Daniel B Nothaft
email: daniel.nothaft@colorado.edu

Alexis S Templeton
email: alexis.templeton@colorado.edu

Jeemin H Rhim

David T Wang

Jabrane Labidi

Hannah M Miller

Eric S Boyd

Juerg M Matter

Shuhei Ono

Edward D Young

Sebastian H Kopf

Peter B Kelemen

Mark E Conrad

Geochemical, biological and clumped isotopologue evidence for substantial microbial methane production under carbon limitation in serpentinites of the Samail Ophiolite, Oman

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

At temperatures and pressures near the Earth's surface (< 400 • C, < 100 MPa), ultramafic rocks such as peridotite in contact with water are thermodynamically driven to hydrate and oxidize, forming variable amounts of serpentine, magnetite, brucite, hydrogen (H 2 ), and other phases [START_REF] Evans | Metamorphism of alpine peridotite and serpentinite[END_REF][START_REF] Frost | On the Stability of Sulfides, Oxides, and Native Metals in Serpentinite[END_REF]McCollom & Bach, 2009;Klein & Bach, 2009;Klein et al., 2009[START_REF] Klein | Abiotic methane synthesis and serpentinization in olivine-hosted fluid inclusions[END_REF]. This process, often called "serpentinization", can produce H 2 at temperatures at least as low as 55 • C [START_REF] Miller | Low temperature hydrogen production during experimental hydration of partially-serpentinized dunite[END_REF].

The resultant H 2 can be thermodynamically favored to reduce carbon dioxide (CO 2 ) to methane (CH 4 ) [START_REF] Shock | Chemical Environments of Submarine Hydrothermal Systems[END_REF]. The reduction of CO 2 by H 2 to form CH 4 can be catalyzed on mineral surfaces as in the Sabatier reaction [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF][START_REF] Klein | Abiotic methane synthesis and serpentinization in olivine-hosted fluid inclusions[END_REF], or enzymatically through microbial methanogenesis [START_REF] Whiticar | Carbon and hydrogen isotope systematics of bacterial formation and oxidation of methane[END_REF].

In continental settings undergoing serpentinization, where fluid-rock reactions typically occur at low temperatures (< 150 • C), there is disagreement regarding the ori-gin of CH 4 . Three key potential CH 4 sources have been identified in these environments.

One potential source is the abiotic reduction of CO 2 to CH 4 at warmer-than-present temperatures in fluid inclusions within crystals that can store CH 4 and subsequently release it. Another potential source is the abiotic, mineral-catalyzed reduction of CO 2 to CH 4 at the low temperatures that prevail in the present-day weathering environment. A third potential source is microbial methanogenesis.

Storage of CH 4 produced at temperatures of 270 • C to 800 • C in fluid inclusions in minerals such as olivine and the release of this CH 4 through subsequent chemical/physical alteration are the dominant processes contributing to CH 4 fluxes from sediment-poor seafloor hydrothermal vents [START_REF] Kelley | Methane-rich fluids in the oceanic crust[END_REF][START_REF] Kelley | Abiogenic methane in deep-seated midocean ridge environments: Insights from stable isotope analyses[END_REF][START_REF] Mcdermott | Pathways for abiotic organic synthesis at submarine hydrothermal fields[END_REF]D. T. [START_REF] Wang | Clumped isotopologue constraints on the origin of methane at seafloor hot springs[END_REF][START_REF] Labidi | Methane thermometry in deep-sea hydrothermal systems: Evidence for re-ordering of doubly-substituted isotopologues during fluid cooling[END_REF]. In continental, low-temperature serpentinizing settings, however, debate continues as to whether fluid inclusions can sustain observed CH 4 fluxes [START_REF] Etiope | Abiotic methane in continental ultramafic rock systems: Towards a genetic model[END_REF][START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF].

Abiotic reduction of CO 2 to CH 4 can occur at temperatures at least as low as 20 • C when catalyzed by the transition metal ruthenium (Ru) [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF]. Ru is present in considerable abundance in chromitite bodies in ultramafic rock accumulations [START_REF] Etiope | Widespread abiotic methane in chromitites[END_REF], but it has only been shown to catalyze CO 2 hydrogenation under conditions where free gas phases exist [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF]. The prevalence of this process, particularly in aquifers whose fluid compositions appear to be dominantly influenced by aqueous reactions with harzburgite, is another matter of ongoing debate [START_REF] Etiope | Methane origin in the Samail ophiolite: Comment on "Modern water/rock reactions in Oman hyperalkaline peridotite aquifers and implications for microbial habitability[END_REF][START_REF] Miller | Reply to "Methane origin in the Samail ophiolite: Comment on 'Modern water/rock reactions in Oman hyperalkaline peridotite aquifers and implications for microbial habitability[END_REF].

Low-temperature CH 4 production can also be mediated by microbes called "methanogens".

Microbial CH 4 has traditionally been viewed as a minor/negligible source of CH 4 in serpentinizing settings. This is due in large part to the relatively 13 C-enriched composition of CH 4 in serpentinizing settings (δ 13 C commonly -20 VPDB to 5 VPDB), which contrasts with the more 13 C-depleted composition of CH 4 in sedimentary settings dominated by microbial methanogenesis (δ 13 C commonly -90 VPDB to -50 VPDB) [START_REF] Etiope | Methane origin in the Samail ophiolite: Comment on "Modern water/rock reactions in Oman hyperalkaline peridotite aquifers and implications for microbial habitability[END_REF][START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of ¿20,000 samples[END_REF][START_REF] Etiope | Abiotic methane in continental ultramafic rock systems: Towards a genetic model[END_REF]. However, cultures of methanogens can produce CH 4 with minimal C isotope fractionation in H 2 -rich, CO 2poor fluids simulating serpentinizing systems [START_REF] Miller | Large carbon isotope variability during methanogenesis under alkaline conditions[END_REF]. In these cultures, it has been inferred that the net C isotope effect of methanogenesis was attenuated due to microbial conversion of a large proportion of available CO 2 to CH 4 when CO 2 was the limiting substrate. Such results illustrate that 13 C-enriched CH 4 in natural serpentinizing settings does not necessarily derive from non-microbial sources. Still, the quantity and isotopic composition of microbial CH 4 in serpentinizing settings remains uncertain.

In this study, we assessed sources and sinks of CH 4 in the Samail Ophiolite of Oman, a site of active, low-temperature serpentinization and carbonation. Fluids and particulates in groundwaters accessed via wells in the Samail Ophiolite have been sampled for biogeochemical studies annually from 2014 through 2018 from January to March. Microbiological and geochemical data from sampling campaigns in 2014 through 2017 and a limited number of C and H bulk stable isotope analyses of CH 4 sampled in 2014 have been previously reported (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF] Here, we present new geochemical and 16S rRNA gene amplicon sequencing data from samples acquired in 2018. We also present new bulk stable isotope data on CH 4 , ethane (C 2 H 6 ), and propane (C 3 H 8 ) from samples obtained from 2015 through 2018. Further, we report analyses of multiply-substituted "clumped" isotopologues of CH 4 , 13 CH 3 D and 12 CH 2 D 2 , for the first time on samples from this ophiolite.

Leveraging one of the largest longitudinal data sets on CH 4 biogeochemistry in an ophiolite, we have identified robust trends across years and hydrogeologic settings. We observed a wide range of C isotopic compositions of CH 4 and short-chain alkanes, intramolecular isotopologue disequilibrium in CH 4 , and widespread occurrence of gene sequences affiliated with methanogens, which collectively indicate that substantial quantities of microbial CH 4 are produced and mix with abiotic CH 4 released from fluid inclusions in the manuscript submitted to JGR: Biogeosciences Samail Ophiolite. Our finding that microbial methanogenesis proceeds even in hyperalkaline fluids lends insight into the functioning of microbial ecosystems that leverage reactions between water and ultramafic rocks to power metabolic processes on Earth and perhaps on other rocky bodies of the Solar System [START_REF] Ménez | Abiotic Hydrogen and Methane: Fuels for Life[END_REF][START_REF] Glein | Hydrogen, Hydrocarbons, and Habitability Across the Solar System[END_REF].

Geologic Setting

The Samail Ophiolite (Figure 1) consists of pelagic sedimentary rocks (< 0.1 km), volcanic rocks (0.5 km to 2.0 km), sheeted dikes (1 km to 1.5 km), gabbro and igneous peridotite (0.5 km to 6.5 km), residual mantle peridotites, (8 km to 12 km), and a metamorphic sole of greenschist-to granulite-facies metamorphic rocks (< 0.5 km) [START_REF] Glennie | Late Cretaceous nappes in Oman Mountains and their geologic evolution[END_REF][START_REF] Clark | Stable isotope disequilibria in travertine from high pH waters: Laboratory investigations and field observations from Oman[END_REF]Lippard et al., 1986;[START_REF] Nicolas | Structures of Ophiolites and Dynamics of Oceanic Lithosphere[END_REF][START_REF] Nicolas | Accretion of Oman and United Arab Emirates ophiolite-discussion of a new structural map[END_REF]. The ophiolite crust formed from 96.12 Ma to 95.50 Ma, and convergence began at about the same time [START_REF] Rioux | Synchronous formation of the metamorphic sole and igneous crust of the Semail ophiolite: New constraints on the tectonic evolution during ophiolite formation from high-precision U-Pb zircon geochronology[END_REF], or up to 10 Myr earlier [START_REF] Guilmette | Forced subduction initiation recorded in the sole and crust of the Semail Ophiolite of Oman[END_REF][START_REF] Soret | Slow subduction initiation forces fast ophiolite formation Soret[END_REF]. Ophiolite emplacement continued until 78 Ma to 71 Ma [START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF]. Part of the ophiolite was subaerially eroded in the Late Cretaceous, then became covered in parts by Maastrictian to Eocene limestones due to subsidence and transgression [START_REF] Nolan | Maastrichtian to early Tertiary stratigraphy and palaeogeography of the Central and Northern Oman Mountains[END_REF][START_REF] Skelton | The Maastrichtian transgression onto the northwestern flank of the Proto-Oman Mountains: sequences of rudist-bearing beach to open shelf facies[END_REF].

The mantle section of the ophiolite is mainly composed of highly depleted, residual mantle harzburgites, together with 5 % to 15 % dunite, which both contain a few percent chromian spinel [START_REF] Godard | Relationships between geochemistry and structure beneath a palaeo-spreading centre: a study of the mantle section in the Oman ophiolite[END_REF][START_REF] Hanghøj | Composition and Genesis of Depleted Mantle Peridotites from the Wadi Tayin Massif, Oman Ophiolite; Major and Trace Element Geochemistry, and Os Isotope and PGE Systematics[END_REF][START_REF] Boudier | Cross section through the peridotite in the Samail Ophiolite, southeastern Oman Mountains[END_REF][START_REF] Collier | Spatial-Statistical Properties of Geochemical Variability as Constraints on Magma Transport and Evolution Processes at Ocean Ridges[END_REF]. The extent of serpentinization is typically 30 % to 60 %, reaching 100 % in some cases [START_REF] Dewandel | Seismic wave velocity and anisotropy of serpentinized peridotite in the Oman ophiolite[END_REF][START_REF] Boudier | Serpentine Mineral Replacements of Natural Olivine and their Seismic Implications: Oceanic Lizardite versus Subduction-Related Antigorite[END_REF]Miller et al., 2016;[START_REF] Kelemen | & the Oman Drilling Project Science Team[END_REF]. Chromitites are most often found in association with dunites near the crust-mantle transition, possibly representing bases of cumulate piles, but are also found dispersed throughout the mantle section [START_REF] Rollinson | Chromite in the mantle section of the Oman ophiolite: A new genetic model[END_REF].

Geologic reservoirs of C underlying the ophiolite include Mid Permian to Late Cretaceous shallow marine carbonates, which host oil and gas fields in parts of northern Oman and the United Arab Emirates [START_REF] Terken | The Natih Petroleum System of North Oman[END_REF][START_REF] Alsharhan | PETROLEUM GEOLOGY OF THE UNITED ARAB EMIRATES[END_REF]Etiope et al., 2015). Maastrictian to Eocene limestones that partially overly the ophiolite have been shown to transfer inorganic C to peridotites where they are in contact (de Obeso & [START_REF] De Obeso | Fluid rock interactions on residual mantle peridotites overlain by shallow oceanic limestones: Insights from Wadi Fins, Sultanate of Oman[END_REF].

C is also stored within the ophiolite, primarily in the form of carbonate minerals [START_REF] Neal | Past and present serpentinisation of ultramafic rocks; an example from the Semail Ophiolite Nappe of Northern Oman[END_REF]; P. B. [START_REF] Kelemen | In situ carbonation of peridotite for CO 2 storage[END_REF]; P. B. [START_REF] Kelemen | Rates and mechanisms of mineral carbonation in peridotite: natural processes and recipes for enhanced, in situ CO 2 capture and storage[END_REF][START_REF] Noël | Evidence of polygenetic carbon trapping in the Oman Ophiolite: Petro-structural, geochemical, and carbon and oxygen isotope study of the Wadi Dima harzburgite-hosted carbonates (Wadi Tayin massif, Sultanate of Oman)[END_REF]. Hydration and carbonation of > 20 000 km 3 of peridotite continue today in the Samail Ophiolite, largely at < 60 • C [START_REF] Neal | Hydrogen generation from mantle source rocks in Oman[END_REF][START_REF] Frost | On the Stability of Sulfides, Oxides, and Native Metals in Serpentinite[END_REF][START_REF] Kelemen | In situ carbonation of peridotite for CO 2 storage[END_REF][START_REF] Kelemen | Rates and mechanisms of mineral carbonation in peridotite: natural processes and recipes for enhanced, in situ CO 2 capture and storage[END_REF][START_REF] Streit | Coexisting serpentine and quartz from carbonate-bearing serpentinized peridotite in the Samail Ophiolite, Oman[END_REF][START_REF] Paukert | Reaction path modeling of enhanced in situ CO 2 mineralization for carbon sequestration in the peridotite of the Samail Ophiolite, Sultanate of Oman[END_REF]Chavagnac, Ceuleneer, et al., 2013;Chavagnac, Monnin, et al., 2013;[START_REF] Mervine | Carbonation rates of peridotite in the Samail Ophiolite, Sultanate of Oman, constrained through 14 C dating and stable isotopes[END_REF][START_REF] Falk | Controls on the stable isotope compositions of travertine from hyperalkaline springs in Oman: Insights from clumped isotope measurements[END_REF]Miller et al., 2016;[START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF].

