Elucidating carbon sources of hydrothermal vent animals using natural 14C abundances and habitat water temperature

Deep‐sea hydrothermal vents host exceptional ecosystems with lush animal communities primarily relying on organic matter (OM) produced by chemoautotrophic microbes. Though energy sources and food webs at vents have been extensively studied, the exact carbon sources of chemosynthetic primary production, such as methane (CH4) and carbon dioxide (CO2) in the vent fluid or bottom water, have not been elucidated quantitatively across spatial scales. Here, we investigate carbon and nitrogen sources of 12 vent animal species at the Iheya North field, Okinawa Trough inhabiting different distances from the central venting area and with various feeding ecologies using natural‐abundance radiocarbon (Δ14C) in combination with conventional stable carbon and nitrogen isotope ratios (δ13C and δ15N). Our results show that generally, animals living closer to vent orifices were more depleted in 14C, indicating they assimilate more carbon from vent fluid CO2. Those relying on methanotrophs, however, exhibited low Δ14C values regardless of distance due to the lack of methane in the non‐vent‐influenced bottom water. Organisms with low Δ14C values also tend to exhibit low δ15N values, implying NH4+ assimilation into biomass in environments with high NH4+ concentrations. Our results demonstrate that 14C can clearly distinguish between chemoautotrophically fixed carbon originating from the vent fluid and detrital OM derived from surface primary production, and also discriminate between CO2‐ and CH4‐based chemoautotrophy. Although vent animals rely on vent fluid energetically, our results highlight that the dependency on vent fluids as carbon source varies greatly depending on habitat, as well as carbon fixation pathways of microbial primary producers.

mass in environments with high NH 4 + concentrations.Our results demonstrate that 14 C can clearly distinguish between chemoautotrophically fixed carbon originating from the vent fluid and detrital OM derived from surface primary production, and also discriminate between CO 2 -and CH 4 -based chemoautotrophy.Although vent animals rely on vent fluid energetically, our results highlight that the dependency on vent fluids as carbon source varies greatly depending on habitat, as well as carbon fixation pathways of microbial primary producers.
Deep-sea hydrothermal vent ecosystems harbor unique communities of benthic animals, powered by chemoautotrophic microbes utilizing energy from the oxidation of molecules contained in the hydrothermal fluid (e.g., Fisher et al. 1989;Markert et al. 2007) to produce organic matter (OM).Many animals live in symbiosis with these bacteria and rely on them for nutrition (e.g., Cavanaugh et al. 1981;Felbeck 1985;Belkin et al. 1986;Duperron et al. 2006;Watsuji et al. 2014).The rest, however, feed on microbial mats or other animals.Different vent animals have varying levels of tolerance to the physicochemical conditions, leading to a zonation away from the vent orifices (Johnson et al. 1994;Marsh et al. 2012;Gollner et al. 2015).
While the available energy sources (i.e., the coupling of reducing and oxidizing compounds in the vent fluid and bottom seawater, respectively) for chemoautotrophic carbon production have been investigated in detail, their carbon and nitrogen sources for biomass build-up have not been a focus.Metabolic pathways have typically been studied using the stable isotope ratio of carbon (δ 13 C), based on the different carbon fixation pathways and hence different isotope fractionations.This includes the reductive tricarboxylic acid (rTCA) cycle, the Calvin-Benson-Bassham (CBB) cycle, and methane oxidation pathways (House et al. 2003;Pearson 2010).However, both the different degrees of isotope fractionation from carbon dioxide to biomass or from methane to biomass, together with the variable δ 13 C values of the substrate at each vent field (Kawagucci et al. 2013), complicate the efforts to identify carbon sources of organisms using δ 13 C values.This also makes it particularly difficult to apply an isotope mixing model for animal food source analyses without accurate isotopic compositions of each endmember source.Furthermore, the mixotrophic diet of many organisms further hampers the estimation of chemosynthetic carbon contribution.
The stable nitrogen isotope ratio of nitrogen (δ 15 N) of organisms from chemosynthesis-based ecosystems has often been reported to exhibit low values down to À12‰ (Van Dover 2002; Yamanaka et al. 2015).Some studies suggested that the availability of inorganic N sources for chemoautotrophs may change the isotopic fractionations during microbial assimilations (Liao et al. 2014;Reid et al. 2020), but the reason behind this has not been resolved.
Natural-abundance radiocarbon (Δ 14 C) has recently become an increasingly used tool for investigating carbon flows in marine ecosystems (Ishikawa et al. 2021).It is useful for investigating carbon sources of vent organisms (Williams et al. 1981;Nomaki et al. 2019) since it can discriminate between carbon sources such as detrital OM derived from surface primary production (ca.40‰) and chemosynthetic OM.Chemoautotrophic microbes at deep-sea hydrothermal vents exhibit depleted 14 C concentrations because they utilize either 14 C-depleted bottom water (ca.À200‰ in the Pacific Ocean, Stuiver et al. 1983) or totally 14 C-depleted dissolved inorganic carbon from the vent fluid (À1000‰) as their inorganic carbon source (Williams et al. 1981).The Δ 14 C values of meio-and macro-fauna on the Izu-Ogasawara Arc ranged between À695‰ and À236‰, clearly showing that the contribution of the vent fluid CO 2 varies among vent animals, depending on their proximity to the vent orifices (Nomaki et al. 2019).
