0.7* À8.1* 1.5* +5.3** 0.3** 3 À8.6 to nr +6.8 to *Van Dover et al. (2001), Indian Knoll À8.7*** +7.0** **Yamanaka et al. (2003a), Ridge ***Gamo et al. (2001) Asterisks on the right shoulder of isotope values indicate cited data appeared in Reference column at the same line. nr not reported, nd not determined ... 117 Table 10.3 Carbon, nitrogen, and sulfur isotope compositions of benthic animal bodies sampled around methane seeps and associated environmental geochemical data 118 Soft body parts Issuing fluids Hydrogen Carbon Nitrogen Sulfur Methane Ammonium sulfide 13 15 34 13 34 Genus (Order δ CVPDB δ NAir δ SCDT δ CVPDB δ SCDT 15 Area Field Site or Family) Species Tissues (‰)SD(‰)SD(‰)SDNumber (‰) δ NAir (‰) (‰) Note References Ryukyu Kuroshima Bathymodiolus hirtus Gill À37.6 À1.5 +17.2 1 À43.3 to nr À22.5 to Separate *Tsunogai et al. (2010) arc knoll À38.6* À21.2 type Foot À39.5 À1.3 +17.4 1 (See text) Mantle À43.1 À1.4 +17.6 1 Adductor À36.9 À1.0 +17.5 1 Bathymodiolus hirtus Gill À43.1 0.0 +10.6 1 Mixed type Foot À41.5 +0.8 +10.7 1 (See text) Mantle À45.8 nd +9.8 1 Adductor À45.3 nd +9.1 1 Bathymodiolus securiformis Gill À41.6 +0.9 +20.2 1 Separate type Foot À39.9 +0.5 +20.2 1 (see text) Mantle À41.5 +1.4 +20.5 1 Adductor À40.7 +1.9 +20.7 1 Bathymodiolus securiformis Gill À38.3 À0.6 +7.2 1 Mixed type Foot À36.4 nd +7.9 1 (See text) Mantle À42.8 nd +6.2 1 Adductor À37.5 nd +4.1 1 Calyptogena kawamurai Gill À34.1 0.7 +5.3 0.5 À3.2 6.5 4 Foot À33.5 0.2 +6.9 0.2 +2.0 8.1 4 Mantle À34.2 0.5 +6.3 0.3 À0.5 3.9 4 Adductor À33.9 0.3 +7.3 0.5 À4.9 15.8 4 Kyushu Kagoshima Wakamiko Lamellibrachia satsuma Trophosome À20.5 0.9 +6.4 1.2 À10.0 14.5 8 À27.2 2.7 À33.6 to *Miura et al. (2002), Mizota Island Bay À27.1*, and Maki (1998), Yamanaka Trophosome nd nd À21.2 0.5 4 +6.8, +13.8 et al. (2013b) Sagami off Alvinocaris longirostris Muscle À31.1 1.0 +0.9 0.5 nd 3 À72.0 to nr À22.7** *Hattori et al. (1996), **Sakai Trough Hatsushima À70.0* et al. (1987) (Sagami Bathymodiolus platifrons Gill À58.9 3.2 À7.6 0.7 +18.7 1.9 3 Bay) Bathymodiolus japonicus Gill À60.4 0.0 À7.7 1.0 +16.4 3 Calyptogena okutanii Gill À69.2 1.1 À11.2 1.1 nd 2 Calyptogena okutanii Gill À36.6 0.3 À9.7 0.2 +16.8 6.5 3

Calyptogena soyoae Gill À32.1 nd À23.4* 4.9* 3 *Mizota and Yamanaka al. et Yamanaka T. (2003) Calyptogena soyoae Gill À36.6 0.9 À6.6 2.2 nd 6 Acharax johnsoni Gill À30.4 À11.1 nd 1 Acharax johnsoni Gill À30.0 0.0 À4.8 0.3 nd 2 0ACmiaino h tbeIooi opstoso abn irgn n Sulfur and Nitrogen, Carbon, of Compositions Isotopic Stable the of Compilation A 10