Methods

Fluid sampling and field measurements

Wells were drilled into the Samail Ophiolite by the Ministry of Regional Municipalities and Water Resources of the Sultanate of Oman prior to 2006 ("WAB" and "NSHQ" wells in this study) and by the Oman Drilling Project in 2016 through 2018 ("CM") (Parsons International & Co., 2005; P. [START_REF] Kelemen | Scientific drilling and related research in the samail ophiolite, sultanate of Oman[END_REF]. Information on well location, construction, and water level are given in Table 1. In sampling campaigns in 2014 and 2015, a 12 V submersible Typhoon R pump (Proactive Env. Products, Bradenton, FL, USA) with typical flow rates of 5 L•min -1 was used. This pump was used in all years of sampling at well NSHQ04 due to partial obstruction of this well. In all other sampling from 2016 onwards, a larger submersible pump (Grundfos SQ 2-85) with typical flow rates of 20 L•min -1 was used. The pumping depths are reported in Tables 1 and2. For fluids sampled in 2018, temperature, conductivity, and pH were measured using a ColeParmer PC100 -OH -Figure 1. Study area in Samail Ophiolite, Sultanate of Oman. Geologic map data from [START_REF] Nicolas | Accretion of Oman and United Arab Emirates ophiolite-discussion of a new structural map[END_REF]. Inset: overview of Samail Ophiolite (shaded in brown) with study area (larger map) indicated by the red shaded box. A topographic map of the study area is provided in Supporting Information Figure S1.

Meter, while Eh was measured using a Mettler Toledo SG2 SevenGo meter. The analytical uncertainties for temperature, conductivity, pH, and Eh are 0.5 • C, 1.0 % of measured value, 0.01 µS•cm -1 , and 1 mV, respectively. Each well was pumped for ≥ 20 min prior to sampling. Sampling commenced once fluid pH and conductivity measurements stabilized.

Chemical and isotopic analyses of fluids

To analyze aqueous concentrations (c) of non-carbonaceous chemical species, samples were collected by passing groundwater through a 0. Aqueous concentrations of Cl -, Br -, F -, and SO 2- 4 were measured on a Dionex IC25 All isotopic values reported in units. δ 13 C and δD reported in the VPDB and VSMOW reference frames, respectively. Data from 2014 previously reported by Miller et al. (2016). Abbreviations: n.d., not determined; mbct, meters below casing top.

manuscript submitted to JGR: Biogeosciences ion chromatograph with an AS9-HC IonPac column, with the exception of NO - 3 , which was measured on a Dionex 4500I ion chromatograph with an IonPac AS14 column using EPA method 300.0 (analytical uncertainty of 2 %).

The concentration and δ 13 C of dissolved inorganic C ( CO 2 ) were measured by acidification of water samples and transfer of resultant CO 2 (g) via a Thermo Fisher Gas-Bench II to a Thermo Delta V Plus isotope ratio mass spectrometer. We optimized the methods of [START_REF] Assayag | Improved method for isotopic and quantitative analysis of dissolved inorganic carbon in manuscript submitted to JGR: Biogeosciences natural water samples[END_REF] for the wide range of c CO2 observed in ophiolite groundwaters. Complete methodological details are available in [START_REF] Nothaft | Dissolved inorganic carbon concentration and 13 C/ 12 C. Retrieved 2021[END_REF]. Sample δ 13 C values were converted to the VPDB reference frame using measured δ 13 C values of international reference materials (Harding Iceland Spar and LSVEC). Isotopic reference frame calculations were performed using the Isoverse suite of packages [START_REF] Kopf | Isoreader: An R package to read stable isotope data files for reproducible research[END_REF] for the statistical programming language, R (R Core Team, 2019).

Water δ 18 O and δD were measured on a Picarro L2120-i cavity ring down spectrometer. The instrument analyzed each sample six times, excluding the first three analyses to avoid memory effects. Reported precision is the standard deviation of the last three measurements. Reported accuracy is the mean difference between accepted values and measured values of standards. Mean precision in the run was 0.06 for δ 18 O and 0.23 for δD; mean accuracy was 0.04 for δ 18 O and 0.47 for δD.

Gases dissolved in pumped groundwaters were sampled by injecting water into N 2 purged vials for headspace gas analysis using methods described by Miller et al. (2016) in field campaigns occurring from 2014 to 2017. In addition, the bubble strip method (modified from [START_REF] Kampbell | DETERMINING DISSOLVED HYDROGEN, METHANE, AND VINYL CHLORIDE CONCENTRATIONS IN AQUEOUS SOLUTION ON A NANOMOLAR SCALE WITH THE BUB-BLE STRIP METHOD[END_REF]) was used from 2016 to 2018. Details on bubble strip gas sampling are available in D. B. [START_REF] Nothaft | Bubble strip aqueous gas sampling[END_REF]. The gas concentrations reported in this study were determined from bubble strip samples. These concentrations were measured on an SRI 8610C gas chromatograph (GC) with N 2 as the carrier gas.

H 2 , CO, CH 4 , and CO 2 were separated with a 2 mm by 1 mm ID micropacked ShinCarbon ST column, whereas alkanes of 2 to 6 C atoms ("C 2 -C 6 short-chain alkanes") were separated with a PORAPAK Q 6 ft by 0.085 in ID column. Peak intensities were measured concurrently using a thermal conductivity detector (TCD) and a flame ionization detector (FID) and calibrated with standard gas mixes (Supelco Analytical, Bellefonte, PA, USA; accuracy of ±2 % of reported concentration). Measurement repeatability expressed as relative standard deviation was 5 % over most of the calibrated range. The limit of quantitation was defined as the signal at which the relative standard deviation increased to 20 %. In 2018, H 2 and CO were analyzed on a Peak Performer 1 gas chromatograph equipped with a reducing compound photometer (RCP). Due to the high sensitivity of the RCP, the signal at limit of quantitation (S LQ ) for these analyses was defined as S LQ = S b + 10 • σ b , where S mb is the mean signal of blanks prepared in field and σ b is the population standard deviation of these blanks, in accordance with American Chemical Society guidelines [START_REF] Macdougall | Guidelines for data acquisition and data quality evaluation in environmental chemistry[END_REF]. Gaseous concentrations were converted to aqueous concentrations using gas solubilities [START_REF] Sander | Compilation of Henry's law constants (version 4.0) for water as solvent[END_REF] and corrected for temperature and volume changes between sampling and analysis.

Prior to 2017, bulk stable isotope analyses of CH 4 were conducted at the Center for Isotope Geochemistry at the Lawrence Berkeley National Laboratory (LBNL) by gas chromatography/combustion/pyrolysis isotope-ratio mass spectrometry (GC/C/Pyr/IRMS) using methods described by Miller et al. (2016). The measurement repeatability expressed as 1 sample standard deviation (s) for these analyses is ±0. Sequences were demultiplexed with idemp [START_REF] Wu | yhwu/idemp[END_REF]. The resultant fastq files were quality filtered using Figaro v1.1.1 [START_REF] Weinstein | Zymo-Research/figaro[END_REF] and the DADA2 v1.16 R package [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF]. Amplicon sequence variants were assigned taxonomy to the genus level using the RDP classifier (Q. [START_REF] Wang | Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy[END_REF] trained on the Silva SSU 138 reference database [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF] using the DADA2 assignTaxonomy function. Species level assignments were based on exact matching between amplicon sequence variants and sequenced reference strains using the DADA2 addSpecies function. Sequences assigned to mitochondria, chloroplast, and Eukaryota, or not assigned at the domain level (collectively < 1 % of sequences), were removed. After all of the above filtering, 24 000 to 40 000 reads per sample remained for the samples presented here obtained in 2018. In addition, 16S rRNA gene sequencing data from previous Oman sampling campaigns [START_REF] Paukert | Mineral Carbonation in Mantle Peridotite of the Samail Ophiolite, Oman: Implications for permanent geological carbon dioxide capture and storage[END_REF]through 2017;(Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]) were re-processed in accordance with the methods outlined here to facilitate comparisons across the data sets. The complete data processing pipeline for samples across all years, from raw data provided by the sequencing facility through to taxonomic assignment, are available in D. B. [START_REF] Nothaft | danote/Samail 16S compilation: First release of Samail 16S data processing repository[END_REF]. Additional analyses and plotting can be found in D. B. Nothaft, Templeton, et al. (2021). For samples presented in this study, demultiplexed fastq files (without additional processing) are also accessible on the NCBI Short Read Archive under accession PRJNA655565.

Thermodynamic calculations

Oxidation-reduction potential, pH, and concentrations of major ions and CO 2

were used as inputs for the modeling software PHREEQC [START_REF] Charlton | Modules based on the geochemical model PHREEQC for use in scripting and programming languages[END_REF][START_REF] Parkhurst | Description of input and examples for PHREEQC version 3-A computer program for speciation, batch-reaction, one-dimensional transport, and inverse geochemical calculations[END_REF], with which fluids were speciated using the LLNL database.

Activities of formate and acetate were separately calculated according to the Debye-Hückel equation. Activities of the aqueous gases were assumed equivalent to their concentrations, which is reasonable for neutral species in low ionic strength solutions. Standard

Gibbs free energies (∆G • r ) of the CH 4 -forming reactions were calculated using the program SUPCRTBL [START_REF] Johnson | SUPCRT92: A software package for calculating the standard molal thermodynamic properties of minerals, gases, aqueous species, and reactions from 1 to 5000 bar and 0 to 1000[END_REF]Zimmer et al., 2016) using conditions of 1 bar and 35 • C to approximate in situ conditions. Gibbs free energies were then calculated as ∆G r = ∆G • r + RT ln Q r , where R is the universal gas constant, T is temperature, and Q r is the reaction quotient. All of the above calculations and software inputs and outputs can be found in D. B. Nothaft, Templeton, et al. (2021).

4 Results and discussion

Controls on groundwater chemistry

To assess the source and reaction histories of Samail Ophiolite groundwaters, we measured their stable isotopic compositions and solute concentrations. Groundwater δD and δ 18 O plotted near local and global meteoric water lines [START_REF] Weyhenmeyer | Isotope study of moisture sources, recharge areas, and groundwater flow paths within the eastern Batinah coastal plain, Sultanate of Oman[END_REF][START_REF] Terzer | Global isoscapes for δ 18 O and δ 2 H in precipitation: improved prediction using regionalized climatic regression models[END_REF], indicating that the groundwaters derive from rain (Table 3; Supporting Information Figure S2; [START_REF] Matter | Recharge areas and geochemical evolution of groundwater in an alluvial aquifer system in the Sultanate of Oman[END_REF]Miller et al., 2016;[START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF]). The sampled groundwaters included oxidized and moderately alkaline Mg 2+ -HCO - 3 waters, typical of reaction with peridotite in communication with the atmosphere, and reduced and hyperalkaline Ca 2+ -OH -waters, typical of extensive hydration and oxidation of peridotite in closed-system conditions with respect to the atmosphere (Table 3; [START_REF] Barnes | Geochemical evidence of present-day serpentinization[END_REF][START_REF] Barnes | The relationship between fluids in some fresh alpine-type ultramafics and possible modern serpentinization, western United States[END_REF][START_REF] Neal | Past and present serpentinisation of ultramafic rocks; an example from the Semail Ophiolite Nappe of Northern Oman[END_REF][START_REF] Bruni | Irreversible water-rock mass transfer accompanying the generation of the neutral, Mg-HCO3 and high-pH, Ca-OH spring waters of the Genova province, Italy[END_REF][START_REF] Cipolli | Geochemistry of high-pH waters from serpentinites of the Gruppo di Voltri (Genova, Italy) and reaction path modeling of CO 2 sequestration in serpentinite aquifers[END_REF][START_REF] Kelemen | Rates and mechanisms of mineral carbonation in peridotite: natural processes and recipes for enhanced, in situ CO 2 capture and storage[END_REF][START_REF] Paukert | Reaction path modeling of enhanced in situ CO 2 mineralization for carbon sequestration in the peridotite of the Samail Ophiolite, Sultanate of Oman[END_REF]). Ca 2+ -OH -waters had higher conductivities (930. µS•cm -1 to 3350 µS•cm -1 ) than Mg 2+ -HCO - 3 waters (498 µS • cm -1 to 1183 µS • cm -1 ) (Table 1). The increase in conductivity from Mg 2+ -HCO - 3 waters to Ca 2+ -OH -waters is driven by enrichments in Ca 2+

derived from dissolution of primary silicate minerals in addition to Na + and Cl -derived from mineral dissolution, sea spray, and/or leaching of sea salts introduced during subseafloor alteration and/or ophiolite emplacement [START_REF] Neal | Past and present serpentinisation of ultramafic rocks; an example from the Semail Ophiolite Nappe of Northern Oman[END_REF][START_REF] Stanger | The hydrogeology of the Oman Mountains (Doctoral dissertation[END_REF][START_REF] Murad | Factors controlling groundwater quality in Eastern United Arab Emirates: a chemical and isotopic approach[END_REF][START_REF] Paukert | Reaction path modeling of enhanced in situ CO 2 mineralization for carbon sequestration in the peridotite of the Samail Ophiolite, Sultanate of Oman[END_REF][START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF]. The increase in pH from Mg 2+ -HCO - 3 waters (pH 8.66 to 9.62) to Ca 2+ -OH -waters (10.51 to 11.39) was accompanied by a shift to lower f O2 and Eh (∼ 10 -51 bar and -174 mV to -253 mV, respectively, in most Ca 2+ -OH -waters) (Table 1), indicating reduced conditions in Ca 2+ -OH -waters.

Concentrations of CO 2 were relatively high in Mg 2+ -HCO - 3 waters and gabbro waters (up to 3490 µmol • L -1 ), but below the limit of quantitation (< 12 µmol • L -1 ) in most Ca 2+ -OH -waters (Table 3). This is consistent with water-harzburgite reaction path modeling that terminates at chrysotile-brucite-diopside-calcite equilibrium, corresponding to a c CO2 of 8 µmol • L -1 at 25 • C and 1 bar [START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. Literature values for c CO2 in ophiolitic Ca 2+ -OH -waters are often higher than those predicted by reaction path modeling, but the lower range of reported values approaches [START_REF] Barnes | Geochemical evidence of present-day serpentinization[END_REF][START_REF] Barnes | The relationship between fluids in some fresh alpine-type ultramafics and possible modern serpentinization, western United States[END_REF][START_REF] Barnes | Present day serpentinization in New Caledonia, Oman and Yugoslavia[END_REF][START_REF] Neal | Past and present serpentinisation of ultramafic rocks; an example from the Semail Ophiolite Nappe of Northern Oman[END_REF][START_REF] Bruni | Irreversible water-rock mass transfer accompanying the generation of the neutral, Mg-HCO3 and high-pH, Ca-OH spring waters of the Genova province, Italy[END_REF][START_REF] Cipolli | Geochemistry of high-pH waters from serpentinites of the Gruppo di Voltri (Genova, Italy) and reaction path modeling of CO 2 sequestration in serpentinite aquifers[END_REF][START_REF] Paukert | Reaction path modeling of enhanced in situ CO 2 mineralization for carbon sequestration in the peridotite of the Samail Ophiolite, Sultanate of Oman[END_REF][START_REF] Falk | Controls on the stable isotope compositions of travertine from hyperalkaline springs in Oman: Insights from clumped isotope measurements[END_REF][START_REF] Brazelton | Metagenomic identification of active methanogens and methanotrophs in serpentinite springs of the Voltri Massif, Italy[END_REF][START_REF] Canovas | Geochemical bioenergetics during low-temperature serpentinization: An example from the Samail ophiolite, Sultanate of Oman[END_REF][START_REF] Crespo-Medina | Methane Dynamics in a Tropical Serpentinizing Environment: The Santa Elena Ophiolite, Costa Rica[END_REF][START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF][START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF]. This spread in the data could reflect groundwater mixing, atmospheric contamination during sampling, differences in reaction temperature and progress, and/or kinetic inhibitions to carbonate mineral precipitation. In Mg 2+ -HCO - 3 waters and waters from gabbroic aquifers, δ 13 C CO2 ranged from -13.54 VPDB to -10.88 VPDB (Table 3), which is comparable to δ 13 C CO2

1 µmol • L -1 (
of Mg 2+ -HCO - 3 waters elsewhere in the ophiolite (-15.56 VPDB to -13.60 VPDB;[START_REF] Matter | Recharge areas and geochemical evolution of groundwater in an alluvial aquifer system in the Sultanate of Oman[END_REF]D. Nothaft et al., 2021)).