Hydrothermal vent ecosystems exhibit wide ranges of total dissolved inorganic carbon (TDIC) and CH 4 concentrations depending on geological settings (Gamo et al. 2006;Kawagucci et al. 2015).The Iheya North hydrothermal field in the Okinawa Trough (Watanabe and Kojima 2015) is an ideal site for investigating the natural-abundance radiocarbon of organisms inhabiting back-arc basin vents (Supporting Information Fig. S1).The vent fluid here exhibits high TDIC, CH 4 , and NH 4 + concentrations (up to 63 mmol L À1 , 869 μmol L À1 , and 1978 μmol L À1 , respectively) due to the coverage of thick sediment layers around the vents (Kawagucci et al. 2011;Miyazaki et al. 2017).This should result in diagnostic signatures of 14 C in the vent communities, even after a substantial mixing of the vent fluid with the surrounding bottom water.Particularly, high CH 4 concentrations should provide 14 C-depleted OM for methanotrophs, which were not examined at Izu-Ogasawara Arc (Nomaki et al. 2019).As for the Δ 14 C values of the potential sources, the bottom water TDIC at Iheya North is À170‰ (at around 1000 m depth; Honda et al. 2000), while the vent fluid TDIC and CH 4 value are À1000‰ (Kawagucci et al. 2020).
Iheya North hosts several faunal zonations roughly corresponding to distances from the vent orifices (Fig. 1), typical for Okinawa Trough vents (Watanabe and Kojima 2015;Thornton et al. 2016).Thriving closest to the vent orifice (a few to tens of cm away) are the sulfide worm Paralvinella aff.hessleri (Fig. 1B) and the vent shrimp Shinkaicaris leurokolos (Fig. 1C; Yahagi et al. 2015).Paralvinella worms graze on microbial mats (Grelon et al. 2006) and though feeding ecology of Shinkaicaris is unclear, some alvinocaridid shrimps have various degrees of reliance on epibiotic symbionts (Methou et al. 2020).Next (tens of cm to a few m away), the squat lobster Shinkaia crosnieri (Fig. 1D) is found with epibionts on the ventral setae dominated by methanotrophic and thioautotrophic bacteria (Watsuji et al. 2015) aggregates.
Diffuse flow venting (tens of m away from the main orifice) is accompanied by the mussels Bathymodiolus platifrons and Bathymodiolus japonicus (Fig. 1E) that have methanotrophic endosymbionts but can also filter-feed (Barry et al. 2005).Many small animals live around them, such as the mixotrophic limpet Lepetodrilus nux (Bates 2007), the alvinocaridid shrimp Alvinocaris longirostris, and the barnacle Neoverruca intermedia which possesses epibiotic symbionts but may also carry out suspension feeding (Nomaki et al. 2019).The whelk Enigmaticolus nipponensis feeds on Bathymodiolus mussels (Chen et al. 2020).The kleptoparasitic scale-worm Branchipolynoe pettiboneae lives inside the mussels and apparently feed on both particulate organic matter (POM) filtered by the mussels and their tissues (Britayev et al. 2007).At peripheral assemblages on sediments (several hundreds of m away), the tubeworm Lamellibrachia columna (Fig. 1F) and the vesicomyid clam Phreagena okutanii (Fig. 1G) are most conspicuous, both relying on thioautotrophic endosymbionts (Li et al. 2019;Breusing et al. 2020;Ip et al. 2021).The barnacle Ashinkailepas seepiophila is commonly found on tubes of L. columna and is a suspension feeder (Watanabe et al. 2021).
Here, we measured Δ 14 C values of the above-mentioned animals collected from Iheya North to evaluate their carbon sources, which in turn reflects the carbon sources of their symbionts or food (e.g., bacterial mat).By combining the δ 13 C, δ 15 N, and Δ 14 C values of organisms with the TDIC, CH 4 , and NH 4 + sources in the vent field, we further discuss the mechanism leading to low Δ 14 C and δ 15 N values in vent animals, as well as how much each species depends on vent-sourced nutrition (i.e., carbon and nitrogen).

Materials
Animal samples from the Iheya North vent field Animal samples for isotopic measurements were collected from the Iheya North hydrothermal vent field, Okinawa Trough during research cruises conducted between 1997 and 2018 (Supporting Information Table S1).Samples were collected from the three known active areas of Iheya North (Nakamura et al. 2015) et al. 2015), $ 30 km southeast of Iheya North, were also prepared for the purpose of comparing Δ 14 C values of frozen and ethanol-preserved samples.Once recovered on-board, the animals were either frozen in À20 C or preserved in 99.5% ethanol until use.