(Thyasiridae) ? Gill À37.6 À7.0 nd 1 Conchocela bisecta Gill À36.0 0.1 À8.5 0.1 nd 2 Provanna glabra Bulk À30.7 0.6 À2.3 0.6 nd 2 Margarites shinkai Bulk À32.9 0.1 À1.7 0.1 nd 2 Phymorhynchus buccinoides Bulk À58.6 4.4 À4.0 1.2 +13.8 6 Bathyacmaea nipponica Bulk À25.7 À3.7 nd 1 Oenopota sagamiana Bulk À30.9 3.6 +2.0 1.5 nd 6 (Ophiuroidea) ? Bulk À32.8 1.3 À2.6 1.1 nd 4 (Actinaria) ? Bulk À15.2 0.7 +15.0 0.7 nd 2 Lamellibrachia ? Trophosome À25.4 0.9 +4.1 0.9 +7.8 3 Lamellibrachia ? Trophosome À20.2 1.5 À0.1 1.8 À27.9, À17.9 Alaysia ? Trophosome À19.5 1.1 +4.2 1.1 nd 2 Nicomache ohtai Bulk À33.3 +1.1 nd 1 Northern South Bathymodiolus ? Gill À18.9* +1.8* +10.6*1À14.6*nr À32.3* *Yamanaka et al. (2003b) Mariana Chamorro Foot nr nr +10.4*1 arc seamount Mantle À21.4* +3.1* +10.2*1 Adductor À19.1* +2.7*nr1 Viscera À20.2* +2.9*nr1 Southern Shinkai Calyptogena mariana Gill À34.6* 0.5* +4.2* 1.4* À9.0 1.0 3 nr nr nr *Ohara et al. (2012) Mariana Seep Field arc Sunda off Calyptogena garuda Gill À35.5 0.3 À1.5 4.2 À12.3 4.2 2 nr nr nr Trough southwest Foot À34.4 1.2 +2.2 4.4 nd 2 corner of Java Mantle À34.8 0.8 +1.3 3.6 nd 2 Adductor À34.5 1.1 +2.7 3.9 nd 2 Asterisks on the right shoulder of isotope values indicate cited data appeared in Reference column at the same line.

nr not reported, nd not determined ... 119 120 T. Yamanaka et al.

Fig. 10.2 Plot of carbon vs. nitrogen isotopic ratios for animal Calyptogena Common marine soft bodies from hydrothermal Bathymodiolus animals Tr fields. Numerical data are shown bernacle Sm Ym in Table 10.2. Barnacles include Shinkaia Kt Ashinkailepas and Neoverruca. Alvinocaris Tr Ym Crustacean 1 includes two Ht Sn limpet IzJ shrimps, Lebbeus and Opaepele, Br Kt My and three crabs, Munidopsis, crustacean 1 IhR IzJ IhN Sm Gandalfus, and Leptodius. crustacean 2 Ht IzJ Ht IzJ Crustacean 2 includes the crab, Tubeworm IzJ Ar Ht IzH Paralomis. Others include sea IhN Ht Br (‰) Alviniconca IhR IhR IzJ IhR anemones, soft corals, and sea Fr IzJ IhN Air others sponges. Abbreviations in the IzJ Yo IzJ N MEn My IzJ

figures indicate the locations of 15 Yo IhN IzJ* the samples (red-colored d IzJ abbreviations indicate sediment- MEn MEn starved hydrothermal fields, see IzJ Tr Table 10.1) Sy IhN* IhN* Sy

Ht* Sm Sy IzJ Yo* RtK

13 d C VPDB (‰)

Fig. 10.3 Plot of carbon vs. Common sulfur isotopic ratios for animal Tr marine MEn animals soft bodies from hydrothermal IhN* Ht* fields. Numerical data are shown MEn Yo* in Table 10.2. Barnacles include IhN* Ht IzJ* IzJ Ashinkailepas and Neoverruca. IzJ IzJ IhR Fr Ht Crustacean 1 includes two Sn Tr IzJ* IzJ shrimps, Lebbeus and Opaepele, IhN IzJ MEnHt IzJ and three crabs, Munidopsis, Sy IzJ Ym RtK Yo IzJ Yo Gandalfus, and Leptodius. Ar IhR My Tr IhN Crustacean 2 includes the crab, IhN Paralomis. Others include sea Sy IzH

(‰) IhR anemones, soft corals, and sea IzJ sponges. Abbreviations in the CDT Calyptogena Sm IzJ figures indicate the locations of S Bathymodiolus the samples (red-colored 34

d bernacle abbreviations indicate sediment- IhN starved hydrothermal fields, see Shinkaia Table 10.1) Alvinocaris limpet Br crustacean 1 crustacean 2 IhR Tubeworm IhR Alviniconca Sm others