Variable concentrations of H 2 and CH 4 across wells suggest spatial heterogeneities in sources and sinks of these gases in the ophiolite. In some Ca 2+ -OH -waters, c H2 was high (up to 253 µmol•L -1 ), but c H2 was below limits of quantitation in other Ca 2+ -OH -waters (Figure 2; Table 4). In Mg 2+ -HCO - 3 waters and waters from gabbroic aquifers, c H2 was generally below limits of quantitation. However, up to 0.992 µmol•L -1

H 2 was measured in well WAB188, which is in gabbro near a faulted contact with peridotites that contain Ca 2+ -OH -waters (Figure 1; Table 1). This suggests production of H 2 within the gabbro host rock or migration of H 2 from peridotites into gabbros surrounding WAB188. In most Ca 2+ -OH -waters, c CH4 was high (up to 483 µmol•L -1 ;

Figure 2, Table 4). However, wells with high c CH4 did not always have high c H2 (Figure 2; Table 4). In Mg 2+ -HCO - 3 waters and gabbro waters, c CH4 was typically lower To assess which CH 4 -forming aqueous reactions might occur within the Samail Ophiolite, ∆G r 's were calculated for the following reactions:

(≤ 0.1 µmol • L -1 ), although c CH4 reached 1.83 µmol • L -1 in well WAB188,
CO 2 (aq) + 4H 2 (aq) = CH 4 (aq) + 2H 2 O(l) (hydrogenotrophic methanogenesis) (2) CH 3 COO -(aq) + H + (aq) = CH 4 (aq) + CO 2 (aq) (acetoclastic methanogenesis) (3) 4HCOO -(aq)+4H + (aq) = CH 4 (aq)+3CO 2 (aq)+2H 2 O(l) (formatotrophic methanogenesis).
(4) Gas-phase, abiotic reactions are also possible [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF][START_REF] Etiope | Widespread abiotic methane in chromitites[END_REF], but measurements of partial pressures of relevant gases in unsaturated zones of the subsurface in the study area are absent. Thus, ∆G r 's of gas-phase reactions were not calculated. In addition to the common hydrogenotrophic and acetoclastic modes of methanogenesis, formatotrophic methanogenesis (Equation 4) was considered because formate can be produced abiotically in serpentinizing settings [START_REF] Mccollom | Experimental constraints on the hydrothermal reactivity of organic acids and acid anions: I. Formic acid and formate[END_REF]Mc-Dermott et al., 2015;[START_REF] Miller | Low temperature hydrogen production during experimental hydration of partially-serpentinized dunite[END_REF] and has been suggested as an important substrate for microbial metabolism in these settings [START_REF] Lang | Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field[END_REF], including for methanogenesis [START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF].

Rather than calculate ∆G r 's of the above reactions for each individual groundwater chemical analysis, we investigate a range of generalized cases to highlight the most important factors controlling ∆G r 's and to assess energetic states of the system that lay beyond our analytical limits. For instance, CO 2 was below the limit of quantitation for the majority of the Ca 2+ -OH -groundwaters sampled in 2018 (< 12 µmol • L -1 ;

Table 3). H 2 was also below the limit of quantitation for several Ca 2+ -OH -and Mg 2+ -HCO - 3 groundwaters (< 0.048 nmol • L -1 in 2017 and < 0.598 nmol • L -1 in 2018; Table 4). Further, formate and acetate were not measured explicitly for this study, but were measured on groundwaters from the studied wells sampled in 2015 [START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF].

Thus, while robust constraints on the above parameters are available for the study area, complete sets of these parameters were generally not directly or simultaneously measured.

In light of this, we considered a representative Mg 2+ -HCO - 3 groundwater and a representative Ca 2+ -OH -groundwater, made informed assumptions when direct con- 2); A, acetoclastic (Equation 3); F, formatotrophic methanogenesis (Equation 4). 1 and3). Since measured c CO2 was below the limit of quantitation in the water sample from NSHQ14, 8 µmol • kg -1 was taken as the c CO2 of the representative Ca 2+ -OH -water, corresponding to the value at chrysotile-brucitediopside-calcite equilibrium at 25 • C and 1 bar obtained from water-harzburgite reaction path modeling [START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. Concentrations of formate and acetate were both assumed to be 1 µmol•kg -1 , which is consistent with their concentrations in earlier samples from wells in Samail Ophiolite [START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF]. Concentrations of CH 4 were assumed to be 100 µmol • kg -1 and 0.1 µmol • kg -1 for the representative Ca 2+ -OH - and Mg 2+ -HCO - 3 waters, respectively, reflecting typical concentrations for these fluids (Table 4, Figure 2). H 2 concentrations vary widely between and within fluid types (Table 4, Figure 2), so calculations were performed for multiple H 2 concentrations (1 mmol•kg -1 , 1 µmol•kg -1 , and 1 nmol•kg -1 ) encompassing the range of concentrations observed in Ca 2+ -OH -fluids. The 1 mmol•kg -1 H 2 case was omitted for the Mg 2+ -HCO - 3 fluid, where such high H 2 concentrations are not observed. The log activities (a) of all relevant species are tabulated in Table 5.

manuscript submitted to JGR: Biogeosciences Table 4. Aqueous gas concentrations, reported in µmol • L -1 . Well Sample year H CO CH C H C H i -C H n-C H i -C H n-C H C H a WAB103 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 1.45 • 10 -1 < 9.88 • 10 -4 < 7.60 • 10 -4 < 4.61 • 10 -4 6.05 • 10 -3 < 3.43 • 10 -4 8.73 • 10 -4 < 2.81 • 10 -4 WAB188 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 9.17 • 10 -1 < 9.88 • 10 -4 < 7.60 • 10 -4 < 4.61 • 10 -4 < 5.78 • 10 -4 < 3.43 • 10 -4 < 3.81 • 10 -4 < 2.81 • 10 -4 2017 9.92 • 10 -1 < 2.79 • 10 -1 1.83 < 1.01 • 10 -3 < 7.79 • 10 -4 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 WAB104 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 < 1.53 • 10 -2 < 9.88 • 10 -4 < 7.60 • 10 -4 4.82 • 10 -4 < 5.78 • 10 -4 7.56 • 10 -4 < 3.81 • 10 -4 < 2.81 • 10 -4 2017 < 4.80 • 10 -2 < 2.79 • 10 -1 2.30 • 10 -2 < 1.01 • 10 -3 < 7.79 • 10 -4 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 WAB105 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 < 1.53 • 10 -2 < 9.88 • 10 -4 < 7.60 • 10 -4 3.70 • 10 -2 < 5.78 • 10 -4 < 3.43 • 10 -4 < 3.81 • 10 -4 < 2.81 • 10 -4 2017 < 4.80 • 10 -2 < 2.79 • 10 -1 2.01 • 10 -2 < 1.01 • 10 -3 < 7.79 • 10 -4 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 WAB55 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 1.15 • 10 -1 1.55 • 10 -3 < 7.60 • 10 -4 2.25 • 10 -3 7.91 • 10 -4 1.60 • 10 -3 < 3.81 • 10 -4 5.52 • 10 -3 2017 < 4.80 • 10 -2 < 2.79 • 10 -1 1.06 • 10 -1 < 1.01 • 10 -3 < 7.79 • 10 -4 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 WAB56 2017 2.40 • 10 -1 < 2.79 • 10 -1 1.60 • 10 -1 < 1.01 • 10 -3 < 7.79 • 10 -4 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 NSHQ04 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 1.44 • 10 2 2.45 • 10 -2 2.22 • 10 -3 < 4.61 • 10 -4 < 5.78 • 10 -4 < 3.43 • 10 -4 < 3.81 • 10 -4 < 2.81 • 10 -4 2017 < 4.80 • 10 -2 < 2.79 • 10 -1 4.83 • 10 2 < 1.01 • 10 -3 b 1.03 • 10 -3 < 4.72 • 10 -4 < 6.01 • 10 -4 < 3.50 • 10 -4 < 3.91 • 10 -4 < 2.88 • 10 -4 WAB71 2018 < 5.98 • 10 -1 < 1.32 • 10 -1 7.76 1.00 • 10 -3 < 7.60 • 10 -4 < 4.61 • 10 -4 < 5.78 • 10 -4 < 3.43 • 10 -4 < 3.81 • 10 -4 < 2.81 • 10 -4 2017 5.92 • 10 -1 < 2.79 • 10 -1 1.48 • 10 1 < 1.01 • 10 -3 < 7.
The calculated ∆G r 's (Table 5) indicate that all of the CH 4 -forming reactions considered here can have sufficient chemical potential to sustain microbial life in certain states of the system. That is, ∆G r > ∆G min , where ∆G min (also known as the Biological Energy Quantum) is the minimum free energy that must be available to sustain life in a given environment (thought to be around -9 kJ•mol -1 to -20 kJ•mol -1 ; [START_REF] Schink | Energetics of syntrophic cooperation in methanogenic degradation[END_REF][START_REF] Hoehler | Biological energy requirements as quantitative boundary conditions for life in the subsurface[END_REF][START_REF] Schink | Syntrophism among Prokaryotes [Article[END_REF]). Acetoclastic methanogenesis had the most negative ∆G r in all conditions tested. formatotrophic methanogenesis had more negative ∆G r than hydrogenotrophic methanogenesis in all Ca 2+ -OH -conditions tested, but formatotrophic methanogenesis had less negative ∆G r than hydrogenotrophic methanogenesis in the Mg 2+ -HCO - 3 case at 1 µmol•kg -1 H 2 . Hydrogenotrophic methanogenesis had sufficient chemical potential to sustain microbial life only when a H2 was high enough, with the threshold a H2 being higher in Ca 2+ -OH -waters, where a CO2(aq) is lower. These calculations are generally consistent with those of [START_REF] Canovas | Geochemical bioenergetics during low-temperature serpentinization: An example from the Samail ophiolite, Sultanate of Oman[END_REF], who found that hydrogenotrophic methanogenesis had modest potential energy yields in waters from surface seeps in the Samail Ophiolite at pH ranging from 8 to 12.

Several additional factors should be considered when interpreting the ∆G r results.

First, reactions proceeding in environmental systems are often drawn towards equilibrium, and thus a large negative ∆G r of a given reaction may indicate that that reaction is not actively occuring, but only has the potential to occur. Second, substrate transport into the cell is not addressed in our calculations. A more complete model would account for rates of CO 2 diffusion across the cell membrane and/or energy expended to transport charged species such as formate and acetate into the cell [START_REF] Hoehler | Biological energy requirements as quantitative boundary conditions for life in the subsurface[END_REF]. Third, mixing is not explicitly accounted for in our calculations. Mixing has been suggested as a key factor controlling energetic favorability of various reactions in the Samail Ophiolite. This is especially pertinent to hydrogenotrophic methanogenesis because c CO2 is so much lower in endmember hyperalkaline fluids than in near-surface, atmosphere-influenced fluids [START_REF] Canovas | Geochemical bioenergetics during low-temperature serpentinization: An example from the Samail ophiolite, Sultanate of Oman[END_REF][START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. The c CO2 used for the example Ca 2+ -OH -fluid in our calculations is representative of a minimum value for the system [START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. Mixing would tend to inject CO 2 into the fluids and increase the energetic favorability of hydrogenotrophic methanogenesis.

In . This notion is generally supported by our calculations in that formatotrophic methanogenesis had more negative ∆G r than hydrogenotrophic methanogenesis in all Ca 2+ -OH -conditions tested, whereas the reverse was true for the Mg 2+ -HCO -

3 case at 1 µmol • kg -1 H 2 .
Remarkably, although acetoclastic methanogenesis had the most negative ∆G r of the investigated CH 4 -forming reactions (Table 5), it has the least microbiological evidence of being a major methanogenic pathway in the Samail Ophiolite. Conversion of isotopically labeled acetate ( 13 CH 3 OO -) to 13 CH 4 , has, however, been documented in cultures from serpentinite springs in the Voltri Massif, Italy [START_REF] Brazelton | Metagenomic identification of active methanogens and methanotrophs in serpentinite springs of the Voltri Massif, Italy[END_REF], indicating that acetoclastic methanogenesis can operate in some serpentinizing settings.

In the aquifers sampled via wells in the Samail Ophiolite, methanogens may be out-competed for acquisition of acetate by other groups of microbes, such as sulfate reducers. Indeed, geochemical evidence of microbial acetate oxidation coupled to sulfate reduction has been reported in alkaline, H 2 -rich, crystalline rock aquifers inhabited by microbial communities dominated by sulfate reducing bacteria and methanogens [START_REF] Moser | Desulfotomaculum and Methanobacterium spp. Dominate a 4-to 5-Kilometer-Deep Fault[END_REF]. 4; Figure 2). NSHQ14 waters also had high c CH4 (106 µmol • L -1 and 71.2 µmol • L -1 in 2017 and 2018, respectively).