Temperature measurements and water sampling
While at the seafloor, temperature measurements of each faunal assemblage were taken using either the temperature probe on the WHATS III fluid sampling system (Miyazaki et al. 2017) or a stand-alone dissolved oxygen/temperature probe (RINKO I, JFE Advantech, Japan) for a duration of 3 min.During measurement, the temperature probe was pressed against the animals being measured.As animal distributions around the vent depend on tolerance capacity in each assemblage and that it was difficult to keep the sensor at the desired microhabitat during measurement, here we take the maximum temperature recorded as the habitat temperature.The ambient temperature of the bottom water was also measured using the same probes while away from areas of venting activity.
To measure the concentrations and nitrogen isotopic compositions (δ 15 N) of NH 4 + , hydrothermal vent fluids were collected using a peristaltic pump sampler through titanium and silicon tubing into the gas-tight samplers WHATS II or WHATS III (Kawagucci et al. 2011;Miyazaki et al. 2017) at both Iheya North Original Site (cruise NT07-13) and Iheya North Aki Site (cruise NT15-13).Hydrothermal fluids were subsampled into polyethylene bottles from the WHATS II or WHATS III samplers.Detailed methods for the measurements of concentrations and δ 15 N values of NH 4 + are described in the Supporting Information Methods.

C analyses of organisms
The frozen animal samples were thawed once and dissected to remove their carbonate or chitinous exoskeletons (where present).Isolated muscle tissues were frozen again at À80 C, then freeze-dried and ground into fine powders.The ethanol-preserved animal samples were gradually transferred to lower concentrations of ethanol/milliQ water solution, from 90% to 0% at every 10% concentration intervals.These were finally transferred to pure milliQ water and changed twice to remove any remaining ethanol.
We evaluated the potential ethanol preservation effects on Δ 14 C values of organisms using the mussel B. platifrons preserved using either 99% ethanol (three individuals) or frozen at À80 C (three individuals), all from the same collecting event at Sakai field (Supporting Information Table S1).Since no statistically significant differences were found in Δ 14 C values between the two treatments (t-test, p > 0.1), we regard the two preservation methods as having no substantial effects on the Δ 14 C values and producing directly comparable data.
We placed samples in pre-combusted glass cups and dried them on a hot plate to remove water, then they were decalcified with 0.1 mol L À1 HCl and completely dried again.The samples were graphitized by the modified methods of Yokoyama et al. (2007Yokoyama et al. ( , 2010)).Briefly, dried samples (2.08-13.14mg) were combusted in an evacuated quartz tube with copper oxide at 500 C for 30 min and at 850 C for 2 h.The CO 2 gas was cryogenically purified in a vacuum line and reduced to graphite with hydrogen and an iron catalyst at 630 C for 6 h.We measured the Δ 14 C values with a single stage accelerator mass spectrometer (AMS) at the Atmosphere and Ocean Research Institute, University of Tokyo (Chiba, Japan; AMS lab code YAUT; Yokoyama et al. 2019).
Radiocarbon data reported here is corrected for radioactive decay between 1950 and the year when the samples were measured.The equation used is written as follows: The Δ 14 C value of the international standard (Ox-1: oxalic acid-1) takes into account radioactive decay since 1950 (Stuiver and Polach 1977).
Radiocarbon measurements were conducted with six to nine sets of 6 min measurements.Numbers of measurements depended on the samples, since we aimed to collect more than 20,000 counts to achieve accurate measurements.We also took into account all possible mass fractionations by measuring δ 13 C values using AMS instead of offline IRMS.We used four international standards, namely IAEA-C1, IAEA-C3, IAEA-C6, and NIST SRM 4990C, with each standard having different radiocarbon concentrations.The measured values were calibrated with the calibration curve obtained from the measurements of these standards.

Analyses of total organic carbon, total nitrogen contents, and their stable isotopic compositions
The stable carbon and nitrogen isotopic compositions were determined using an isotope ratio mass spectrometer (Delta Nomaki et al. plus XP; Thermo Finnigan) connected to an elemental analyzer (FlashEA1112; Thermo Finnigan) through a continuous flow interface (ConFloIII; Thermo Finnigan), which was adapted for small sample sizes (Ogawa et al. 2010).The isotope data were calibrated using five to six inter-laboratory determined standards (BG-T, BG-P, BG-A, CERKU-01, L-glutamic acid, and L-valine; Tayasu et al. 2011, Indiana University, Shoko Science) in the range of 5.70‰ to +60.40‰ for δ 15 N and À28.86‰ to +0.18‰ for δ 13 C, respectively.The L-tyrosine listed above was used as a standard for the quantification of total organic carbon and total nitrogen contents.Errors for the repeated analyses (from 5 to 20) of the abovementioned standards of δ 13 C and δ 15 N were AE 0.12‰ (SD, 1σ) and AE 0.33‰ (SD, 1σ), respectively.
Calculation of vent fluid volume ratio, vent fluid TDIC ratio, and habitat TDIC Δ 14 C value Based on the measured animal habitat temperatures (Table 1), we calculated the mixing ratio of vent fluid (Table 1; Miyazaki et al. 2017) and bottom water at each habitat (Table 1) using the following equation: where T h is the habitat temperature, T b is the temperature of bottom water, and T v is the temperature of vent fluid.