13 d C VPDB (‰) 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 121

Fig. 10.4 Plot of carbon vs. nitrogen isotopic ratios of animal Common marine soft bodies from methane seep animals fields. Numerical data are shown in Table 10.3. Other bivalves Calyptogena include Thyasiridae, Solemyidae, Bathymodiolus and Thyasiridae groups. These Annelida bivalves harbor thioautotrophic other bivalves endosymbiots in their gill tissues. limpet Others include Alviniconca others KgW shrimp, Actiniaria sea anemone, Kr and Ophiuroidea starfish. (‰) SmS Abbreviations in the figures Air indicate the locations of the N Kr* SCh 15

samples (see Table 10.1), and d Kr* plots without abbreviations Kr* Jv represent collections off Kr* Hatsushima, in Sagami Bay

SbH*SbH*

SbH*

13 d C VPDB (‰)

Fig. 10.5 Plot of carbon vs. sulfur isotopic ratios of animal Common marine soft bodies from methane seep animals fields. Numerical data are shown Kr* in Table 10.3. Other bivalves SbH* include Thyasiridae, Solemyidae, and Thyasiridae groups. These SbH* Kr* bivalves harbor thioautotrophic endosymbiots in their gill tissues. Kr* Others include Alviniconca SCh shrimp, Actiniaria sea anemone, Kr* and Ophiuroidea starfish. (‰) Abbreviations in the figures indicate the locations of the CDT samples (see Table 10.1), and S Kr 34 plots without abbreviations d represent collections off Calyptogena KgW Hatsushima, in Sagami Bay SmS Bathymodiolus Jv Annelida other bivalves limpet others

13 d C VPDB (‰) 122 T. Yamanaka et al.

Fig. 10.6 Apparent differences 10 between the δ34S values of animal Methanotroph soft bodies and issuing hydrogen IhR sulfide from hydrothermal fields. IzJ 5 Ht The two dotted lines indicate the IzJ IhNIhN IzJ δ34 possible range of variation in S Sn Ht Sy values of hydrogen sulfide and Yo kinetic isotope fractionation 0 (‰) involved with its assimilation. Sy Abbreviations indicate the sample Ht Yo RtK IhN My IzJ locations shown in Table 10.1 IhR Sy Sy -5 IzJ IzH Yo Yo IhN -10 IzJ IhR soft body – hydrogen sulfide

S -15 34 Δ

-20

IhR IhR -25 Shinkaia Alvinocaris Bathymodiolus Acharax Polychaeta Alviniconcha Calyptogena

Fig. 10.7 Apparent differences Methanotroph between the δ34S values of animal soft bodies from hydrothermal 0 fields and seawater sulfate-sulfur (δ34S ¼ +21 ‰). The dotted line indicates the possible range of Ht kinetic isotope fractionation IhNIhNYo -10 involved in the incorporation and Ht IzJ Yo Sn subsequent assimilation of IzJ IzJ Ht IhR sulfates. Abbreviations indicate IhR the sample locations shown in Yo RtK (‰) Yo Sy Table 10.1 My IzJ -20 IzJ IhN Sy IzH Sy Sy IhN IzJ

soft body – sulfate -30 S 34 Δ

-40

IhR IhR IhR

-50 Shinkaia Alvinocaris Bathymodiolus Acharax lviniconcha Polychaeta A Calyptogena 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 123

Fig. 10.8 Apparent differences Methanotroph between the δ34S values of animal 0 soft body parts obtained from methane seep areas and seawater Kr 34 SbH sulfate-sulfur (δ S ¼ +21 ‰). SbH Kr The dotted line indicates the SbH possible range of kinetic isotope SbH fractionation involved with -10 Kr SCh assimilation of sulfate. SbH Abbreviations indicate the sample (‰) Kr locations shown in Table 10.1