4
CH 4 has ranged in δ 13 C from -6.89 VPDB to +3.7 VPDB in fluid samples from NSHQ14, with a mean weighted by sample year of -0.8 VPDB (Figure 3a; Table 2). These δ 13 C values are generally higher than those of CH 4 emanating from sedimentpoor seafloor hydrothermal vents, where a dominantly abiotic origin has been proposed

( [START_REF] Welhan | Methane, Hydrogen and Helium in Hydrothermal Fluids at 21 • N on the East Pacific Rise[END_REF][START_REF] Merlivat | Hydrothermal vent waters at 13 • N on the East Pacific Rise: isotopic composition and gas concentration[END_REF][START_REF] Charlou | Mineral and gas chemistry of hydrothermal fluids on an ultrafast spreading ridge: East Pacific Rise, 17 • to 19 • S (Naudur cruise, 1993) phase separation processes controlled by volcanic and tectonic activity[END_REF][START_REF] Charlou | Compared geochemical signatures and the evolution of Menez Gwen (37 • 50 N) and Lucky Strike[END_REF][START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 • 14 N, MAR)[END_REF][START_REF] Proskurowski | Abiogenic hydrocarbon production at Lost City hydrothermal field[END_REF][START_REF] Kumagai | Geological background of the Kairei and Edmond hydrothermal fields along the Central Indian Ridge : Implications for the distinct chemistry between their vent fluids[END_REF][START_REF] Mcdermott | Pathways for abiotic organic synthesis at submarine hydrothermal fields[END_REF]D. T. Wang et al., 2018); represented by Mid-Cayman Rise, Lost City, and Ashadze II in Figure 3a), higher than typical mantle values [START_REF] Deines | The carbon isotope geochemistry of mantle xenoliths[END_REF], and similar to marine carbonate [START_REF] Schidlowski | Carbon isotopes as biogeochemical recorders of life over 3.8 Ga of Earth history: evolution of a concept[END_REF]. CH 4 δ 13 C at NSHQ14 is generally higher than δ 13 C of carbonate veins in NSHQ14 (-7.05 VPDB to -4.69 VPDB; (Miller et al., 2016)), which is opposite to that which would be expected at equilibrium [START_REF] Bottinga | Calculated fractionation factors for carbon and hydrogen isotope exchange in the system calcite-carbon dioxide-graphite-methanehydrogen-water vapor[END_REF], indicating that CH 4 is not in isotopic equilibrium with co-existing carbonate minerals.

manuscript submitted to JGR: Biogeosciences δ 13 CCH 4 . Shaded fields of typical gas origin after [START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of ¿20,000 samples[END_REF]. Abbreviations: PM, primary microbial; SM, secondary microbial; T, thermogenic; A, abiotic. (c) Plot of ratio of methane (C1) to the sum of ethane (C2) and propane (C3) vs. δ 13 CCH 4 . Only analyses for which C2 was above limit of quantitation are plotted. If C3 was below limit of quantitation, its contribution to C1/ (C2 + C3) was assumed to be negligible, and therefore C1/C2 is plotted. error for analyses performed at UCLA. Contextual data from ophiolites: Oman/UAE (Fritz et al., 1992;Etiope et al., 2015;[START_REF] Boulart | Differences in gas venting from ultramafic-hosted warm springs: the example of Oman and Voltri ophiolites[END_REF]Miller et al., 2016;[START_REF] Vacquand | Reduced gas seepages in ophiolitic complexes: evidences for multiple origins of the H 2 -CH 4 -N 2 gas mixtures[END_REF], the Philippines [START_REF] Abrajano | Geochemistry of reduced gas related to serpentinization of the Zambales ophiolite[END_REF][START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF] CH 4 is accompanied by C 2 -C 6 alkanes in fluids from NSHQ14 (Table 4). These alkanes had C 1 / (C 2 + C 3 ) ratios of 1240 in 2017 and 881 in 2018, which are similar to fluid samples and rock crushings from other ophiolites and sediment-poor seafloor hydrothermal vents [START_REF] Abrajano | Geochemistry of reduced gas related to serpentinization of the Zambales ophiolite[END_REF][START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF][START_REF] Mcdermott | Pathways for abiotic organic synthesis at submarine hydrothermal fields[END_REF][START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF]), but 10 2 times higher than those of Kidd Creek mine, Canada, for which a low-temperature, abiotic origin of alkanes has been proposed [START_REF] Sherwood Lollar | Abiogenic formation of alkanes in the Earth's crust as a minor source for global hydrocarbon reservoirs[END_REF], 2008;[START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF] (Figure 3c). Thus, C 1 / (C 2 + C 3 ) ratios could reflect differences in alkane formation mechanisms or extents of reaction in Precambrian shield sites like Kidd Creek versus ophiolites and sediment-poor seafloor hydrothermal vents.

C 2 H 6 and C 3 H 8 at NSHQ14 are strongly 13 C-enriched (δ 13 C of -6.0 VPDB and +3.3 VPDB, respectively; Table 2; Figure 4). The observed δ 13 C values are ∼ 15 higher than those in the most mature (and therefore most 13 C-enriched) thermogenic C 2 H 6 and C 3 H 8 samples from confined systems [START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of ¿20,000 samples[END_REF][START_REF] Fiebig | Abiogenesis not required to explain the origin of volcanichydrothermal hydrocarbons[END_REF]. Increases in δ 13 C C3 of ∼ 15 have been attributed to microbial oxidation of shortchain alkanes, which enriches the residual in 13 C [START_REF] Martini | Microbial production and modification of gases in sedimentary basins: A geochemical case study from a Devonian shale gas play, Michigan basin[END_REF]. However, shortchain alkane oxidizing microbial species [START_REF] Shennan | Utilisation of C2-C4 gaseous hydrocarbons and isoprene by microorganisms[END_REF][START_REF] Singh | Anaerobic Oxidation of Ethane, Propane, and Butane by Marine Microbes: A Mini Review[END_REF][START_REF] Laso-Pérez | Anaerobic Degradation of Non-Methane Alkanes by "Candidatus Methanoliparia" in Hydrocarbon Seeps of the Gulf of Mexico[END_REF] were not detected in 16S rRNA gene sequences of DNA obtained from NSHQ14.

Thus, there is not strong evidence to suggest that δ 13 C C2 and δ 13 C C3 at NSHQ14 re-manuscript submitted to JGR: Biogeosciences sult from post-genetic microbial alteration. Rather, δ 13 C C2 and δ 13 C C3 should reflect formation conditions and C source(s).

C 2 H 6 and C 3 H 8 at NSHQ14 are not likely to derive from nearby organic matter.

Hydrocarbon-rich sedimentary formations in northern Oman not only lack a clear structural connection to the ophiolite aquifer, but also yield oils with δ 13 C values [START_REF] Terken | The Natih Petroleum System of North Oman[END_REF] at least 20 lower than those of C 2 H 6 and C 3 H 8 at NSHQ14. Furthermore, total organic C in peridotites exposed to alteration at the seafloor, a proxy for organic C endogenous to the Samail Ophiolite, is also relatively 13 C-depleted (approximately -25±

5 VPDB; [START_REF] Alt | The role of serpentinites in cycling of carbon and sulfur: Seafloor serpentinization and subduction metamorphism[END_REF][START_REF] Alt | Recycling of water, carbon, and sulfur during subduction of serpentinites: A stable isotope study of Cerro del Almirez, Spain[END_REF]Alt, Shanks, et al., 2012;[START_REF] Delacour | Carbon geochemistry of serpentinites in the Lost City Hydrothermal System (30 • N, MAR)[END_REF]). Closed-system thermal cracking of these organic matter sources is unlikely to have produced the comparatively 13 C-enriched C 2 H 6 and C 3 H 8 at NSHQ14

and previously reported elsewhere in the ophiolite (Figure 4; [START_REF] Fritz | Deuterium and 13 C evidence for low temperature production of hydrogen and methane in a highly alkaline groundwater environment in Oman[END_REF]).

Thermal cracking of organic matter and open-system degassing can enrich late-produced short-chain alkanes in 13 C due to kinetic isotope effects associated with the cleavage of precursor sites in the parent organic matter and the resultant Rayleigh distillation of these sites [START_REF] Rooney | Modeling thermogenic gas generation using carbon isotope ratios of natural gas hydrocarbons[END_REF][START_REF] Fiebig | Abiogenesis not required to explain the origin of volcanichydrothermal hydrocarbons[END_REF]. Thermogenic gas production can proceed slowly at temperatures as low as 60 • C, but substantial thermogenic gas production typically occurs at reservoir temperatures above 120 • C [START_REF] Burnham | A simple kinetic model of petroleum formation and cracking[END_REF][START_REF] Hunt | Catalytic production of methane from CO 2 and H 2 at low temperature: Insight on the reaction mechanism[END_REF][START_REF] Stolper | The utility of methane clumped isotopes to constrain the origins of methane in natural gas accumulations[END_REF][START_REF] Cumming | Potential sources of dissolved methane at the Tablelands, Gros Morne National Park, NL, CAN: A terrestrial site of serpentinization[END_REF][START_REF] Fiebig | Abiogenesis not required to explain the origin of volcanichydrothermal hydrocarbons[END_REF]. These temperatures are higher than temperatures along groundwater flow paths intersecting the wells in this study. Measured groundwater temperatures in the study area are ∼ 35 [START_REF] Kelemen | Rates and mechanisms of mineral carbonation in peridotite: natural processes and recipes for enhanced, in situ CO 2 capture and storage[END_REF][START_REF] Mervine | Carbonation rates of peridotite in the Samail Ophiolite, Sultanate of Oman, constrained through 14 C dating and stable isotopes[END_REF]Miller et al., 2016). Geothermal gradients derived from geophysical logs of NSHQ14 are 25 [START_REF] Paukert | Mineral Carbonation in Mantle Peridotite of the Samail Ophiolite, Oman: Implications for permanent geological carbon dioxide capture and storage[END_REF][START_REF] Matter | Oman Drilling Project Phase I Borehole Geophysical Survey[END_REF], which is typical of near-surface, continental settings (Lowell et al., 2014). At the low temperatures and ordinary geothermal gradients within the active alteration zone of the Samail Ophiolite, thermal cracking of organic matter is unlikely to proceed at sufficient rates to attain the high extents of reaction progress necessary to explain the observed 13 C enrichments in short-chain alkanes at NSHQ14 over relevant timescales.

• C• km -1 (A.
Alternatively, short-chain alkanes in NSHQ14 fluids may have an abiotic source.

Several studies have demonstrated storage of large quantities of CH 4 and associated shortchain alkanes in fluid inclusions in ophiolites [START_REF] Sachan | Preservation of methane generated during serpentinization of upper mantle rocks: Evidence from fluid inclusions in the Nidar ophiolite, Indus Suture Zone, Ladakh (India)[END_REF][START_REF] Klein | Abiotic methane synthesis and serpentinization in olivine-hosted fluid inclusions[END_REF][START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF]. However, the findings of these studies disagree with those of Etiope et al. 2). This CH 4 is D-enriched with respect to coexisting H 2 (δD H2 = -685 VSMOW; (Miller et al., 2016)) and D-depleted with respect to coexisting water (δD H2O = +0.2 VSMOW in 2018; Table 3). Although H 2 and water reflect H isotopic equilibrium at ∼ 50 • C (Miller et al., 2016), both H 2 and water are in H isotopic disequilibrium with CH 4 (Figure 3b). type anaerobic methane oxidation is suggested to be a highly reversible metabolic pathway [START_REF] Knittel | Anaerobic Oxidation of Methane: Progress with an Unknown Process[END_REF][START_REF] Timmers | Reverse methanogenesis and respiration in methanotrophic archaea[END_REF]. This reversibility has been proposed

to bring ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 towards equilibrium at low temperatures (70 [START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF][START_REF] Sherwood Lollar | Abiogenic formation of alkanes in the Earth's crust as a minor source for global hydrocarbon reservoirs[END_REF], 2008)) and laboratory experiments with synthetic Ru catalysts (E. [START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF][START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF].

To independently assess the potential influences of microbial processes on CH 4 concentration and isotopic composition, DNA was extracted from biomass in pumped groundwaters and subjected to amplification and sequencing of 16S rRNA genes. 16S rRNA gene sequences of biomass collected in 2018 were searched for matches to known CH 4 -cycling taxa, as compiled previously by [START_REF] Crespo-Medina | Methane Dynamics in a Tropical Serpentinizing Environment: The Santa Elena Ophiolite, Costa Rica[END_REF]. Sequences closely affiliated with both methanogenic and methanotrophic taxa were found to be widespread in the aquifer (Figure 5). Based on phylogenetic inference, the dominant methanogenic taxon was related to the genus Methanobacterium, whose members can produce CH 4 from H 2 and CO 2 , CO, or formate [START_REF] Balch | Methanogens: reevaluation of a unique biological group[END_REF]. Methanobacterium comprised a high proportion (24 %) of 16S rRNA gene sequences at NSHQ14 in 2018. Relative abundances of Methanobacterium 16S rRNA gene reads were similarly high in 2017 (12 %) and 2016 (28 %), but lower (< 1 %) in 2015 and 2014 (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]. The increase in the relative abundance of 16S rRNA genes affiliated with Methanobacterium in samples collected in 2016 and onwards versus those collected in 2014 and 2015 coincided with a change in sampling methods from smaller, lower-flow pumps (maximum depth 20 m) prior to 2016 to larger, higher-flow pumps (maximum depth 90 m). The obligate anaerobic nature of this methanogen genus [START_REF] Boone | Methanobacterium. In Bergey's manual of systematics of archaea and bacteria (p. 1-8)[END_REF] is consistent with its higher relative gene abundances in fluids sampled from greater depths, which presumably receive less input of atmospheric O 2 than do shallower fluids. reported as percentages rounded to the ones place. Cases when a taxon was detected in a sample and was < 1 % read relative abundance after rounding are labeled "< 1". Cases when no reads of a taxon were detected in a sample are labeled "n.r." Data shown are from unique field samples.

Previous 16S rRNA gene sequencing studies that obtained field samples in triplicate from Samail

Ophiolite groundwaters through similar methods to those used here have found typical standard deviations of relative abundances less than or equal to 25 % of the mean relative abundance [START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF].

Consortia capable of anaerobic oxidation of CH 4 coupled to SO 2- 4 reduction, including ANME, were not detected by 16S rRNA gene sequencing of samples obtained from NSHQ14 in 2018 (Figure 5), 2016, or 2014 (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF], although sequences affiliated with order ANME-1b were detected in low abundance (< 1 % of reads) in samples obtained from NSHQ14 in 2017 and 2015 [START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]. This scarcity of ANME may result from metabolic inhibition by high c H2 in groundwaters at NSHQ14 and elsewhere in the Samail Ophiolite. It has been proposed that the thermodynamics of "reverse methanogenesis" require low c H2 (e.g. ≤ 1 nM in a marine cold seep environment [START_REF] Boetius | A marine microbial consortium apparently mediating anaerobic oxidation of methane[END_REF]). Indeed, the bioenergetics of SO 2- 4 -driven oxidation of CH 4 are less favorable than SO 2- 4 -driven oxidation of H 2 or non-CH 4 organics, or other metabolisms such as methanogenesis or acetogenesis in the Samail Ophiolite [START_REF] Canovas | Geochemical bioenergetics during low-temperature serpentinization: An example from the Samail ophiolite, Sultanate of Oman[END_REF] and in deep continental settings where radiolytic H 2 accumulates [START_REF] Kieft | Geochemically Generated, Energy-Rich Substrates and Indigenous Microorganisms in Deep, Ancient Groundwater[END_REF][START_REF] Moser | Desulfotomaculum and Methanobacterium spp. Dominate a 4-to 5-Kilometer-Deep Fault[END_REF][START_REF] Kieft | Microbiology of the Deep Continental Biosphere[END_REF].

While 16S rRNA gene sequences affiliated with anaerobic CH 4 oxidizing microbes have only occasionally been detected at NSHQ14, 16S rRNA gene sequences affiliated with the genus Methylococcus, which contains aerobic methanotrophs [START_REF] Hanson | Methanotrophic bacteria[END_REF], have been detected in all samples from NSHQ14, ranging from 1 % to < 1 % of reads in samples obtained from 2014 to 2018 (Figure 5; (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]). Since the aerobic lifestyle of Methylococcus is at odds with that of the obligate anaerobe, Methanobacterium, it seems most likely that these two taxa are spatially separated in the aquifer, and that waters containing each of them

were mixed during open borehole pumping. Still, the > 10 times higher abundances of Methanobacterium-related 16S rRNA genes relative to those of Methylococcus at NSHQ14 in samples from 2016 to 2018 suggest that the microbial CH 4 cycle at this well is dominated by CH 4 production, rather than consumption.