Based on V vf and TDIC concentrations of vent fluid (Table 1; Miyazaki et al. 2017), contribution of vent fluid TDIC to bottom water TDIC at the habitat was then determined as follows: where C v is the TDIC concentration of vent fluid, C b is the TDIC concentration of bottom water (Table 1).The Δ 14 C values of TDIC at each habitat were then calculated based on the vent fluid TDIC ratio and Δ 14 C values of both vent fluid (Δ 14 C vf ) and bottom water (Δ 14 C bw ) using a following equation: where Δ 14 C bw is À170‰ (Honda et al. 2000).The depletions of 14 C in both TDIC and CH 4 observed at other Okinawa Trough sites (Kawagucci et al. 2020) and the Rebecca's Roost vent site, Guaymas Basin (Pearson et al. 2005) provide reasonable grounds to postulate that TDIC and CH 4 at Iheya North hydrothermal vent field are also depleted in 14 C (i.e., Δ 14 C vf = À1000‰).
To verify the vent fluid TDIC ratio calculated from habitat water temperature, we also calculated the relative contribution of TDIC based on the animal tissue Δ 14 C value using the following equation: Vent fluid TDIC ratio animal ¼ Note that the calculated ratio assumes the C sources of animals are TDIC of vent fluid or bottom water only, and thus any contribution of C from methane or photosynthesis-based OM are not considered.

Habitat temperature
The in situ habitat temperature of the assemblages generally decreased from the center of main venting orifices toward the peripheral assemblages on soft sediments (Table 1).The highest temperature was recorded in the sulfide worm Paralvinella aff.
hessleri colony (25.60 C), followed by the shrimp Shinkaicaris leurokolos (16.12 C), then the squat lobster Shinkaia crosnieri (13.40 C).The diffuse flow assemblage dominated by the mussel Bathymodiolus platifrons was much cooler at 4.80 C, which was similar to both peripheral assemblages in sedimented areas, including the tubeworm Lamellibrachia columna and the vesicomyid clam Phreagena okutanii colony (4.70 C and 4.28 C, respectively).The ambient seawater (i.e., bottom water with no influence from vent fluid) temperatures were 4.16 C, 4.16 C, and 4.13 C at the Iheya North Original site, Iheya North Natsu site, and Iheya North Aki site, respectively (Table 1).It should be noted that the habitat temperatures were based on measurements carried out for 3 min, and there may be temporal fluctuation at longer timescales that can potentially cause variabilities on vent fluid mixing ratio and the reconstructed habitat Δ 14 C values of TDIC mentioned below.
The highest vent fluid volume contribution (7.0%) was recorded at the habitat of the sulfide worm Paralvinella aff.Nomaki et al.

Natural-abundance radiocarbon
The calculated habitat Δ 14 C values of TDIC matched well with the measured Δ 14 C values of animals for the sulfide worm Paralvinella aff.hessleri, the shrimp Shinkaicaris leurokolos, the squat lobster Shinkaia crosnieri, the stalked barnacle Ashinkailepas seepiophila, and the vesicomyid clam Phreagena okutanii (Table 1).
Nitrogen isotopic compositions of NH 4 + in the vent fluids exhibited a narrow range of δ 15 N values, from 2.8‰ to 4.0‰ among five different water samples collected from four different chimneys (Supporting Information Table S2).These values were intermediate of the entire animal samples examined (Fig. 3B,C).

Discussion
C sources of chemosynthesis revealed by Δ 14 C values The natural-abundance radiocarbon concentrations in the vent animals investigated were generally associated with the distance from the central vent orifice (Fig. 1)-which can be explained by the 14 C-dead vent fluid (À1000‰; Kawagucci et al. 2020) being diluted by the bottom water (À170‰ at depths around 1000 m; Honda et al. 2000).Chemoautotrophic microbes that fix CO 2 would therefore exhibit more depleted 14 C values (below À800‰ in this study; Fig. 2; Supporting Information Table S1) near the venting orifice and show similar Δ 14 C values to bottom water in diffuse flow or peripheral habitats further away.
If all of the organic carbon in the soft tissue is synthesized from DIC, 74.8% of the habitat seawater DIC comes from the vent fluid DIC in the sulfide worm Paralvinella aff.hessleri (Δ 14 C values: À791‰ AE 77‰) (Table 1).This corresponds well to the vent fluid TDIC contribution calculated from the habitat temperature (68.4%;Table 1).Despite the mixing ratio of vent fluid to the habitat seawater being 7% based on the habitat temperature, the vent fluid in Iheya North is approximately 30 times higher in TDIC concentration (63 mmol L À1 ) compared to the surrounding bottom water (2.2 mmol L À1 ; Gamo et al. 2006), resulting in a high (74.8%AE 9.2%) contribution of vent fluid TDIC to habitat seawater TDIC and consequently biomass.This mixing ratio of vent fluid to bottom water is similar to that of this species' habitat at the Myojin Knoll vent field on the Izu-Ogasawara Arc (7.7%), based on measured Δ 14 C values and vent fluid TDIC concentrations at the site (Nomaki et al. 2019).Even though the mixing ratios were similar between the two vent sites, the higher TDIC concentration in the vent fluid of Iheya North would result in diagnostic Δ 14 C values (À791‰ AE 77‰ at Iheya North, À695‰ and À661‰ at Myojin Knoll).Similarly high δ 13 C values in this study (À16.1‰AE 0.4‰; Fig. 3) and Myojin Knoll (À11.5‰AE 2.3‰; Nomaki et al. 2019) indicate that they mainly feed on thioautotrophic microbes producing OM via the rTCA cycle on the chimney, as previously suggested (Yorisue et al. 2012).