-20

Kr soft body – sulfate S

34 -30 Δ KgW Jv

-40

SbH SbH

-50 Calyptogena Bathymodiolus Tubeworm Phymorhynchus

10.3.3.2 Methane Figs. 10.3 and 10.5, many animal tissue samples had δ34S The δ13C values of dissolved methane in the venting fluids values lower than +15 ‰, suggesting that the animals rely ranged from À54 to À4 ‰ (Tables 10.2 and 10.3). nearly all or in part on thioautotrophic nutrition. Some Methanotrophic species, which are known to harbor animals (mostly mussels, such as Bathymodiolus japonicas, methanotrophic endsymbionts, are expected to show δ13C B. platifrons, B. hirtus,andB. securifornis), which have values close to that of the associated methane. Apparent higher δ34Svalues( +15 ‰), are known to harbor differences between δ13C values of animal soft tissues and methanotrophic endosymbionts in their gill tissue the associated methane are shown in Figs. 10.9 and 10.10. (Fujiwara et al. 2000; Fujikura et al. 2003). These results Most animal samples showed higher δ13C values relative to are quite reasonable, since some of the methanotrophic those of the associated methane. mussels inhabiting the Okinawa Trough have δ34Svalues of slightly less than +14 ‰. Bathymodiolus mussels are expected to have preserved their ability to feed via filtration 10.4 Discussion (Page et al. 1990, 1991). Therefore, these results may suggest positive assimilation of thioautotrophic nutrition 10.4.1 The Contribution of Thioautotrophic via filter feeding. Mussels from South Chamorro serpentine 34 Nutrition to the Benthic Animal seamount also had low δ Svalues(~+10 ‰). The mussels Community are considered to harbor both thioautotrophic and methanotrophic endosymbionts (i.e., dual symbiosis) For benthic communities inhabiting the areas around (Yamanaka et al. 2003b). The other heterotrophs, except δ34 hydrothermal vents and methaneseeps,i.e.,thedischarge for vent and seep endemic species, also have Svaluesof < ‰ zone of TAIGA, major mechanisms that exploit their lim- slightly +15 (Figs. 10.3 and 10.5), indicating that ited sulfur isotopic fractionation are considered to be a thioautotrophic nutrition at the vent and seep fields prevails positive assimilation of sulfide-sulfur, since δ34Svalues not only with the endemic animals, but also with diverse of sulfide are usually lower than +12 ‰.Asshownin animals inhabiting areas around the fields. 124 T. Yamanaka et al.

40 IhN IhN

30 Br IhR Ht Tr Br Ht IzJ Ht IzJ IhN 20 IzJ Tr IhR IzJ Methanotroph Tr IhN IzJ (‰) IhR IzJ IhR IzJ Kt IzH IzJ 10 IhN Kg Yo IhR Ht Yo Yo Sy 0 My soft body – methane C 13 Δ

-10 MEn

-20 My

-30 Shinkaia other Bathymodiolus fish other animals crustacean

Acharax bernacle other molluscan

Alvinocaris Polychaeta Calyptogena

Fig. 10.9 Apparent differences between the δ13C values of animal soft body parts and associated methane obtained from hydrothermal areas. Abbreviations indicate the sample locations shown in Table 10.1

Fig. 10.10 Apparent isotopic 15 fractionation of δ13C values of animal soft bodies and issuing SbH methane from methane seep fields. The mussels harbor methanotrophic symbionts in their gill tissues. Abbreviations 10 IzJ IhN

indicate the sample locations (‰) shown in Table 10.1. “Mix” indicates that the sample specimens form a mixed colony of B. hirtus and B. securiformis 5 Kr Ht Yo soft body – methane C 13

Δ SCh 0 Mix

Mix

-5 B. platifrons B. ?

B. hirtus

B. platifrons B. japonicus

B. securiformis 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 125

One group of Calyptogena soyoae from the off methane during biological oxidation (e.g., Silverman and Hatsushima site showed quite high δ34S values Oyama 1968) before delivery to methanotrophs.