16S rRNA gene sequencing of subsurface biomass from NSHQ14 is complemented by other observations that suggest that methanogens are not only prevalent, but active.

Genes involved in methanogenesis are enriched [START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF] and actively transcribed in waters sampled from NSHQ14 [START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]. Transformation of both 14 C-labeled HCO - 3 and 14 C-labeled formate to CH 4 have been shown to occur in water samples from NSHQ14 at significantly higher rates than in killed controls, with formatotrophic methanogenesis greatly outpacing hydrogenotrophic methanogenesis [START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF]. Taken together with a cell abundance of 1.15 • 10 5 cells • mL -1 in groundwater at NSHQ14 [START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF], these data suggest that aquifer regions accessed by NSHQ14 host abundant active methanogenic cells (thousands per mL, assuming ∼ 24 % of cells are methanogens based on 16S rRNA gene data). These active cells could influence CH 4 concentration and isotopic composition.

The genomic and cultivation data of [START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF] [START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. In addition to direct uptake of CO 2 , carbonate minerals may serve as a C source for methanogenesis in carbonated peridotites [START_REF] Miller | Large carbon isotope variability during methanogenesis under alkaline conditions[END_REF]. Another potential C source is carbon monoxide (CO). CO has always been below limits of quantitation in Oman wells (< 132 nmol • L -1 in 2018; Table 4), but it is unclear whether this indicates minimal CO production or rapid CO turnover.

The microbiological data from NSHQ14 fluids are compatible with δD CH4 , ∆ 13 CH 3 D, and ∆ 12 CH 2 D 2 values that collectively indicate a substantial addition of microbial CH 4 to an otherwise abiotic pool of CH 4 . Although the data presented here do not enable us to precisely determine the mole fractions and isotopic compositions of the microbial and abiotic components of CH 4 at NSHQ14, the δD CH4 data alone suggest that perhaps the majority of CH 4 at NSHQ14 formed through non-equilibrium processes, which include microbial methanogenesis. Thus, the high δ 13 C of CH 4 at NSHQ14 suggests that the microbial component is more 13 C-enriched than microbial CH 4 formed in sedimentary environments, which typically ranges from -90 VPDB to -50 VPDB ( [START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of ¿20,000 samples[END_REF]; Figure 3a). In cultures of a hydrogenotorophic strain of Methanobacterium provided CaCO 3 (s) as a C source at pH ∼ 9, [START_REF] Miller | Large carbon isotope variability during methanogenesis under alkaline conditions[END_REF] observed suppressed apparent isotope effects during methanogenesis (α CO2/CH4 = 1.028). The authors attributed this to the slow kinetics of carbonate dissolution at high pH and the neartotal conversion of the resultant CO 2 (aq) to CH 4 by Methanobacterium. If the primary mode of methanogenesis at NSHQ14 is in fact formatotrophic methanogenesis and abiotic formate production is the rate-limiting step in the overall process through which CO 2

is converted to CH 4 , similar isotopic bottlenecks could apply. Cellular formate uptake and enzymatic conversion processes whose isotope effects remain unknown could be important drivers of the isotopic composition of CH 4 in hyperalkaline, serpentinizing set-tings. In such settings, the suppression of C isotope fractionation during methanogenesis is supported by observations of high δ 13 C values (up to +14 VPDB) of lipid biomarkers thought to be produced by methanogens at Chimaera, Turkey (Zwicker et al., 2018) and at Lost City (Bradley et al., 2009). Evaluation of these hypotheses will require further physiological studies of methanogens aimed at understanding substrate selection and limitation systematics in hyperalkaline, low-C conditions and the isotopic implications of these factors.

While the data support substantial microbial CH 4 and abiotic, fluid inclusion-derived CH 4 in NSHQ14 fluids, we find less evidence for abiotic CH 4 production at the low temperatures that pervade the modern weathering horizon in the ophiolite. Below 100 • C, access of gas-phase H 2 and CO 2 or CO to the catalytic metals Ru or Rh is required for CH 4 to form at appreciable rates [START_REF] Thampi | Methanation and photomethanation of carbon dioxide at room temperature and atmospheric pressure[END_REF][START_REF] Hunt | Catalytic production of methane from CO 2 and H 2 at low temperature: Insight on the reaction mechanism[END_REF][START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF][START_REF] Mccollom | Abiotic methane formation during experimental serpentinization of olivine[END_REF]. It has been proposed that the spatial concentration of potentially-catalytic Ru-rich chromites in chromitites is important for catalysis of lowtemperature CO 2 reduction to CH 4 in ophiolites [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF][START_REF] Etiope | Widespread abiotic methane in chromitites[END_REF]. While peridotites in Oman ubiquitously contain a few percent distributed chromite [START_REF] Hanghøj | Composition and Genesis of Depleted Mantle Peridotites from the Wadi Tayin Massif, Oman Ophiolite; Major and Trace Element Geochemistry, and Os Isotope and PGE Systematics[END_REF], massive chromitites were not reported in lithologic descriptions of cores or drill cuttings from NSHQ14 or any of the six additional wells ranging from 300 m to 400 m depth that have been drilled in the same catchment by the Oman Drilling Project (P. [START_REF] Kelemen | & the Oman Drilling Project Science Team[END_REF]. Nor are chromitites notably abundant in outcrop within this catchment. Further, although some flow paths of meteoric water through the ophiolite may result in saturation in H 2 and separation of a free gas phase [START_REF] Canovas | Geochemical bioenergetics during low-temperature serpentinization: An example from the Samail ophiolite, Sultanate of Oman[END_REF], the depth to water is < 20 m in all wells in the catchment of NSHQ14, suggesting water-saturated conditions in the subsurface. Moreover, if free H 2 (g) were generated at high extents of reaction progress, co-existing CO 2 (g) would be extremely scarce due to precipitation of carbonate minerals and high pH [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF][START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF]. It has been proposed that CH 4 in ophiolites can form through reduction of CO 2 (g) from non-atmospheric sources such as magma, the mantle, or sedimentary carbonate formations [START_REF] Etiope | Low-temperature catalytic CO 2 hydrogenation with geological quantities of ruthenium: a possible abiotic CH 4 source in chromitite-rich serpentinized rocks[END_REF]. A magmatic/mantle CO 2 source

is not supported at NSHQ14 because excess He above air saturation in groundwaters from this well has a dominantly radiogenic isotopic composition that is distinct from mantlederived He [START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF]. Further, although sedimentary carbonates are present in the vicinity of NSHQ14 and elsewhere in the ophiolite [START_REF] Boudier | Cross section through the peridotite in the Samail Ophiolite, southeastern Oman Mountains[END_REF][START_REF] De Obeso | Fluid rock interactions on residual mantle peridotites overlain by shallow oceanic limestones: Insights from Wadi Fins, Sultanate of Oman[END_REF], there is no clear mechanism to liberate CO 2 (g) from mineral carbonates and transfer that CO 2 (g) to catalytic sites of reaction on chromites where H 2 (g) is also present. Thus, the apparent lack of massive chromites and free gaseous potential reactants suggest that the subsurface surrounding NSHQ14 is not conducive to low-temperature abiotic CH 4 production. While substantial low-temperature CH 4 production in the catchment of NSHQ14 seems unlikely, NSHQ14 groundwaters could be mere carriers of CH 4 that was produced elsewhere in the ophiolite under gaseous conditions and that has subsequently migrated into the aquifer. Some studies of CH 4 origin in other peridotite bodies have favored such a hypothesis [START_REF] Etiope | Abiotic methane seepage in the Ronda peridotite massif, southern Spain[END_REF][START_REF] Marques | Linking serpentinization, hyperalkaline mineral waters and abiotic methane production in continental peridotites: an integrated hydrogeological-bio-geochemical model from the Cabeço de Vide CH 4 -rich aquifer (Portugal)[END_REF]. However, it is not clear how this hypothesis could be tested in the case of the NSHQ14, nor how it addresses the issue of CO 2 source.

In summary, isotopic and microbiological data lead us to conclude that the high concentrations of CH 4 (10 2 µmol•L -1 ) in groundwaters accessed by NSHQ14 primarily result from microbial methanogenesis and the release of abiotic CH 4 from fluid inclusions. 1). As of 2017, the well is obstructed at 8 m below the casing top, precluding deeper sampling (Section 3.1; Table 1).

Primary differences in fluid composition between NSHQ04 and NSHQ14 include lower pH by ∼ 1 and higher c Ca and c Si at NSHQ04 (Tables 1 and3; (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF]). These differences could be related to the scarcity of fresh, near-surface olivine at NSHQ04, which may result in a greater influence of pyroxene serpentinization at NSHQ04 (Miller et al., 2016). Low-temperature pyroxene serpentinization generally continues after olivine is exhausted, and leads to higher c Si and, depending on pyroxene chemical composition, can also lead to higher c Ca and lower pH [START_REF] Bach | Unraveling the sequence of serpentinization reactions: petrography, mineral chemistry, and petrophysics of serpentinites from MAR 15 • N (ODP Leg 209, Site 1274)[END_REF][START_REF] Leong | Thermodynamic constraints on the geochemistry of low-temperature, continental, serpentinization-generated fluids[END_REF].

The relatively low pH and high c Si could also stem from mixing of Ca 2+ -OH -waters with gabbro-or atmosphere-influenced fluids.

Compared to NSHQ14, NSHQ04 has generally had lower c H2 (detected in 2014, but not in 2018, 2017, 2015[START_REF] Collier | Spatial-Statistical Properties of Geochemical Variability as Constraints on Magma Transport and Evolution Processes at Ocean Ridges[END_REF]Table 4; Figure 2; (Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Paukert Vankeuren | Multitracer determination of apparent groundwater ages in peridotite aquifers within the Samail ophiolite, Sultanate of Oman[END_REF]). The relatively low c H2 measured in waters pumped from NSHQ04 is probably due at least in part to microbial H 2 oxidation. Although there are multiple enzymes with which which a diversity of microbes oxidize H 2 [START_REF] Peters | FeFe]-and [NiFe]-hydrogenase diversity, mechanism, and maturation[END_REF], aerobic H 2 oxidation by bacteria of the genus Hydrogenophaga has been identified as a particularly prevalent process in serpentinizing settings, including the Samail Ophiolite [START_REF] Suzuki | Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site[END_REF][START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Marques | Linking serpentinization, hyperalkaline mineral waters and abiotic methane production in continental peridotites: an integrated hydrogeological-bio-geochemical model from the Cabeço de Vide CH 4 -rich aquifer (Portugal)[END_REF]. Sequences affiliated with Hydrogenophaga accounted for 20 % of 16S rRNA gene reads in DNA extracted from biomass in waters pumped from NSHQ04 in 2018, which is similar to previous years of sampling at NSHQ04 (6 % to 18 % in 2014, 2015, and 2017; inter-annual mean of 12 %) and higher than all other studied wells (Supporting Information Figure S3; [START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF]Miller et al., 2016;[START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]). 3c), further supporting a major component of microbial CH 4 at NSHQ04.

Microbial CH 4 production at NSHQ04 is also indicated by microbiological data.

16S rRNA gene sequences affiliated with Methanobacterium have been detected in DNA extracted from biomass filtered from waters pumped from NSHQ04, albeit in low relative abundance (< 1 % of reads in 2018; Figure 5; also detected in < 1 % of reads in 2014, but not detected in 2015 and2017;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF]Miller et al., 2016;[START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]). The apparent low relative abundance of Methanobacterium at NSHQ04 could have resulted from the relatively shallow depth from which samples were collected at NSHQ04 due to well obstruction and the consequential sampling of groundwaters that may have experienced atmospheric O 2 infiltration. High relative read abundances of sequences affiliated with aerobes and transient H 2 across years of sampling NSHQ04 suggest that zones of the aquifer that are not always anoxic were accessed. These conditions may restrict methanogen abundance to greater depths than were sampled, but not constrain the upward diffusion of the product of their metabolism, CH 4 . Nevertheless, fluids obtained from NSHQ04 have yielded robust cultures of Methanobacterium [START_REF] Miller | Large carbon isotope variability during methanogenesis under alkaline conditions[END_REF]. In addition, high relative abundances of 16S rRNA gene reads of DNA extracted from biomass in waters sampled from NSHQ04 were related to an aerobic methanotroph of the genus Methylococcus (8 % of reads in 2018; inter-annual mean of 11 %; Figure 5;(Miller et al., 2016;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF]). Greater aerobic methanotrophy at NSHQ04 relative to NSHQ14 may have contributed in part to the lower ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 and higher δ 13 C and δD of CH 4 sampled from NSHQ04.

Methanotrophic activity at NSHQ04 is consistent with the observed 13 C-depletion in CO 2 at NSHQ04 (-29.7 VPDB δ 13 C; Table 2) relative to the other studied wells because environments of active methanotrophy often have 13 C-depleted CO 2 [START_REF] Barker | Carbon isotope fractionation during microbial methane oxidation[END_REF][START_REF] Michaelis | Microbial Reefs in the Black Sea Fueled by Anaerobic Oxidation of Methane[END_REF]. Indeed, δ 13 C CO2 at NSHQ04 is compatible with aerobic oxidation of CH 4 of ∼ 0 VPDB δ 13 C [START_REF] Barker | Carbon isotope fractionation during microbial methane oxidation[END_REF][START_REF] Feisthauer | Different types of methane monooxygenases produce similar carbon and hydrogen isotope fractionation patterns during methane oxidation[END_REF]. Alternatively, 13 C-depletion in CO 2 could be explained by kinetic isotope fractionation during hydroxylation of atmospheric CO 2 upon contact with Ca 2+ -OH -water, which has been interpreted as the cause of δ 13 C as low as -27.21 VPDB in Carich carbonates from hyperalkaline seeps in the Samail Ophiolite [START_REF] Clark | Stable isotope disequilibria in travertine from high pH waters: Laboratory investigations and field observations from Oman[END_REF][START_REF] Kelemen | Rates and mechanisms of mineral carbonation in peridotite: natural processes and recipes for enhanced, in situ CO 2 capture and storage[END_REF][START_REF] Falk | Controls on the stable isotope compositions of travertine from hyperalkaline springs in Oman: Insights from clumped isotope measurements[END_REF]. Considering the relatively shallow sampling depth at NSHQ04 in 2018 ( [START_REF] Mccollom | Methanogenesis as a potential source of chemical energy for primary biomass production by autotrophic organisms in hydrothermal systems on Europa[END_REF][START_REF] Hoehler | Biological energy requirements as quantitative boundary conditions for life in the subsurface[END_REF], but may also be affected by inputs of fresh rainwater and/or H 2 -bearing Ca 2+ -OH -water flowing from the peridotite aquifer into the gabbro aquifer across a fault at depth. Flows of water from higher-head, lower-permeability peridotite aquifers into gabbro aquifers in the Samail Ophiolite have been proposed on the basis of physical hydrologic data [START_REF] Dewandel | A conceptual hydrogeological model of ophiolite hard-rock aquifers in Oman based on a multiscale and a multidisciplinary approach[END_REF] [START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF] and that genes involved in methanogenesis are transcribed in groundwater samples obtained from WAB188 [START_REF] Kraus | Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman[END_REF].