The general correlation between animal tissue and habitat TDIC Δ 14 C values observed for P. aff.hessleri, Alvinocaris longirostris, Shinkaia crosnieri, Ashinkailepas seepiophila, and Phreagena okutanii suggested they are using chemosynthesisbased OM originating from a mixed-origin TDIC of vent fluid and bottom water (Fig. 4).For about half of our examined animals, however, the above-mentioned trends did not apply.Particularly, animals inhabiting the diffuse flow habitat on the chimney (Fig. 2) exhibited obviously lower animal tissue Δ 14 C values (À772‰ AE 67‰ to À510‰ AE 51‰) than the Δ 14 C value of TDIC (À217‰) reconstructed from habitat temperature (Fig. 4).This can be explained by the utilization of CH 4 as the carbon source in the mussel Bathymodiolus platifrons (Fig. 4) which houses methane-oxidizing endosymbionts (Barry et al. 2005).The concentration of CH 4 in the bottom water is > 10 6 lower than that of the vent fluid (Gamo et al. 2006), and thus the available C source for methane oxidation is from the 14 C-depleted CH 4 in vent fluid, regardless of the distance from the main vent orifice and the mixing ratio with the bottom water.The low Δ 14 C values of other animals may indicate subsequent utilization of such carbon around the mussels.The similar low δ 13 C values of Enigmaticolus nipponensis, Branchipolynoe pettiboneae, and Alvinocaris longirostris (À40‰ to À32‰) further support these nutritional insights (Fig. 3).The higher δ 13 C values observed for the limpet Lepetodrilus nux (À26.3‰AE 1.2‰) and the barnacle Neoverruca intermedia (À21.5‰AE 1.2‰) may reflect the mixotrophic and suspension-feeding ecology of these animals, respectively (Bates 2007;Nomaki et al. 2019).Overall, the dependency of animals on CH 4 -derived carbon is clearly indicated by comparing the Δ 14 C values of animals and TDIC at their habitats (Fig. 4).This finding is the key highlight of our study in comparison to previous work by Nomaki et al. (2019), which was performed at the Myojin Knoll vent field on the Izu-Ogasawara Arc characterized by low CH 4 concentrations.
Habitat, symbiont metabolism, and feeding ecology estimated by Δ 14 C, δ 13 C, and δ 15 N The high depletion of 14 C in the soft tissue of the mussel Bathymodiolus platifrons (À772‰ AE 67‰) despite its habitat distant from the central orifice can be explained by its reliance on methane-oxidizing symbionts (Barry et al. 2005).Deviation of its values from that of the vent fluid methane (Δ 14 C = À1000‰) may be attributed to additional nutritional input from filter feeding, known to augment its symbiosis energetically (Page et al. 1991;Laming et al. 2018), and/or CO 2 fixation through anaplerotic pathways.Assuming that the C source of B. platifrons is solely derived from methaneoxidizing symbionts and suspended SOM, the contribution of filter-feeding in this species is 22% AE 6% if SOM consists of photosynthesis-based OM (Δ 14 C = 40‰) and 28% AE 8% if SOM consists solely of chemosynthesis-based OM originating from the bottom water TDIC (Δ 14 C = À170‰).Nevertheless, considering the TDIC-Δ 14 C values can vary greatly (Fig. 4), these estimations must be treated with caution.The whelk Enigmaticolus nipponensis, which is considered to feed primarily on B. platifrons (Chen et al. 2020), also exhibited low Δ 14 C values (À 755‰ AE 72‰) as expected (Supporting Information Table S1).In support of this, the δ 15 N values of E. nipponensis (1.8‰ AE 3.0‰) was on average 3‰ higher than that of B. platifrons (À 1.3‰ AE 3.4‰; Fig. 3B,C; Supporting Information Table S1), corresponding to one trophic level increase ($ 3.4‰; Minagawa and Wada 1984).The δ 13 C values of E. nipponensis (À 37.9‰ AE 1.4‰) were comparable to those of B. platifrons (À 35.9‰AE 4.4‰), which do not contradict their predator/prey relationship (Fig. 3B).The kleptoparasitic scale worm Branchipolynoe pettiboneae lives in the mantle cavity of the mussel Bathymodiolus platifrons and is known to feed on both POM filtered by the mussel as well as directly on the tissue of the host mussel (Britayev et al. 2007).However, the in seawater of the animal habitats.The latter was calculated with the mixing ratio of bottom water and vent fluid based on the habitat temperature measurements.See Table 1 and text for more details.Δ 14 C values for potential C sources are indicated using dotted circles.