(~ +16.5 Æ 6.5 ‰). Masuzawa (1996) reported similar In the former case (1), chemoautotrophs fix CO2 and high δ34S values (~ +14 ‰) from other Calyptogena soyoae therefore do not directly assimilate 13C-depleted methane. in the area, although the δ34S value of hydrogen sulfide from Dissolved inorganic carbon (mainly bicarbonate) is usually that habitat has not been documented. The spatial distribu- abundant in the bottom seawater. This means that the signa- 34 13 13 tion of δ S values for sedimentary sulfide is often heteroge- ture of C-depleted CO2 originating from C-depleted neous within the anoxic environment (e.g., Yamanaka et al. methane oxidation is difficult to detect. In the latter case 1999). Paired analysis of sulfur in clams and substrate (2), methane emitted at the sediment-hosted systems sediments is required for further study. undergoes biological oxidation and incorporation during The above results also suggest that the sediment-hosted penetration of the sediments, resulting in methane that is hydrothermal vent and methane-rich seep fields provide enriched in 13C relative to the primary source to be delivered thioautotrophic nutrition (i.e., sulfur-oxidizing bacteria and to methanotrophic animals (mainly Bathymodiolus mussels). heterotrophic organisms that rely on them) derived from the Another plausible explanation for the methanotrophic suspended particles and detritus in the surface sediments. In species that are enriched in 13C, relative to the associated fact, significant amounts of sulfur-oxidizing microbe cells methane, is the heterotrophic assimilation of organic matter have been identified in the bottom water around hydrother- via filter feeding, as demonstrated by Page et al. (1990). The mal fields in the Okinawa Trough (Yanagawa et al. Chap. 6). δ13C values of methane originating from pyrolysis of Such high biomass flux of the sulfur-oxidizing microbes is organic matter and microbial methanogenesis, relative to supported by microbial sulfide production, suggesting that that of common organic matter, are usually depleted in sub-seafloor bacterial sulfate reduction using methane or 13C. Heterotrophic and thiotrophic animals are enriched in sedimentary organic matter as electron donors (Masuzawa 13C compared to methane from these sources. In these cases, et al. 1992) is quite active at the discharge zone of TAIGA. the soft tissue is probably depleted in 34S due to the depen- From the above-mentioned stable isotopic data, it is pos- dence on thioautotrophic nutrition. In fact, the mussels sible to estimate the contribution of thioautotrophic nutrition inhabiting the Okinawa Trough were slightly depleted in to benthic animal communities. Nevertheless, the primary 34S, suggesting a positive incorporation into thioautotrophic source of hydrogen sulfide is not only hydrothermal but also nutrition, as mentioned above. bacterial. The δ34S values of bacterial hydrogen sulfide vary On the other hand, some animal soft tissues depleted in widely (e.g., Thode 1988). Therefore, it is difficult to accu- 13C, relative to the associated methane (Figs. 10.9 and rately estimate the contribution of thioautotrophic nutrition. 10.10), were found at the field where emitted methane The δ34S values for sulfur of animal soft tissue are less than showed quite high δ13C values (À10 ‰) in an abiotic +10 ‰, indicative of a significant contribution from source (the sediment-starved Myojin and Suiyo seamounts thioautotrophic nutrition. and serpentine-hosted South Chamorro seamount). Further- more, the mussels of South Chamorro harbor thioautotrophic endsymbionts in addition to methanotrophs, which assimi- late abiotic methane enriched in 13C. Therefore, resulting 10.4.2 Variations in the Carbon Isotopic Ratios δ13C values of the mussels decreased relative to those of the of the Benthic Animal Community associated methane. Its dual symbiosis has also been supported by their low δ34S values (+10.6 ‰ for gill The carbon isotopic ratios for the animal soft tissues varied tissues), relative to seawater-sulfate (Table 10.3). widely (Figs. 10.2, 10.3, and 10.4, and Tables 10.2 and 10.3). Some thiotrophic vestimentiferan tubeworms have This may reflect the variation in δ13C values of the methane slightly higher δ13C values (À20 ‰). It is known that (À71 to À6 ‰) issuing from hydrothermal vents and cold this animal has another carbon fixation pathway instead of seeps. In particular, δ13C values of methanotrophic the Calvin cycle (Nelson and Fisher 1995). For example, the Bathymodiolus mussels varied widely, ranging from À70 rTCA cycle is a process that accompanies the insignificant to À20 ‰. The δ13C values for most animal soft tissue isotopic