The investigation was carried out during TS42 cruise between 7 July and 18 August 2024 by RV Tan Suo Yi Hao with the full-ocean-depth human-occupied vehicle Fendouzhe, which was fitted with hydraulically powered manipulators on two swing arms. Under the guidance of operators in the human-occupied vehicle, the arms efficiently acquired the samples and stored them safely in a biological box and a geological box of the vehicle.
Processing of benthic fauna and sea floor video footage
Upon retrieval of the submersible, all collected specimens were promptly transferred from the biological collection box and slurp sampler to the shipboard laboratory. The specimens were then sorted into main taxonomic groups of different levels using visual inspection or under stereomicroscopes. Each organism was counted and preserved in pre-cooled, non-denatured 95% ethanol or in a 4% buffered formaldehyde solution depending on the taxon. Following initial preservation, certain taxonomic groups were further transferred to 70% ethanol for long-term storage.
Visual assessment of species identification, density and spatial structure of the macro-epifauna and mega-epifauna of the seep communities was carried out on the basis of the analysis of video footage recorded by two high-definition cameras mounted on the human-occupied vehicle. For each dive, between three and ten representative screenshots showing the densest cold-seep communities were selected from the video footage. The area of each image was estimated using the submersible’s laser scale, which projects two parallel laser points 10 cm apart onto the sea floor. This provided a reliable spatial reference for calculating the area of the sea floor captured in each image. A standardized quadrat (for example, 50 × 50 cm) was drawn near the laser dots by using this laser scale as a reference in the image. The calculated area was then converted into square metres for standardized density calculations. Animals visible in each quadrat were manually counted, and faunal density was expressed as the number of individuals per square metre. For each dive, density values from the selected images were used to calculate the mean faunal density, and the standard deviation was computed to quantify the variability among replicate images.
Phylogenetic analyses of the coxI gene sequences
Up to 0.5 cm3 of fauna tissue was cut into tiny pieces and subjected to DNA extraction using the PowerSoil DNA Isolation kit (MoBio Laboratories). The extracted DNA was quantified using a Qubit dsDNA HS Assay Kit with Qubit 2.0 fluorometer (Invitrogen). A metagenomic library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina v.4 and sequenced on the NovaSeq X Plus platform (Illumina) to generate 2 × 150 bp pair-ended reads. Raw sequencing reads were qualified using Fastp v.0.23.2 and assembly into contigs using MEGAHIT v.1.2.9. The coxI gene encoding cytochrome c oxidase subunit I of the fauna was retrieved from metagenomic contigs. The retrieved coxI was searched against the National Center for Biotechnology Information GenBank database for preliminary taxonomy identification.
Gas and pore-water sampling
During each dive, 6–12 sediment pushcores were collected using the manipulators of the submersible. Upon recovery, these pushcores were immediately transported to the ship’s cold room, which is kept at around 4 °C, to facilitate subsequent processing. Among them, one or two pushcores were allocated for gas concentration analysis onboard, with only subsamples exhibiting high methane concentrations being prepared for further carbon and deuterium isotope analysis. In addition, one or two pushcores were used to extract pore water samples for geochemical analysis.
Pore-water sampling was conducted using Rhizon samplers (as described in ref. 51). These samplers were inserted into the cores at 2-cm intervals and connected to 50-ml evacuated disposable syringes fitted with three-way Luer-lock stopcocks. The first approximately 1 ml of extracted pore water was discarded to remove any contaminants. Subsequently, around 15 ml of pore water was collected within a 2-h time frame. One portion of the pore water was preserved with a 20% zinc acetate solution for subsequent hydrogen sulfide analysis. The remaining pore water samples were transferred to 15-ml centrifuge tubes and frozen for subsequent ion analysis.
For the analysis of gas composition and isotopes, sediment samples were collected using a 2.5-ml cutoff plastic syringe, which was inserted through pre-drilled holes in the pushcore tube at depth intervals of 4 cm. A 2.5-ml sediment sample was then transferred to a 20-ml gas-tight glass vial, which was filled with 2 M NaOH solution and sealed with a crimp cap containing butyl rubber stoppers. The vials were vigorously shaken and stored in an upside-down position at 4 °C until analysis, which was conducted onboard in a single day. Before analysis, the vials were shaken again, and 2 ml of the NaOH solution was replaced with helium gas to create a headspace. The headspace gas from the push core was also directly extracted using a 20-ml syringe equipped with a three-way stopcock and was immediately transferred to a 12-ml vacuum Labco vial for further analysis.