While subsurface fluids sampled at WAB188, NSHQ14, and NSHQ04 all bear evidence of methanogenic activity, the conditions under which methanogenesis proceeds at WAB188 are fundamentally distinct. In contrast to the Ca 2+ -OH -fluids from NSHQ14 and NSHQ04, the circumneutral fluids from WAB188 have ∼ 10 2 to ∼ 10 3 times higher c CO2 (inter-annual mean of 2910 µmol•L -1 , s = 620 µmol•L -1 , n = 3; Table 3) and ∼ 75 lower δ 13 C CH4 (inter-annual mean δ 13 C = -73 VPDB, s = 13 , n = 3; Table 2; Figure S5). Since WAB188 fluids contain relatively 13 C-depleted CH 4 that is not associated with substantial concentrations of C 2 -C 6 alkanes (Table 4), a standard interpretation [START_REF] Bernard | A geochemical model for characterization of hydrocarbon gas sources in marine sediments[END_REF][START_REF] Milkov | Revised genetic diagrams for natural gases based on a global dataset of ¿20,000 samples[END_REF] 5). Further, the apparent α CO2/CH4 at WAB188 (based on measured δ 13 C CO2 of -13.52 VPDB;

Table 3) is compatible with that of Methanobacterium cultures grown hydrogenotrophically with excess HCO - 3 (aq), which was greater than the α CO2/CH4 observed for parallel cultures under CO 2 -poor conditions [START_REF] Miller | Large carbon isotope variability during methanogenesis under alkaline conditions[END_REF]. In sum, the conditions at manuscript submitted to JGR: Biogeosciences WAB188 contrast starkly with those that prevail in Ca 2+ -OH -fluids, where C substrates for methanogenesis are often more scarce than H 2 . These differences may be reflected in the inverse relationship between c CO2 and δ 13 C CH4 across fluids from wells WAB188, NSHQ14, and NSHQ04 (Figure S5), which is consistent with an effect of C availability on the apparent C isotope effect of microbial methanogenesis. et al., 2017). This lends geochemical support to recent microbiological findings that independently indicate that the activity of formatotrophic methanogens increases relative manuscript submitted to JGR: Biogeosciences to hydrogenotrophic methanogens as groundwater pH increases in the ophiolite [START_REF] Fones | Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation[END_REF].

Conclusions

In addition, an abiotic, fluid inclusion-derived source of CH in the ophiolite, which is considered unusual for CH 4 with a substantial microbial component.

This study supports the premise that H 2 produced from water/rock reaction can fuel microbial life, even under challenging conditions of high pH and low oxidant availability. By identifying where and how microbial methanogenesis can reasonably be expected to occur in H 2 -rich, subsurface environments, this work complements theoretical models in guiding the search for rock-hosted life, including extraterrestrial life. For example, our findings substantiate predictions that microbial methanogenesis could occur in the reduced, alkaline ocean of Saturn's moon, Enceladus [START_REF] Mckay | The Possible Origin and Persistence of Life on Enceladus and Detection of Biomarkers in the Plume[END_REF][START_REF] Glein | The pH of Enceladus' ocean[END_REF][START_REF] Waite | Cassini finds molecular hydrogen in the Enceladus plume: Evidence for hydrothermal processes[END_REF] and in the Martian subsurface [START_REF] Kral | Potential use of highly insoluble carbonates as carbon sources by methanogens in the subsurface of Mars[END_REF].

Open Research

Data (in Excel format) and source code (in R Markdown format) used to produce the figures, data tables and analyses for this paper (as well as additional data on analytical uncertainties and trace element concentrations) are available online in D. B. Nothaft, Templeton, et al. (2021). Additional DNA sequence data processing codes are available in D. B. [START_REF] Nothaft | danote/Samail 16S compilation: First release of Samail 16S data processing repository[END_REF]. The sequences are accessible on the NCBI Short Read Archive under accession PRJNA655565.

Figure 2 .

 2 Figure 2. Aqueous concentrations of CH4 and H2 in Oman groundwater samples from 2017and 2018. Left and down carrots denote "below limit of quantitation" for CH4 and H2, respectively, with the adjacent point plotted at the limit of quantitation for that gas and year of analysis.

Figure 3 .

 3 Figure 3. Molecular and isotopic compositions of natural gases. (a) Plot of δDCH 4 vs.

  abbreviations same as in(a). In (a) and (c), uncertainties are smaller than plotted symbols. (b) Plot of ε methane/water vs. ∆ 13 CH3D. X and Y axes are swapped with respect to original publication of this type of plot[START_REF] Wang | Nonequilibrium clumped isotope signals in microbial methane[END_REF] so that (b) is comparable against (d). The data from (b) are plotted in the D. T.[START_REF] Wang | Nonequilibrium clumped isotope signals in microbial methane[END_REF] orientation in Supporting Information FigureS4. Equilibrium line from[START_REF] Horibe | DH fractionation in the system methane-hydrogenwater[END_REF] and E.[START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF]. Abbreviations: LTA-KC, low-temperature abiotic (Kidd Creek-type); M, microbial. Green dot-dashed lines in (b) and (d) indicate a range of CH4 isotopic compositions that have been attributed to either low cell-specific rates of methanogenesis or anaerobic oxidation of methane; that is, they start at isotopic compositions produced by methanogen cultures and end at isotopic equilibrium between 5 • C and 70 • C, which is the range of temperatures over which anaerobic oxidation of methane has been documented (D. T.[START_REF] Wang | Nonequilibrium clumped isotope signals in microbial methane[END_REF][START_REF] Stolper | Distinguishing and understanding thermogenic and biogenic sources of methane using multiply substituted isotopologues[END_REF][START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF][START_REF] Ash | Exchange catalysis during anaerobic methanotrophy revealed by 12 CH 2 D 2 and 13 CH 3 D in methane[END_REF][START_REF] Giunta | Methane sources and sinks in continental sedimentary systems: New insights from paired clumped isotopologues 1 3CH 3 D and 1 2CH 2 D 2[END_REF]. (d) Plot of ∆ 13 CH3D vs. ∆ 12 CH2D2, after E.[START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF]. Fields, abbreviations, and temperature axis same as in (b). In (b) and (d), error bars represent 95 % confidence interval for analyses performed at MIT, and 1 standard

(

  Figure4), which, in turn, overlap with δ 13 C values of CH 4 from nearby gas seeps at Los Fuegos Eternos and Nagsasa in the Philippines(-7.4 VPDB to -5.6 VPDB; Figure 3a;[START_REF] Abrajano | Geochemistry of reduced gas related to serpentinization of the Zambales ophiolite[END_REF][START_REF] Vacquand | Reduced gas seepages in ophiolitic complexes: evidences for multiple origins of the H 2 -CH 4 -N 2 gas mixtures[END_REF]).[START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF] also crushed

Figure 4 .

 4 Figure 4. Plot of δ 13 C of CH4 and co-occurring n-alkanes vs. the number of C atoms per molecule. Error bars represent uncertainties on δ 13 C analyses performed at CUB. Only samples for which δ 13 CC 2 was determined are plotted. Contextual data from ophiolites: Oman/UAE[START_REF] Fritz | Deuterium and 13 C evidence for low temperature production of hydrogen and methane in a highly alkaline groundwater environment in Oman[END_REF], the Philippines[START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF]; sediment-poor seafloor hydrothermal vents: Mid-Cayman Rise[START_REF] Mcdermott | Pathways for abiotic organic synthesis at submarine hydrothermal fields[END_REF][START_REF] Grozeva | Chemical and isotopic analyses of hydrocarbon-bearing fluid inclusions in olivine-rich rocks [Article[END_REF], Lost City[START_REF] Proskurowski | Abiogenic hydrocarbon production at Lost City hydrothermal field[END_REF], Ashadze II (J. L.[START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF]; and Precambrian Shield: Kidd Creek,

  end member vent fluid temperatures. As a result of the apparent faster re-equilibration of 12 CH 2 D 2 , the Lost City data plot above the 13 CH 3 D-12 CH 2 D 2 equilibrium line (towards higher ∆ 12 CH 2 D 2 ) in Figure 3d. Therefore, isotopic compositions of CH 4 formed in fluid inclusions in the oceanic lithosphere and stored for millions of years at low temperatures may be expected to fall somewhere along a continuum from ∆ 13 CH 3 D, ∆ 12 CH 2 D 2 , and CH 4 -H 2 O isotopic equilibrium at ∼ 330 • C to compositions approaching lower temperature (∼ 70 • C or perhaps even lower) equilibrium, with 12 CH 3 D, 13 CH 3 D, CH 4 -H 2 O isotopic re-equilibration proceeding at varying rates. This is not the case for Samail Ophiolite samples, as detailed below. Across five years of samples from NSHQ14, δD CH4 has ranged from -232 VSMOW to -311.73 VSMOW, with a mean weighted by sample year of -275 VSMOW (Figure 3a; Table

  Moreover, NSHQ14 fluids exhibit intra-CH 4 disequilibrium, as indicated by ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 values (Table 2) plotting below the equilibrium line in Figure 3d. These non-equilibrium isotopic compositions indicate that post-genetic alteration of CH 4 must have occurred or that fluid inclusions are not the only source of CH 4 at NSHQ14. One potential post-genetic alteration mechanism is diffusion. However, CH 4 at NSHQ14 cannot be the diffusion residual of CH 4 that was originally at intramolecular equilibrium (or with ∆ 12 CH 2 D 2 above the apparent ∆ 13 CH 3 D equilibrium temperature) because the diffusion slope (change in ∆ 12 CH 2 D 2 over change in ∆ 13 CH 3 D) is shallower than the equilibrium line slope over the relevant temperature range (E. Young et al., 2017). Another potential alteration mechanism is microbial CH 4 oxidation. Two types of microbial CH 4 oxidation have been studied for their effects on CH 4 clumped isotopologue relative abundances: anaerobic methane oxidation of the ANME type and aerobic CH 4 oxidation. ANME-

Figure 5 .

 5 Figure 5. 16S rRNA gene read relative abundances of DNA extracted from Samail Ophiolite groundwaters sampled in 2018 affiliated with CH4-cycling taxa. Read relative abundances are

While H 2

 2 has only been transiently detected at NSHQ04, c CH4 at this well has consistently been the highest among our sample sites (144 µmol•L -1 in 2018 and 483 µmol• L -1 in 2017. In comparison to NSHQ14, CH 4 at NSHQ04 is more 13 C-and D-enriched (mean weighted by sample year δ 13 C = +3.3 VPDB, s = 1.8 ; δD = -220 VSMOW, s = 11 ; n = 4;

Figure 6 .

 6 Figure 6. Conceptual model of CH4 dynamics in Samail Ophiolite. Cross section after Neal and Stanger (1985), Dewandel et al. (2005), and Rempfert et al. (2017). Groundwater flow is depicted with blue arrows. Cross-hatching illustrates fissured zone of aquifer, extending to ∼ 50 m depth. A deep tectonic fracture hosting upward groundwater flow is shown as a black line. Yellow dashed line indicates proposed transition between conditions where methanogenesis is limited by H2 versus C availability. Isotopic systematics are written in black text. Abbreviations: eq., equilibrium; T , temperature; FTT, Fischer-Tropsch-type.

  Figure 1.

  Figure 6.

Table 1 .

 1 Well data and field measurements.

	f O2 /	[bar] b	2.99 • 10 -36	2.01 • 10 -34		1.23 • 10 -37	2.99 • 10 -36	7.17 • 10 -25			2.81 • 10 -37	5.14 • 10 -51		2.52 • 10 -51		n.d.			1.19 • 10 -51
	Eh/	[mV]	167 a	214 a		133	162	269 a			20.2 a	-174		-229		n.d.			-253 a
		pH		8.51	8.16		8.79	8.66	9.62 a			10.61 a	10.51 a		11.22		11.32			11.39
	Temperature /	[ • C]	34.9	35.6		33.7	33.7	36.2 a			35.6 a	33.4		34.9		33.6			36.7
	Conductivity /	µS • cm -1	1410	1120		548	498	1183 a			930. a	3350		1970		2860			2670
	Pump	depth /	[mbct]	70.	50.		85	60.	50. a			30. a	8		70.		75			85
	Water	level /	[mbct]	15	9.5		40.	16.5	7.5 a			7.62 a	4.7		8.3		13.4			9.2
	Screen	interval /	[mbct]	90 -98	34.5 -51		100.8 -104	110 -117	8 -97			7 -27	open > 5.8		128 -131		open > 23.7			open > 5.8
	Well	depth /	[mbgl]	101	78		120.4	120.5	102			106	304		136.5		400.			304
	UTM coordinates	Well Geologic description (WGS-84)	easting northing	WAB103 648 577 2 530 362 Gabbro	WAB188 671 123 2 529 798 Gabbro, near contact with	harzburgite	WAB104 643 099 2 541 124 Harzburgite	WAB105 644 678 2 536 524 Harzburgite	WAB55 634 777 2 506 101 Harzburgite with abundant	carbonate veins, near	contact with gabbro	WAB56 634 851 2 501 617 Harzburgite	NSHQ04 670 971 2 531 699 Harzburgite, near fault	with gabbro	WAB71 670 322 2 533 981 Dunite, near fault with	harzburgite	CM2A 636 988 2 534 284 Mostly dunite with	occasional gabbro and	harzburgite	NSHQ14 675 495 2 529 716 Harzburgite

Measurements refer to sampling

February-March, 2018, unless

noted. Well elevations are given in Supporting Information Figure S1. Abbreviations: n.d., not determined; mbgl, meters below ground level; mbct, meters below casing top. Casings extend ∼ 1 m above ground level. a Not determined during 2018 sampling, so most recent prior data is reported (2015 to 2017; (Rempfert et al., 2017; Fones et al., 2019)). b Calculated from temperature, pH, and Eh. Where one or more of these parameters were obtained during different sampling years, f O2 should be considered a representative estimate.

Table 2 .