fractionation during carbon fixation (Markert et al. samples were higher than those of the associated methane 2007). Some heterotrophic animals that have high δ13C (Figs. 10.9 and 10.10). This can be explained by the follow- values may feed via mechanisms similar to the tubeworms. ing two scenarios: (1) the contribution of methane as a One species of limpet, Phymorhynchus buccinoides, carbon source for chemosynthesis-based animals, except obtained from off Hatsushima, has been reported to feed for methanotrophic species, is insignificant at many dis- predominantly on the dead bodies of Bathymodiolus mussels charge zones or (2) the δ13C values of methane are increas- (Fujikura et al. 2009). Their δ13C values are significantly ing due to the selective incorporation of isotopically light lower (~ À60 ‰, Table 10.3) than those of the other 126 T. Yamanaka et al. heterotrophs. Although such specific heterotrophs relying on methane seeps off Hatsushima showed a positive correlation a single vent or seep endemic species have rarely been between carbon and nitrogen isotopic ratios. This implies reported until recently (Van Dover 2000), carnivores, that the δ13C and δ15N values of the mussels reflect subsur- scavengers, and parasites relying on a single vent or seep face microbial processes, which preferentially incorporate animals are likely found in the vent and seep communities. isotopically light methane together with ammonium. In fact, parasitical polychaeta, Branchipolynoe pettiboneae, Ammonium is a plausible source of nitrogen nutrition for collected from inside the shell of Bathymodiolus mussels methanotrophic communities. Due to a lack of isotopic data obtained from JADE site, Izena Hole have similar δ13C for ammonium in this habitat, further studies are needed. and δ34S values to the associate mussels with ~6 ‰ enriched in 15 N (Table 10.2). With further study, these animals will continue to be identified. 10.4.4 Competition for Energy Sources and the Role of Filter Feeding by Bathymodiolus Mussels 10.4.3 Nitrogen Isotopic Ratios of Symbiotic Bivalves Two Bathymodiolus species (B. hirtus and B. securiformis) have been observed at the Kuroshima knoll seep site, where Nitrogen isotopic ratios of animals have been used for the they usually form small colonies composed of one species estimation of trophic levels in studies of food web structures (separate-type). Nevertheless, some colonies form a mixture (Minagawa and Wada 1984). Usually, higher trophic levels of both species (mixed-type). Both species are believed to are believed to have elevated δ15N values. Such enrichment harbor methanotrophic bacteria as their sole endosymbionts in 15N with increasing trophic level is also common in vent (Fujikura et al. 2003). In fact, sulfur isotopic compositions and seep communities, while symbiotic mussels, such as for specimens from separate-type colonies show a range Calyptogena clams and Bathymodiolus mussels, still have typical of methanotrophic animals (δ34S  +15 ‰). The significantly low δ15N values (Figs. 10.2 and 10.4). Such δ34S values of specimens from the mixed-type colonies are trends were recognized in early studies of the about 10 ‰ lower than those from the separate-type chemosynthesis-based animal community, and the reasons colonies. Both carbon and nitrogen isotopic ratios for two for such low values have been debated (e.g., Van Dover types of samples are relatively similar. Such low δ34S values 2000), although the detailed mechanism is not well under- may be due to the assimilation of thioautotrophic nutrition stood. Some plausible explanations have been proposed, via filter feeding. Mussels of the mixed-type colonies may e.g., assimilation of ammonium, which prevails in anoxic compete with each other for methane nutrition as an energy sediments, may be an important process. During the assimi- source. They may compensate for the shortage of lation of ammonium nutrition, isotopic fractionation may methanotrophic nutrition derived from the endosymbionts occur (e.g., Hu¨bner 1986; Yoneyama et al. 1993; Lee and by filter feeding. Childress 1994). Isotopic fractionation during the assimila- tion of ammonium depends on its concentration and the types of enzymes used in ammonium assimilation. The 10.5 Summary 15 reported values range from À20 to +4 ‰ (δ Nammonium – 15 δ Norganism) (Hu¨bner 1986; Yoneyama et al. 1993). Benthic animal communities supported by thioautotrophic The δ13C and δ15N values of most animal tissues, especially and methanotrophic nourishment are widely distributed over the Bathymodiolus mussels inhabiting the methane seeps, the discharge area of TAIGA, and they have high biodiver- are highly correlated (R2 ¼ 0.85) (Fig. 10.4). The increasing sity including common marine benthos. As we have δ13C values may be due to isotopic fractionation during summarized the nutritional sources of the benthic animals microbial oxidation and/or assimilation of methane nutrition. discussed in this study in the Supplementary document The δ15N values of source ammonium are also plausibly (Suppl. 