relative contribution of these two nutritional sources has been unclear.We found that the Δ 14 C values of Branchipolynoe pettiboneae (À522‰ AE 4‰) were on average 250‰ higher than those of Bathymodiolus platifrons (À772‰ AE 67‰; Fig. 2), showing that it indeed does not solely rely on host tissue for nutrition.Assuming that the other food source is suspended OM originating from the photic zone (Δ 14 C values of 40‰), the contribution of carbon from the host mussel is about 70%.Nevertheless, in reality, the SOM contains both photosynthesis-based and chemosynthesis-based OM around the vents, and data on their ratio would be necessary to make an accurate calculation.The slightly higher δ 13 C values of Branchipolynoe pettiboneae (À34.7‰AE 0.0‰, n = 3) relative to Bathymodiolus platifrons (À35.9‰AE 4.4‰, n = 4) coincide with the contribution of other carbon sources for the kleptoparasitic scale worm.
The vent shrimp Shinkaicaris leurokolos exhibited large intraspecific variations in the Δ 14 C values (À755‰ to À506‰; Fig. 2).The volume ratio of vent fluid to bottom water at their habitat was 3.9% based on bottom water temperature, and resulting vent fluid TDIC contribution is 54%, which matched well to the animal Δ 14 C-based calculation (Table 1).Though not known for this species, some alvinocaridid shrimps rely on photosynthesis-based nutrition as juveniles and gradually transition into adults relying on chemosynthesis-based nutrition including contributions from epibiotic symbionts (Methou et al. 2020).The carapace length of the four S. leurokolos individuals used in this study ranged between 5.9 and 7.2 mm, smaller than the reported size range of adult shrimps (Komai and Segonzac 2007).As photosynthetic OM exhibits higher Δ 14 C values (ca.40‰) than chemosynthetic OM (Pearson et al. 2005;Nomaki et al. 2019), these individuals are likely undergoing a change in their diet; the variation in Δ 14 C values probably reflects different levels of reliance on chemosynthetic carbon sources or remnants of photosynthetic OM as their biomass, among individuals.Assuming (1) the variations among individuals depend on individual feeding habits rather than habitat heterogeneity, (2) their food sources are either SOM derived from the photic zone and/or OM produced by chemoautotrophic bacteria fixing CO 2 , and (3) the individual exhibiting the lowest Δ 14 C value (À755‰) solely relies on chemosynthetic OM-then the individual exhibiting the highest Δ 14 C values relies on SOM for 30% of its food sources.The observed changes in their diet were not obvious in the δ 13 C and δ 15 N values (À18.2‰ to À14.4‰ for δ 13 C and À2.9‰ to À1.6‰ for δ 15 N; Fig. 3C) probably due to closer δ 13 C values of photosynthesis-based and chemosynthesis-based OM.The Δ 14 C-δ 13 C plot (Fig. 3A) suggests individuals exhibiting higher Δ 14 C value tend to have lower δ 13 C value (down to À18.2‰), closer to photosynthesis-based OM (À25‰ to À22‰ for zooplankton; Nomaki et al. 2019).
The squat lobster Shinkaia crosnieri has been reported to primarily feed on methanotrophic epibionts that it "farms" on its dense ventral setae (Watsuji et al. 2015).Its Δ 14 C value is, however, not as low (À622‰ AE 23‰) compared to Bathymodiolus platifrons with methanotrophic endosymbionts in the gill, and matched closer to the Δ 14 C-TDIC values reconstructed by habitat temperature (À 562‰; Fig. 4; Table 1).This is likely due to S. crosnieri not solely relying on methanotrophs, as the same setae also host a considerable amount of thioautotrophs that fix C from CO 2 (Watsuji et al. 2010).If we assume (1) methanotrophic bacteria on the setae exhibit Δ 14 C value of À1000‰ and (2) S. crosnieri feeds exclusively on methanotrophic and thioautotrophic bacteria on its setae, then at the assumed Δ 14 C value of À562‰ methanotrophic bacteria makes up only 14% of carbon sources in S. crosnieri.Nevertheless, the high δ 15 N values of S. crosnieri (8.3‰ AE 1.1‰; Fig. 3B,C), consistent with a previous study (3.1-5.2‰;Yamanaka et al. 2015), suggests that S. crosnieri may feed on heterotrophic bacteria on the setae or potentially even other animals.If we consider these other food sources can potentially have higher Δ 14 C values, then the contribution of methanotrophic bacteria may be higher than 14% in reality.