 2 Isotopic compositions of CH4, C2H6, and C3H8.

	Well	Sample	Pump depth	laboratory δ 13 CCH 4	δDCH 4	∆ 13 CH3D	∆ 12 CH2D2	δ 13 CC 2H6 δ 13 CC 3H8
		year	/ [mbct]
			50.	CUB	-86.7	n.d.	n.d.	n.d.	n.d.	n.d.
	WAB188		78	CUB	-60.8	n.d.	n.d.	n.d.	n.d.	n.d.
			20.	LBNL	-71.3	n.d.	n.d.	n.d.	n.d.	n.d.
	WAB56		12	LBNL	-83.2	n.d.	n.d.	n.d.	n.d.	n.d.
			8	CUB UCLA	4.7 4.177	-229 -227.396 0.229 ± 0.288 -24.502 ± 0.944 n.d. n.d.	n.d. n.d.	n.d. n.d.
	NSHQ04		5.8	CUB MIT	6.8 3.59	-225 -229.67	n.d. 0.12 ± 0.17	n.d. n.d.	n.d. n.d.	n.d. n.d.
			22	LBNL MIT	0.8 1.60	-209 -230.00	n.d. 0.72 ± 0.29	n.d. n.d.	n.d. n.d.	n.d. n.d.
			18	LBNL	2.4	-205	n.d.	n.d.	n.d.	n.d.
			70.	CUB	3.6	-307	n.d.	n.d.	n.d.	n.d.
	WAB71		50.	CUB	3.9	-313	n.d.	n.d.	n.d.	n.d.
			50.	LBNL	3.0	n.d.	n.d.	n.d.	n.d.	n.d.
			18	LBNL	2.9	n.d.	n.d.	n.d.	n.d.	n.d.
				CUB	-4.3	-206	n.d.	n.d.	n.d.	n.d.
	CM2A		75	MIT	-3.83	-190.32	2.87 ± 0.57	n.d.	n.d.	n.d.
				UCLA	-4.710	-197.73 2.638 ± 0.284 -1.267 ± 0.886	n.d.	n.d.
				CUB	-2.3	-314	n.d.	n.d.	n.d.	n.d.
			85	MIT	-5.02	-311.73	0.77 ± 0.44	n.d.	n.d.	n.d.
				UCLA	-3.352	-293.58 2.074 ± 0.298 -0.204 ± 1.358	n.d.	n.d.
	NSHQ14		85	CUB MIT	0.2 -0.08	-271 -268.82	n.d. 0.69 ± 0.23	n.d. n.d.	-6.0 n.d.	+3.3 n.d.
			70.	LBNL MIT	1.8 -6.89	-273 -308.52	n.d. 0.69 ± 0.17	n.d. n.d.	n.d. n.d.	n.d. n.d.
			20.	LBNL	3.7	n.d.	n.d.	n.d.	n.d.	n.d.
			260.	LBNL	3.0	-232	n.d.	n.d.	n.d.	n.d.

  CH 4 + 12 CH 3 D 12 CH 4 + 13 CH 3 D. (1)Analogous expressions can be written for doubly-deuterated CH 4 , 12 CH 2 D 2 .

	for δD) were used for calibration. Over the range of peak amplitudes of analyses reported
	here, the repeatability expressed as 1 s on analyses of standards is ±0.6 for δ 13 C and
	±7 for δD. The analytical uncertainty (accuracy) expressed as 1 standard error on
	a 3-point calibration was < 0.3 for δ 13 C and < 9 for δD (Supporting Information
	Section S1).
	The relative abundances of CH 4 isotopologues, including the doubly-substituted
	isotopologue, 13 CH 3 D, were measured at the Massachusetts Institute of Technology (MIT)
	by tunable infrared laser direct absorption spectroscopy following the methods described
	by Ono et al. (2014). Abundances of CH 4 isotopologues, including both 13 CH 3 D and 12 CH 2 D 2 ,
	were measured at the University of California, Los Angeles (UCLA) by high-mass-resolution
	gas-source isotope ratio mass spectrometry following the procedure of E. D. Young et
	al. (2016). The abundance of 13 CH 3 D relative to a random (stochastic) distribution of
	isotopes among the isotopologues in a CH 4 sample is described by its ∆ 13 CH 3 D value,
	which is defined as: ∆ 13 CH 3 D = ln Q, where Q is the reaction quotient of the isotope
	exchange reaction:
	3.3 16S rRNA gene sequencing and analysis
	Biomass for DNA extraction was concentrated by pumping 5 L to 20 L of ground-
	water through Millipore polycarbonate inline filters (0.45 µm pore diameter, 47 mm fil-
	ter diameter). At well NSHQ04, a 0.22 µm pore diameter polyethersulfone Millipore Sterivex
	filter was used instead due to the lower-flow pump used at this well (Section 3.1). Fil-
	ters were placed in cryovials, transported frozen in liquid N 2 , and stored in a -70 • C freezer
	until extraction. DNA was extracted from one quarter subsamples of each filter using
	a Qiagen PowerSoil DNA extraction kit. The V4 hypervariable region of the 16S rRNA
	gene was amplified by PCR in duplicate reactions using the 515 (Parada) -806R (Ap-
	prill) primer pair modified to include Illumina adapters and the appropriate error-correcting
	barcodes. Each 25-µL reaction mixture included 12.5 µL of Promega HotStart Master-
	mix, 10.5 µL of PCR-grade water, 1 µL of PCR primers (combined at 10 µM), and 1 µL
	of purified genomic DNA. PCR consisted of an initial step at 94 • C for 3 min followed
	by 35 cycles of 94 • C for 45 s, 50 • C for 1 min, and 72 • C for 1.5 min. PCR concluded with
	a final elongation step at 72 • C for 10 min. No-template controls and DNA extraction
	controls were subjected to PCR to check for potential contamination in our PCR and
	DNA extraction reagents, respectively. Amplification was evaluated via electrophoresis
	in a 2 % agar gel. Amplicons from duplicate reactions were pooled, cleaned, and their
	concentrations normalized using a Thermo Fisher SequalPrep normalization plate kit.
	Amplicons were sequenced on an Illumina MiSeq at the CUB Next-Generation Sequenc-
	ing Facility with 2-by-150 bp paired-end chemistry.
	2 for δ 13 C and ±5 for
	δD.
	From 2017 onwards, bulk stable isotope analyses of CH 4 and co-occurring alkane
	gases were conducted at the University of Colorado -Boulder (CUB) by GC/C/Pyr/IRMS
	using a Trace 1310 GC equipped with an Agilent J & W GS-CarbonPLOT column (30 m
	length, 0.32 mm ID, 3.0 µm film) coupled to a Thermo Scientific MAT253 IRMS. CH 4
	isotope standards purchased from Airgas (uncertainties of ±0.3 for δ 13 C and ±5

13

Table 3 .

 3 Chemical and isotopic composition of water samples.

	Br -		1.39 • 10 2	6.78 • 10 1		3.55	8.60	1.12 • 10 2		1.79 • 10 -1a	1.25 a	1.50 • 10 2	2.48 • 10 2	1.67 • 10 2
	Cl -		6.25 • 10 3	4.22 • 10 3	7.76 • 10 2	8.54 • 10 2	6.54 • 10 3		1.33 • 10 1a	1.82 • 10 4a	1.17 • 10 4	1.85 • 10 4	1.36 • 10 4
	SO 2-4		1.57 • 10 3	1.41 • 10 3	3.80 • 10 2	2.92 • 10 2	8.03 • 10 2		6.00 a	6.83 • 10 2a	6.08 • 10 1	5.56 • 10 2	1.57 • 10 2
	NO -3		4.72 • 10 2	3.21 • 10 2	3.14 • 10 2	3.02 • 10 2	3.02 • 10 2		3.00 a	3.00 a	1.84 • 10 2	1.64 • 10 2	3.60 • 10 2
	Si		4.63 • 10 2	4.77 • 10 2	4.15 • 10 2	2.83 • 10 2	3.58 • 10 1		2.22 • 10 2	3.60 • 10 1a	2.35 • 10 1	2.81 • 10 1	1.03 • 10 1
	Fe		7.35	2.90 • 10 1		3.88	4.83	2.52		n.d.	8.20 • 10 -1a	8.48 • 10 1	4.03 • 10 1	8.48 • 10 1
	Mg		1.87 • 10 3	1.82 • 10 3	2.30 • 10 3	1.87 • 10 3	3.34 • 10 3		1.00 a	1.80 • 10 1a	< 2.06 • 10 -1	9.49	6.23
	Ca		2.58 • 10 2	1.41 • 10 3	1.96 • 10 2	2.58 • 10 2	5.06 • 10 1		5.43 • 10 2a	7.79 • 10 3a	4.14 • 10 3	1.75 • 10 3	3.60 • 10 3
	Na		1.18 • 10 3	4.06 • 10 3	7.53 • 10 2	1.18 • 10 3	4.44 • 10 3		3.56 • 10 3a	1.04 • 10 4a	6.25 • 10 3	2.07 • 10 4	1.03 • 10 4
	δ 13 C CO2		-13.54	-13.52		-13.88	-10.88	-12.63		n.d.	-29.7	n.d.	n.d.	n.d.
	CO 2		2.67 • 10 3	3.48 • 10 3	3.62 • 10 3	3.32 • 10 3	2.40 • 10 3		1.3 • 10 2a	1.8 • 10 1	< 1.2•10 1	< 1.2•10 1	< 1.2•10 1
	Well δD H2O δ 18 O H2O	gabbro-hosted groundwaters	WAB103 -0.5 0.34	WAB188 -2.1 -0.71	Mg 2+ -HCO -3 groundwaters	WAB104 -0.5 -0.53	WAB105 0.4 0.50	WAB55 2.2 0.26	Ca 2+ -OH -groundwaters	WAB56 n.d. n.d.	NSHQ04 -15 a -3.0 a	WAB71 -3.0 -0.40	CM2A 1.7 0.67	NSHQ14 0.2 0.43

Concentrations reported in

µmol • L -1 . indicates the sum of all dissolved species of the element. All δ values reported in units. δ 18 O and δD reported relative to VSMOW. δ 13 C reported relative to VPDB. Samples obtained in February-March 2018, unless noted. Abbreviations: n.d., not determined.

a Not determined during 2018 sampling, so most recent prior data is reported

(2015 to 2017;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF]

).

Table 5 .

 5 Gibbs free energies of potential CH4-forming reactions and log activities of relevant species. Abbreviations: H, hydrogenotrophic (Equation

  centration measurements were lacking, and evaluated ∆G r 's for a range of H 2 concentrations. Measurements of major inorganic dissolved constituents, pH, and Eh from wells WAB105 and NSHQ14 were used for the model Mg 2+ -HCO - 3 and Ca 2+ -OH -flu-

				log (activity)			∆G r / kJ • mol -1
	water type	H +	CO 2 (aq) HCOO -CH 3 COO -CH 4 (aq) H 2 (aq)	H	A	F
							-9.0	64	-115 -90
	Ca 2+ -OH -	-11.1	-11.6	-6.1	-6.1	-4.0	-6.0	-6 -115 -90
							-3.0	-77 -115 -90
	Mg 2+ -HCO -3	-8.7	-4.9	-6.0	-6.0	-7.0	-9.0 -6.0	8 -63 -107 -47 -107 -47

ids, respectively (Tables

  addition to energetic considerations, microbiological approaches can help elucidate which CH 4 -forming reactions occur. Kraus et al. (2021) found higher transcript abundances of carbonic anhydrase and formate dehydrogenase relative to acetate kinase and phosphate acetyltransferase in hyperalkaline groundwaters from wells in the Samail Ophiolite, suggesting that CO 2 /HCO - 3 and formate are more actively used substrates for methanogenesis than acetate in these conditions. Further, Fones et al. (2020) identified two lineages of Methanobacterium in Samail Ophiolite groundwaters that were shown by genomic and microcosm-based radiotracer approaches to use different methanogenic

pathways. Methanobacterium Type I lineage predominated in circumneutral waters and is capable of using either CO 2 or formate for methanogenesis. Methanobacterium Type II lineage, which was more abundant in hyperalkaline waters and which branched from the Type I lineage, was exclusively capable of formatotrophic methanogenesis. It was postulated that gene loss and acquisition in Type II lineage allowed it to be specially suited to the high-pH and low-CO 2 conditions resulting from extensive serpentinization. Thus, microbiological data suggest that hydrogenotrophic or formatotrophic methanogenesis are the most likely pathways for methanogenesis in the Samail Ophiolite and that the relative contributions of each of these pathways to microbial CH 4 production at a given site may depend on local geochemical factors such as a CO2(aq)

  .2.2 Abiotic, 13 C-enriched CH 4 , C 2 H 6 , and C 3 H 8 mixed with microbial CH 4 produced under C-limited conditions in the Ca 2+ -OH -

	waters of well NSHQ14
	Well NSHQ14 is situated in a catchment dominated by partially serpentinized harzbur-
	gite with meter-scale partially serpentinized dunite bands (Figure 1; Supporting Infor-
	mation Figure S1; Table 1). The well is cased to 5.8 meters below ground level (mbgl)
	and drilled to 304 mbgl (Table 1). Groundwaters accessed via NSHQ14 had the highest
	pH (11.39), and lowest Eh (-253 mV) and f O2 (1.19 • 10 -51 bar) among the wells in-
	vestigated (Table 1), indicating that fluids sampled from NSHQ14 have extensively par-

ticipated in serpentinization. This is also reflected in the c H2 of groundwaters sampled at NSHQ14, which was the highest among the studied wells (253 µmol•L -1 and 131 µmol•

  Sources of CH 4 can also be assessed by measuring H isotopic compositions and clumped isotopologue relative abundances of CH 4 and comparing these isotopic compositions to temperature-dependent equilibria. These isotopic equilibria are represented by thick gray lines in Figure3b and d. Intra-CH 4 equilibrium is governed by the increasing relative stability of bonds between two heavy isotopes (more "clumping") at lower temperatures, which is reflected in higher ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 values. However, isotopic equilibrium will only be expressed if kinetics allow it. In the first study to publish clumped isotopologue (∆ 13 CH 3 D) data on CH 4 -and H 2 -rich gases from sediment-poor seafloor hydrothermal vents, D. T.[START_REF] Wang | Clumped isotopologue constraints on the origin of methane at seafloor hot springs[END_REF] found that these gases yielded apparent CH 4 -H 2 O H isotopic and ∆ 13 CH 3 D equilibrium temperatures of 270 • C to 360 • C, despite having a range of effluent fluid temperatures from 96 • C to 370 • C. This was interpreted as evidence for a closure temperature of 270 • C for H isotope exchange in the CH 4 -H 2 O and CH 4 -H 2 systems in seafloor hydrothermal settings (e.g. Mid-CaymanRise in Figure3b and d). However, in a subsequent study that re-analyzed some of the same samples, plus a greater number of samples from low-temperature vents at Lost City (96 • C to 64 • C), and contributed the first ∆ 12 CH 2 D 2 values from these settings, Labidi et al. (2020) found evidence for re-equilibration of clumped isotopologue and CH 4 -H 2 O H isotopic systems at lower temperatures. Of these isotopic systems, that of 12 CH 2 D 2 had the fastest apparent re-equilibration kinetics (approximately twice as fast as 13 CH 3 D), which was explained by differences in symmetry numbers among the isotopologues. The 12 CH 2 D 2 -based temperatures of the Lost City samples, which were as low as 69 +4

[START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF]

; and Precambrian Shield: Kidd Creek, Canada

[START_REF] Sherwood Lollar | Isotopic signatures of CH 4 and higher hydrocarbon gases from Precambrian Shield sites: A model for abiogenic polymerization of hydrocarbons[END_REF]

. manuscript submitted to JGR: Biogeosciences

  Figure3b and d) do not support a major role for anaerobic methane oxidation in the study area. Aerobic CH 4 oxidation is less reversible than ANME-type anaerobic methane oxidation due to differences in the enzymes and electron acceptors used for those respective processes. For this reason, aerobic CH 4 oxidation does not bring CH 4 into isotopic equilibrium, but rather imparts a normal, classical kinetic isotope effect during CH 4 consumption. In a study of the effect of aerobic CH 4 oxidation on ∆ 13 CH 3 D, D. T.[START_REF] Wang | Fractionation of the methane isotopologues 13[END_REF] found that the fractionation factor for 13 CH 3 D was closely approximated by the product of the fractionation factors for13 CH 4 and 12 CH 3 D. Although it has not yet been demonstrated experimentally, it is hypothesized that the fractionation factor for 12 CH 2 D 2 during aerobic CH 4 oxidation may likewise be approximated by the square of the fractionation factor for 12 CH 3 D (E. D. Young, 2020). This "product rule" for isotopic fractionation during aerobic CH 4 oxidation results in decreases in ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 with concomitant increases in δ 13 C and δD in residual CH 4 (D. T. Wang et al., 2016; E. D. Young, 2020). Thus, aerobic CH 4 oxidation could draw ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 values originally near equilibrium down below the equilibrium line in Figure 3d. However, if CH 4 samples from NSHQ14 were originally near H isotope equilibrium with water of SMOW-like isotopic composition, aerobic methane oxidation would push the residual CH 4 towards higher δD (and ε methane/water ) values (above the equilibrium line in Figure 3b), which is inconsistent with the comparatively low δD CH4 observed at NSHQ14. For the reasons outlined above, post-genetic alteration of CH 4 near CH 4 -H 2 O and intramolecular isotopic equilibrium does not explain the observed isotopic compositions of CH 4 sampled from NSHQ14. Therefore, the release of CH 4 stored in fluid inclusions cannot account for all of the CH 4 at NSHQ14. Alternative processes that do produce CH 4 with ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 values lower than equilibrium include micro-

• C to 30 • C) through continuous breaking and reforming of bonds in the CH 4 molecule (E.