10.1), microbial methane and hydrogen sulfide increased by microbial consumption, because isotopically generated within the sedimentary layer are important energy light ammonium in preferentially incorporated. Therefore, sources for microbes, in addition to abiogenic methane and this implies that δ15N values of ammonium increase together hydrogen sulfide dissolved in the issuing fluids. Although with δ13C values of methane due to bacterial isotopic discrim- some hydrothermal vent and methane seep endemic species ination until the ammonium in source fluids reaches the harbor endosymbiotic bacteria and lack digestive organs, mussel habitat. Bathymodiolus mussels still have ability to feed in the Among the seep fields, the carbon isotopic ratios of meth- Okinawa Trough and some other methane seep fields. The ane have reflected the methane origin. However, in our data suggest that the sediment covered discharge area of limited data set, Bathymodiolus mussels obtained from TAIGA is also abundant in suspended and/or free-living 10 A Compilation of the Stable Isotopic Compositions of Carbon, Nitrogen, and Sulfur... 127 microbes, namely thioautotrophs, which support huge bio- the Hatoma and the Kuroshima Knolls, the Nansei-shoto area, mass production and biodiversity on the deep seafloor. JAMSTEC. J Deep Sea Res 22:21–30 (in Japanese with English abstract) Fujikura K, Sasaki T, Yamanaka T, Yoshida T (2009) Turrids whelk, Acknowledgements We are grateful to Drs. K. Fujikura, Y. Fujiwara, Phymorhynchus buccinoides feeds on Bathymodiolus mussels at a S. Tsuchida, H. Watanabe, D. J. Lindsay, F. Inagaki, and Y. Ohara for seep site in Sagami Bay, Japan. Plankton Benthos Res 4(1):23–30 providing the animal samples used in this study. We are grateful to Drs. Fujikura K, Okutani T, Maruyama T (2012) Deep-sea Life: Biological K. Yanagi, S. Kojima, T. Okutani, and T. Yamamoto for identification observations using research submersibles, 2nd edn. Toukai Univer- of the sample animals. We are also indebted to Professor H. Chiba and sity Press, Toukai (in Japanese with English captions) Dr. S. Kawaguchi for their very useful comments. All of the fluid, water, Fujiwara Y, Takai K, Uematsu K, Tsuchida S, Hunt JC, Hashimoto J and chimney samples were obtained through the cooperative efforts of (2000) Phylogenetic characterization of endosymbionts in three the operation teams of the ROVs HyperDolphin, Dolphin 3 k, Kaiko, and hydrothermal vent mussels: influence on host distributions. Mar ROPOS, the DSV Shinkai 2000 and 6500, and the captain and crew of Ecol Prog Ser 208:147–155. doi:10.3354/meps208147 the support ships R/Vs Natsushima, Kaiyo, Yokosuka, and Thomas Gamo T, Sakai H, Kim ES, Shitashima K, Ishibashi J (1991) High Tompson, to whom we extend our heartfelt thanks. This research was alkalinity due to sulfate reduction in the clam hydrothermal field, supported by the Ministry of Education, Culture, Sports, Science and Okinawa trough. Earth Planet Sci Lett 107:328–338 Technology of Japan through a Special Coordination Fund ‘TAIGA’ Gamo T, Chiba H, Yamanaka T, Okudaira T, Hashimoto J, Tsuchida S, project (New Scientific Research on Innovative Areas, 20109005) and Ishibashi J, Kataoka S, Tsunogai U, Kouzuma F, Okamura K, Sano the Archaean Park Project (International Research Project on Interac- Y, Shinjo R (2001) Chemical characteristics of newly discovered tion between Sub-Vent Biosphere and Geo-Environments). black smoker fluids and associated hydrothermal plumes at the Rodriguez Triple Junction, Central Indian Ridge. Earth Planet Sci Open Access This chapter is distributed under the terms of the Lett 193:371–379. doi:10.1016/S0012-821X(01)00511-8 Creative Commons Attribution Noncommercial License, which Glasby GP, Iizasa K, Yuasa M, Usui A (2000) Submarine hydrothermal permits any noncommercial use, distribution, and reproduction in any mineralization on the Izu-Bonin Arc, south of Japan: an overview. medium, provided the original author(s) and source are credited. Mar Georesources Geotechnol 18(2):141–176. doi:10.1080/ 10641190009353785 Hashimoto J, Ohta S, Gamo T, Chiba H, Yamaguchi T, Tsuchida S, Okudaira T, Watabe H, Yamanaka T, Kitazawa M (2001) First hydrothermal vent communities from the Indian Ocean Discovered. 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