Among the three species inhabiting peripheral sedimented areas, the tubeworm Lamellibrachia columna exhibited much lower Δ 14 C values (À609‰ AE 22‰) than the clam Phreagena okutanii (À234‰ AE 31‰) and the barnacle Ashinkailepas seepiophila (À290‰ AE 34‰) (Figs. 2, 3).Another siboglinid tubeworm, the giant tubeworm Riftia pachyptila, acquires sulfides for its thioautotrophic endosymbionts from the surrounding seawater mixed with hydrothermal vent fluid using its plume, and it may also acquire CO 2 via the plume as suggested by its Δ 14 C value (À270‰ AE 20‰) closer to the bottom water (À233.4‰)(Williams et al. 1981).On the other hand, Lamellibrachia tubeworms acquire sulfides via a posterior extension (the "root") that penetrates deep into cracks or sediments to directly access the geofluids there, which can be up to 1 m in length (Cordes et al. 2005;Dattagupta et al. 2006;Li et al. 2019).Our Δ 14 C values indicated that they also acquire CO 2 posteriorly via the root, likely through diffusion as has been previously hypothesized (Cordes et al. 2005), and thus exhibit 14 C-depleted values compared to the values estimated from the habitat temperature measured above the sediment (Figs. 2, 3).The calculated vent fluid/bottom water TDIC ratio based on the measured habitat temperature (4.70 C) was 3.5-5.0%,while those based on animal Δ 14 C value was 52.9% (Table 1).Since hydrothermally influenced sediments show increasing temperature with increasing depth in sediments (Nakajima et al. 2019), the measured in situ temperature on the surface of sediment near its anterior end (the plume) does not reflect the true temperature of the environment where it obtains its carbon source.
Although the intraspecies variations in Δ 14 C value was low for L. columna, a specimen exhibited δ 13 C values $ 7‰ lower than the others (Supporting Information Table S1).Siboglinid tubeworms are reported to have symbionts capable of utilizing Nomaki et al. two distinct carbon fixation pathways (i.e., rTCA and CBB cycles; Li et al. 2019;Breusing et al. 2020).The observed lower δ 13 C value may be attributed to a larger dependency on CBB cycles in this individual.
Vesicomyid clams obtain hydrogen sulfide from the foot, which extends deep into the sediment, but take up both oxygen and CO 2 in its gill from waters drawn in with its siphon situated above the seafloor (Childress and Girguis 2011).The Δ 14 C values of Phreagena okutanii agrees with this and indicate the inorganic carbon used for chemosynthesis is indeed mostly derived from the bottom water (92.2%;Table 1), which differs from the tubeworm L. columna (47.1%).Although both of these species harbor thioautotrophic endosymbionts and inhabit similar habitats away from the central vent orifice, our results show that they actually rely on completely different sources of carbon.The slight depletion in 14 C (Δ 14 C: À234 AE 31‰) compared to the habitat TDIC Δ 14 C value (À179‰) in P. okutanii may be attributed to a minor diffusion of CO 2 from the foot or the vent fluid TDIC diffused to the sediment surface, which accounts for 7.8% contribution of the vent fluid TDIC relative to bottom water (Table 1) and equivalent to a vent fluid to bottom water volume ratio of 0.27%.Ashinkailepas seepiophila has been reported as a suspension feeder that utilizes POM in the bottom water originating from both the photic zone and chemoautotrophic carbon production some distances away (Watanabe et al. 2021).Although we have no POM data around the sampled habitat, it is probable that POM here contains both photosynthesis and chemosynthesis origins.The intermediate δ 13 C values of this species (À24.1‰AE 1.0‰) can be explained by the assimilation of photosynthesis-based OM, or by assimilations of chemosynthetic OM with mixed origins, for example, originating from L. columna symbionts, P. okutanii symbionts, and/or animals living at the diffuse flow area.The high δ 15 N values of A. seepiophila up to 13.4‰ (Supporting Information Table S1) can be explained by the large contribution of POM being derived from the water column, which exhibits δ 15 N values of $ 10‰ around the study area (Minagawa et al. 2001), while δ 15 N values around 6‰ are also concordant with assimilations of OM originating from detritus of the cooccurring L. columna and P. okutanii.Importantly, our Δ 14 C values indicated that there is certainly a contribution of chemosynthetic POM in this species (Figs.3A, 4).Neolepadid barnacles in chemosynthetic systems often exhibit epibiotic symbionts on their cirri (Watanabe et al. 2021), and it is possible that Ashinkailepas does the same.The contribution of OM synthesized by these epibionts would provide the same Δ 14 C value as the habitat DIC (À210‰; Table 1).However, Δ 14 C values of A. seepiophila (À290‰ AE 34‰) are lower than both the habitat DIC and that of P. okutanii (À234‰ AE 31‰), suggesting that a part of POM sources for suspension feeding is derived from OM produced by L. columna symbionts or POM produced at nearby venting area.The contribution of SOM from the photic zone can be as high as 59% when we assume SOM from photic zone (+40‰) and POM from the diffuse flow area (a representative value from B. platifrons is À772‰) as the two endmembers.The contribution of SOM decreases when A. seepiophila also consume chemosynthetic OM with higher Δ 14 C values such as POM originating from L. columna or their epibionts.Environmental POM data is needed for an accurate estimate of the contribution of photosynthetic OM in the nutrition of A. seepiophila.