[START_REF] Young | The relative abundances of resolved 12 CH 2 D 2 and 13 CH 3 D and mechanisms controlling isotopic bond ordering in abiotic and biotic methane gases[END_REF][START_REF] Ash | Exchange catalysis during anaerobic methanotrophy revealed by 12 CH 2 D 2 and 13 CH 3 D in methane[END_REF][START_REF] Giunta | Methane sources and sinks in continental sedimentary systems: New insights from paired clumped isotopologues 1 3CH 3 D and 1 2CH 2 D 2[END_REF]

. Thus, the comparatively low ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 values observed in samples from NSHQ14 and other wells in this study (bial methanogenesis and low-temperature (≤ 90 • C) abiotic reduction of CO 2 or CO through Sabatier or Fischer-Tropsch-type reactions. In Figure 3b and d, microbial methanogenesis is represented by samples from cultures (green shaded areas; (D. T. Wang et al., 2015; D. Stolper et al., 2015; E. Young et al., 2017; Gruen et al., 2018; E. D. Young, 2020)), and low-temperature Sabatier or Fischer-Tropsch-type reactions are represented by field samples from Kidd Creek (gray shaded areas; (E.

  .r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. .r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. . n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. . n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.

				gabbro			Mg 2+ -HCO 3 -			Ca 2+ -OH -
	Deepest taxonomic assignment grouped by	metabolic capability inferred from phylogeny	g. Methylocystis g. Methyloterricola g. Methylococcus g. Ca. Methylomirabilis o. ANME-1b g. Methanobacterium o. Methanomassiliicoccales	1 1 1 1 1 1 1 1 1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1	<1 <1 <1 <1 <1 <1 <1 <1 <1 2 2 2 2 2 2 2 2 2 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 3 3 3 3 3 3 3 3 3 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.	1 1 1 1 1 1 1 1 1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.	n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.	n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.	n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 8 8 8 8 8 8 8 8 8 n.r1 1 1 1 1 1 1 1 1 n.r<1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 24 24 24 24 24 24 24 24 24 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.	Methanotrophy using oxygen Methanotrophy using nitrite Methanotrophy using sulfate Methanogenesis
				WAB103 WAB188	WAB104 WAB105 WAB55	NSHQ04 WAB71	CM2A	NSHQ14
							Well grouped by water type
					Read relative abundance / [%]
								0	5	10 15 20

manuscript submitted to JGR: Biogeosciences nn

  indicate that formate is the dominant substrate for methanogenesis at NSHQ14. Formate concentrations are 1 µmol• L -1 to 2 µmol•L -1 in the studied wells[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF], which are roughly two orders of magnitude lower than formate concentrations at unsedimented seafloor hydrothermal vents impacted by serpentinization at warmer conditions than present in the Samail Ophiolite[START_REF] Mcdermott | Pathways for abiotic organic synthesis at submarine hydrothermal fields[END_REF][START_REF] Lang | Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field[END_REF]. These relatively low formate concentrations in the ophiolite suggest that formate might be the primary limiting substrates for methanogenesis in Ca 2+ -OH -waters, such as at NSHQ14. Coexisting hydrogenotrophic methanogens may produce CH 4 through direct uptake of CO 2 in H 2 -rich Ca 2+ -OH -

water, where kinetic inhibitions to abiotic CO 2 reduction to CH 4 allow for a modest energy yield for hydrogenotrophic methanogens (Section 5; (Leong & Shock, 2020)). Methanogens using CO 2 could benefit from greater chemical disequilibrium if they inhabit zones where deeply-sourced, H 2 -rich Ca 2+ -OH -water mixes with shallow, Mg 2+ -HCO - 3 water (Zwicker et al., 2018;

  The known presence of CH 4 -bearing fluid inclusions in the Samail Ophiolite and our finding of high δ 13 C values of CH 4 , C 2 H 6 , and C 3 H 8 that overlap with values reported from seafloor hydrothermal vents where CH 4 formed at > 270 • C in fluid inclusions predominates suggest a similar source in the ophiolite. However, deficits in 12 CH 3 D, 13 CH 3 D, and 12 CH 2 D 2 relative to equilibrium indicate the production of additional CH 4 at low temperatures. The 13 CH 3 D deficit in particular is more compatible with a microbial origin than a low-temperature abiotic origin. Moreover, genomic, transcriptomic, and phys-Biogeosciences iological data show that methanogens are abundant and active in aquifers accessed via NSHQ14. Organic geochemical and cultivation data from the literature suggest that C isotope effects of methanogenesis may be suppressed under C-limited conditions in serpentinizing settings. That genes associated with methanogens coexist with a smaller abundance of genes associated with methanotrophs (particularly aerobes) in NSHQ14 groundwaters suggests that some of the CH 4 has undergone microbial oxidation, which would further help explain the high δ 13 C values of CH 4 at this well.

	manuscript submitted to JGR: 4.2.3 Abundant microbial CH 4 produced under C-limited conditions and substantial microbial CH 4 oxidation in the Ca 2+ -OH -waters
	of well NSHQ04
	NSHQ04 is situated in partially serpentinized harzburgite 10 m away from a faulted
	contact with crustal gabbros (Figure 1; Supporting Information Figure S1). Surface rock
	exposures surrounding NSHQ04 are dominated by serpentinized harzburgites, with lesser
	dunites, gabbro lenses, and pyroxenite dikes. NSHQ04 is cased to 5.8 mbgl and drilled
	to 304 m depth (Table
	-25-

Table 2 ;

 2 Figure3a). Fluids sampled from NSHQ04 are in CH 4 -H 2 O H isotopic disequilibrium and intra-CH 4 disequilibrium (Figure3b and d), which is also true of fluids from NSHQ14. However, CH 4 sampled from NSHQ04 has distinctly negative ∆ 12 CH 2 D 2 (-24.502 ) and low ∆ 13 CH 3 D (mean weighted by sample year of 0.36 , s = 0.32 , n = 3; Table2). As such, CH 4 from NSHQ04 plots squarely among methanogen culture samples in ∆ 13 CH 3 D/∆ 12 CH 2 D 2 space (Figure3d), suggesting that CH 4 is dominantly microbial at NSHQ04. Moreover, alkane gases dissolved in waters pumped from NSHQ04 exhibited a C 1 / (C 2 + C 3 ) ratio of 5.4•10 3 in 2018, which is higher than other wells in this study (Table4; Figure

Table 1 )

 1 , it is plausible that the sampled groundwaters continuously interact with atmospheric CO 2 . Although the relative influences of methanotrophy and atmospheric CO 2 hydroxylation cannot be determined based on the available data, both processes could affect δ 13 C CO2 at NSHQ04. In summary, low ∆ 13 CH 3 D and ∆ 12 CH 2 D 2 , high C 1 / (C 2 + C 3 ), the presence of Methanobacterium that were readily cultured, and high 16S rRNA gene relative abundances of Methylococcus lead us to conclude that microbial production and consumption of CH 4 are the dominant factors controlling CH 4 concentration and isotopic com-H2 = 0.992 µmol•L -1 in 2017), and CH 4 has consistently been detected at moderate concentrations (c CH4 = 1.83 µmol•L -1 in 2017 and 0.917 µmol•L -1 in 2018)(Table 4;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF]). The high c Ca and moderate but variable pH, Eh, and c H2 in fluids sampled from WAB188 suggest that fluid chemical composition at WAB188 is dominantly controlled by water-rock reaction with gabbro (

	position at NSHQ04.
	4.2.4 H 2 -limited microbial methanogenesis with classic C isotope effect
	expressed at well WAB188
	WAB188 is situated 2 km down-gradient from NSHQ04 and is set in gabbro on the
	opposite side of a fault from NSHQ04 (Figure 1; Supporting Information Figure S1; Ta-
	ble 1). Fluids pumped from WAB188 have had variable pH (8.72 to 5.75) and oxidation-
	reduction potential (f O2 of 10 -61 bar to 10 -34 bar and Eh of -220 mV to +214 mV) across
	four years of sampling (Table 1; (Rempfert et al., 2017; Fones et al., 2019)). WAB188
	has consistently had major ion compositions similar to the gabbro-hosted well WAB103,

except that WAB188 has had higher c Ca

(Table 3;[START_REF] Rabu | Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman[END_REF][START_REF] Fones | Physiological adaptations to serpentinization in the Samail Ophiolite, Oman[END_REF]

). H 2 has occasionally been detected in fluids pumped from WAB188 (c

  would be that the source of CH 4 at WAB188 is dominantly microbial. Such an interpretation is largely based on data from sedimentary settings, where H 2 is typically more scarce than CO 2 . In this re-

	gard, conditions in sedimentary settings are analogous to those at WAB188. Evidence
	that considerable methanogenesis proceeds through a hydrogenotrophic pathway under
	H 2 -limited conditions at WAB188 include microbiological data confirming the capacity
	of Methanobacterium to perform hydrogenotrophic methanogenesis at WAB188 and ther-
	modynamic calculations showing that hydrogenotrophic methanogenesis (with H 2 as lim-
	iting substrate) was more energetically favorable than formatotrophic methanogenesis
	for a fluid with c CO2 and c H2 similar to WAB188 in 2017 (Section 4.2.1; Table

  4 , C 2 H 6 , and C 3 H 8 is inferred from the widespread occurrence of CH 4 in fluid inclusions in peridotites, including those in Oman, and is supported by the relatively 13 C-enriched compositions of CH 4 , C 2 H 6 , and C 3 H 8 measured in gases exsolved from peridotite-hosted groundwaters in this study. The measured δ 13 C values overlap with those of CH 4 , C 2 H 6 and C 3 H 8 from seafloor hydrothermal vents where fluid inclusions are the dominant source of these alkanes, suggesting similar CH 4 sources across these environments. In contrast, abiotic, low-temperature reduction of CO 2 to CH 4 appears less likely to contribute substantially to the CH 4 pool in the study area due to a scarcity of conditions favorable to catalysis, namely, access of gas-phase H 2 and CO 2 /CO to Ru-bearing chromites.Further, we note an inverse relationship between c CO2 and δ 13 C CH4 across groundwaters bearing microbiological evidence of methanogenic activity. This finding supports the hypothesis that the apparent C isotope fractionation between the C substrate used by methanogens and the CH 4 they produce is suppressed when the C substrate is limiting. Thus, our finding that δ 13 C CH4 varies by 90 in the Samail Ophiolite suggests that, in some settings, δ 13 C CH4 may be a powerful indicator of transitions from H 2 -limited to C-limited conditions for microbial methanogenesis, rather than a discriminant between microbial versus abiotic CH 4 . The 16S rRNA gene sequencing data also indicate the presence of microbes capable of CH 4 oxidation, particularly those that can use O 2 as an oxidant. This oxidation may also contribute in part to the 13 C-enriched composition of CH 4

  n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.1 1 1 1 1 1 1 1 1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. .r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 8 8 8 8 8 8 8 8 8 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 1 1 1 1 1 1 1 1 1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. <1 <1 <1 <1 <1 <1 <1 <1 <1 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. .r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. .r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 24 24 24 24 24 24 24 24 24 n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r.

			gabbro		Mg 2+ -HCO 3 -			Ca 2+ -OH -
	Deepest taxonomic assignment grouped by	metabolic capability inferred from phylogeny	g. Methylocystis g. Methyloterricola g. Methylococcus g. Ca. Methylomirabilis o. ANME-1b g. Methanobacterium o. Methanomassiliicoccales	<1 <1 <1 <1 <1 <1 <1 <1 <1	<1 <1 <1 <1 <1 <1 <1 <1 <1	<1 <1 <1 <1 <1 <1 <1 <1 <1	<1 <1 <1 <1 <1 <1 <1 <1 <1		<1 <1 <1 <1 <1 <1 <1 <1 <1	Methanotrophy using oxygen Methanotrophy using nitrite Methanotrophy using sulfate Methanogenesis
			WAB103 WAB188	WAB104 WAB105 WAB55	NSHQ04 WAB71	CM2A	NSHQ14
					Well grouped by water type
			Read relative abundance / [%]
						0	5	10 15 20

n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. n

.r. nnn

manuscript submitted to JGR: Biogeosciences Acknowledgments This research was directly supported by the Rock-Powered Life NASA Astrobiology Institute (NNA15BB02A). This research also used samples and/or data provided by the Oman Drilling Project. The Oman Drilling Project (OmanDP) has been possible through co-mingled funds from the International Continental Scientific Drilling Project (ICDP), the Sloan Foundation -Deep Carbon Observatory (Grant 2014-3-01, Kelemen PI), the National Science Foundation (NSF-EAR-1516300, Kelemen PI), the NASA Astrobiology Institute (NNA15BB02A), the German Research Foundation (DFG), the Japanese Society for the Promotion of Science (JSPS), the European Research Council, the Swiss National Science Foundation, JAMSTEC, the TAMU-JR Science operator, and contri-We thank the Ministry of Regional Municipalities and Water Resources in the Sultanate of Oman (particularly Said Al Habsi, Rashid Al Abri, Salim Al Khanbashi, and Haider Ahmed Mohammed Alajmi) for allowing access to wells and logistical support, Zaher Al Sulaimani and Mazin Al Sulaimani from the Oman Water Centre and AZD Engineering for their technical and logistical support, Jude Coggon for coordinating Oman Drilling Project activities, Benoît Ildefonse for sharing geologic map data, Eric Ellison and Kaitlin Rempfert for their assistance in the field and laboratory, Elizabeth Fones for sharing biomass samples, Emily Kraus for critical discussion of Oman CH 4 cycle processes, and Noah Fierer, Jen Reeves, Corinne Walsh, Matthew Gebert, and Angela Oliverio for assisting with DNA sequencing and interpretation.