Gas concentration and isotope
The concentration of dissolved gases was determined using a gas chromatograph (Trace GC1310; Thermo Scientific) installed onboard the research vessel. Headspace volumes ranging from 100 to 500 μl were sampled and injected into a gas chromatograph equipped with a flame ionization detector. The analytical precision achieved in these measurements is consistently lower than 5%.
The δ13C and δD isotopic compositions of methane were analysed using a gas chromatography–isotope ratio mass spectrometer system, which consisted of a Trace GC1310 connected to a Delta V Advantage Isotope Ratio MS (Thermo Scientific). The analysis was conducted at the Institute of Deep-Sea Science and Engineering (IDSSE), Chinese Academy of Sciences.
Methane was selectively separated from the gas matrix using a gas chromatography column (27 m × 0.3 mm × 20.00 μm; PoraPLOT Q). The separated methane was then combusted in a combustion oven at a temperature of 1,000 °C, which was interfaced with the isotope ratio mass spectrometer (IRMS) for subsequent analysis. The resulting CO 2 was directly introduced into the IRMS for measurement.
The precision of the repeated analyses, expressed as the standard deviation (1σ), was ±0.5‰ for δ13C and ±2‰ for δD. The isotopic compositions of individual carbon compounds were reported as δ-values (‰) relative to the international standards Vienna Peedee Belemnite (VPDB) for δ13C and Vienna Standard Mean Ocean Water (VSMOW) for δD.
Pore-water geochemistry
Hydrogen sulfide concentrations were determined colorimetrically using the methylene blue method (with a limit of detection of approximately 0.5 μM). The concentration of \({{\rm{SO}}}_{4}^{2-}\) in pore water was quantified by ion chromatography using a Dionex ICS-900 system, which was equipped with an AS50 AutoSampler. To ensure that the \({{\rm{SO}}}_{4}^{2-}\) concentrations fell in the optimal analytical range for the ion chromatograph, the anion samples were diluted 70-fold with Milli-Q water. The analytical precision for the determination of \({{\rm{SO}}}_{4}^{2-}\) was ±3.0%. \({{\rm{NH}}}_{4}^{+}\) concentrations were measured using a fluorescence spectrometer (LS55, PE) following the procedure reported in ref. 52, with a relative deviation of 0.5%. DIC concentrations and δ13C-DIC values were analysed using a Gas Bench II IRMS at IDSSE. The samples were pretreated with an acid solution on the Gas Bench II; the resulting carbon dioxide, carried by helium, was separated by a constant-temperature chromatographic column and subsequently analysed for isotope abundance using a MAT253 gas stable IRMS. The analytical precision for DIC was ±0.15% and that for δ13C-DIC was ±0.5‰. Dissolved organic carbon concentrations were measured by a high-temperature catalytic oxidation method using a Multi N/C 3100 (CLD) analyser at IDSSE. Samples were thawed at room temperature and acidified to pH 2 with 2 mol l−1 hydrochloric acid before injection. Salinity was measured in the laboratory using a handheld digital salinity meter (ATAGO PAL-SALT) after the samples were thawed to room temperature.
Methane phase modelling
The phase boundaries and methane hydrate solubility in the methane hydrate–sea water system were calculated using thermodynamic models53,54. The chemical potential of the hydrate phase was determined through the application of the Van der Waals–Platteeuw hydrate model, in conjunction with angle-dependent ab initio intermolecular potentials, as previously described in ref. 55. The Gibbs–Thomson equation, incorporating appropriately parameterized hydrate–water interface values, was used to account for the capillary effects of porous sediments on the hydrate–liquid–vapour equilibrium and the hydrate–liquid equilibrium. The influence of surface textures and mineral components was neglected in this analysis.
The activity coefficients for water and methane in the methane–sea water system were calculated using the Pitzer model. By applying the Poynting correction to the fugacity of methane dissolved in aqueous solution at the hydrate–liquid equilibrium, where methane gas is absent, the extended model for three-phase equilibrium was adapted to predict the solubility of methane in aqueous solution at the hydrate–liquid equilibrium.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.