Relationships between δ 15 N and Δ 14 C values Very low δ 15 N values are well-known from bathymodioline mussels from different vent fields globally (e.g., down to À7.5‰ at Okinawa Trough [Yamanaka et al. 2015], down to À2‰ on Izu-Ogasawara Arc [Yorisue et al. 2012]; down to À10‰ at Central Indian Ridge [Van Dover 2002]).Van Dover (2002) suggested a large variation in δ 15 N values of different inorganic nitrogen sources potentially assimilated by the symbiotic bacteria.For instance, Liao et al. (2014) reported that the δ 15 N values of the Ridgeia vent tubeworm biomass differ from its N source (assumed to be NH 4 + ) to a different extent depending on the environmental NH 4 + concentrations.Ridgeia tubeworms with thioautotrophic symbionts exhibited large δ 15 N differences (27‰) between biomass and N source (NH 4 + ) when the environmental NH 4 + was abundant, whereas the difference was small (4‰) when the environmental NH 4 + was low (Liao et al. 2014).Such a control of biomass δ 15 N has also been reported from both culture and field studies, ranging from 0‰ to 20‰ of isotope effects depending on the NH 4 + concentrations (Sigman and Fripiat 2019).
The links between low δ 15 N values and low Δ 14 C values seen in the entire ecosystem and also among each species in this study (Paralvinella aff.hessleri, Bathymodiolus platifrons, Enigmaticolus nipponensis, and Shinkaia crosnieri) suggest that the low δ 15 N values observed in animals living closer to vent orifice were caused by enhanced isotope fractionation during N assimilation of the chemoautotrophs.The δ 15 N values of NH 4 + in the vent fluid of Iheya North were 2.8-4.0 (3.4‰ AE 0.5‰; Supporting Information Table S2).This is approximately 5‰ (up to 10‰) higher than the δ 15 N values of the shrimp Shinkaicaris leurokolos and the mussel B. platifrons (Supporting Information Table S1).The isotope effects of up to 20‰ (Sigman and Fripiat 2019) or 27‰ (Liao et al. 2014) associated with the NH 4 + assimilation can explain the observed low δ 15 N values for these animals.
In environments where abundant NH 4 + is available, such as the habitats close to vent orifices (typically 1900 μmol L À1 in the vent fluid; Supporting Information Table S2), the NH 4 + incorporated into prokaryotic cells is consumed only partially while the remnant NH 4 + is then again released to the environment.The partial consumption emphasizes the large kinetic isotope effects associated with the enzymatic reaction for NH + 4 consumption, rather than the small effect at NH 4 + transportation across the cell membrane.This results in large 15 N depletion in organisms (Liao et al. 2014), even when the $ 3‰ enrichment in the δ 15 N values of OM occurs through the digestion of chemoautotrophs.
Due to the extremely high concentration of NH + 4 in venting fluids (typically 1900 μmol L À1 ; Supporting Information Table S2) and the high venting flux, the back flux of 15 N-rich NH 4 + has little effect on the environmental δ 15 N values of NH 4

+
. On the other hand, virtually all NH + 4 incorporated into prokaryotic cells can be consumed in environments where only limited NH 4 + is available, such as those habitats further away from the vent orifice.In this case, the δ 15 N values of the biomass are affected only by the small isotope effect associated with the NH + 4 transport across the cell membrane and the $ 3‰ enrichment at digestion.

Fig. 1 .
Fig. 1.Habitats of the relevant vent animals at the Iheya North hydrothermal field.(A) Schematic illustration showing typical positions of animal habitats in relation to the central venting orifice as well as properties of hydrothermal fluid and bottom water, letters correspond to photographs in parts B-G.(B) "Closest to vent" habitat with the sulfide worm Paralvinella aff.hessleri, (C) "closest to vent" habitat with the shrimp Shinkaicaris leurokolos, (D) "near vent" habitat with the squat lobster Shinkaia crosnieri, (E) "diffuse flow" habitat where the mussel Bathymodiolus platifrons, the buccinid whelk Enigmaticolus nipponensis, the scale worm Branchipolynoe pettiboneae, the shrimp Alvinocaris longirostris, the barnacle Neoverruca intermedia, and the limpet Lepetodrilus nux mainly occur, (F) "weak diffuse flow" habitat with the tubeworm Lamellibrachia columna and the stalked barnacle Ashinkailepas seepiophila, and (G) "weak diffuse flow" habitat with the veiscomyid clam Phreagena okutanii.Not to scale.The concentrations and natural-abundance radiocarbon (Δ 14 C) of TDIC for the bottom water cited from Honda et al. (2000).Those of TDIC and CH 4 for hydrothermal fluid are based on Kawagucci et al. (2020), assuming that the Δ 14 C values of Iheya North hydrothermal field can be regarded as 14 C-depleted like the nearby vents in the Okinawa Trough.

Fig. 2 .
Fig. 2. Natural-abundance radiocarbon (Δ 14 C) of animals collected from hydrothermal vent chimneys and the adjacent diffuse vent habitats at the Iheya North hydrothermal field.The animals were aligned based on the habitat distance from the main vent orifice, the ones closer on the bottom and those further away at the top, with notes of maximum habitat temperatures.

Table 1 .
Habitat temperature, mixing ratios of vent fluid to bottom water, mixing ratio of vent fluid TDIC and bottom water TDIC, and calculated Δ 14 C values of TDIC of the habitat for each animal.Equation (Eq.) numbers refer to those in the main text.
Miyazaki et al. (2017)t al. (2017).† Calculated values based on habitat temperature and TDIC concentrations of vent fluid/bottom water.‡ Calculated values based on animal tissue Δ 14 C value.