Chitin is a major structural component of arthropod exoskeletons, and an important carbon and nitrogen source in marine environments. In anoxic sediments, its degradation generates chitooligosaccharides and N-acetylglucosamine (GlcNAc), which are fermented into smaller organic molecules and oxidized anaerobically using soluble electron acceptors or insoluble ones such as metal oxides. To date, many aspects of chitin degradation in deep-sea anoxic sediments have been overlooked, including the potential coupling of insoluble chitin degradation to metal oxide reduction, the involvement of extracellular electron transfer (EET), and the spatial organization of the microorganisms involved. Using anoxic deep-sea sediments recovered from a whale fall site, we developed an innovative workflow based on electrochemical reactors, to characterize chitin degradation in these environments. Sediment samples enriched on poorly crystalline iron oxides, and subsequently transferred into an electrochemical reactor poised at +0.22 V vs SHE, showed active anodic current production when supplied with chitin, which increased 2-fold when amended with GlcNAc. Chitin reactors were dominated byVallitalea(Firmicutes),Spirochaetota,GammaproteobacteriaandDesulfobacterota. Exoenzyme activity assays, metabolite profiling, and continued anodic current production confirmed ongoing chitin degradation linked to EET. We observed metabolic associations between chitin degraders and secondary consumers usingin situimaging (16S rRNA gene FISH coupled with BONCAT and nanoSIMS). These microbial partners, within the electrode-attached community, required close proximity to the poised electrode (≤ 10 µm) to remain metabolically active. Supporting these observations, cultured isolates ofVallitaleasp. andTrichloromonassp. recovered from the whale fall site exhibited chitin degradation and electrochemical activity, respectively. When co-cultured in an bioelectrochemical reactor, the acetate produced byVallitaleasp. during chitin degradation fueledTrichloromonassp., which facilitated EET, hereby demonstrating that syntrophic interactions are used to couple anoxic chitin degradation to EET in deep-sea sediments. These findings exemplify the interspecies interactions and resource optimization occurring in hard-to-reach and largely unknown deep-sea ecosystems.
", "doi": "10.1101/2025.06.30.662270", "publisher": "bioRxiv", "publication_date": "2025-06-30" }, { "id": "authors:1qy57-1g892", "collection": "authors", "collection_id": "1qy57-1g892", "cite_using_url": "https://authors.library.caltech.edu/records/1qy57-1g892", "type": "monograph", "title": "The Ambiguous Genetic Code of Methanogenic Archaea that Grow on Methylamines", "author": [ { "family_name": "Shalvarjian", "given_name": "Katie E." }, { "family_name": "Chadwick", "given_name": "Grayson L.", "orcid": "0000-0003-0700-9350" }, { "family_name": "P\u00e9rez", "given_name": "Paloma I." }, { "family_name": "Woods", "given_name": "Philip H.", "orcid": "0000-0003-1673-8096", "clpid": "Woods-Philip-H" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Nayak", "given_name": "Dipti D." } ], "abstract": "Natural genetic code expansion is a phenomenon wherein an additional amino acid is encoded by a stop codon. These non-standard amino acids are beneficial as they facilitate novel biochemical reactions. However, code expansion leads to ambiguity at the recoded stop codon, which can either be read through or terminated. Pyrrolysine (Pyl) is encoded by the amber codon (TAG/UAG) and is widespread in archaea, where it is required for methylamine-mediated methanogenesis, an environmentally important metabolism. Mechanisms to conditionally suppress the amber stop codon for Pyl installation during protein synthesis have not been identified. Using the model methanogen,Methanosarcina acetivorans,we demonstrate that Pyl-encoding archaea maintain an ambiguous genetic code wherein UAG encodes dual meaning as stop and Pyl. Our data suggest that expression of Pyl biosynthesis and incorporation genes is tuned to the cellular demand for Pyl, which allows these archaea to navigate ambiguous stop decoding in response to environmental cues.
", "doi": "10.1101/2025.06.11.659114", "publisher": "Cold Spring Harbor Laboratory Press", "publication_date": "2025-06-14" }, { "id": "authors:4k9d0-2a571", "collection": "authors", "collection_id": "4k9d0-2a571", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230316-182819000.64", "type": "monograph", "title": "Physiological adaptation of sulfate reducing bacteria in syntrophic partnership with anaerobic methanotrophic archaea", "author": [ { "family_name": "Murali", "given_name": "Ranjani", "orcid": "0000-0003-4073-9910", "clpid": "Murali-Ranjani" }, { "family_name": "Yu", "given_name": "Hang", "orcid": "0000-0002-7600-1582", "clpid": "Yu-Hang-Hank" }, { "family_name": "Speth", "given_name": "Daan R.", "orcid": "0000-0002-2361-5935", "clpid": "Speth-Daan-R" }, { "family_name": "Wu", "given_name": "Fabai", "orcid": "0000-0001-5812-5621", "clpid": "Wu-Fabai" }, { "family_name": "Metcalfe", "given_name": "Kyle S.", "orcid": "0000-0002-2963-765X", "clpid": "Metcalfe-Kyle-S" }, { "family_name": "Cr\u00e9mi\u00e8re", "given_name": "Antoine", "orcid": "0000-0001-7382-2097", "clpid": "Cr\u00e9mi\u00e8re-Antoine" }, { "family_name": "Laso-P\u00e9rez", "given_name": "Rafael", "orcid": "0000-0002-6912-7865", "clpid": "Laso-P\u00e9rez-Rafael" }, { "family_name": "Malmstrom", "given_name": "Rex R.", "orcid": "0000-0002-4758-7369", "clpid": "Malmstrom-Rex-R" }, { "family_name": "Goudeau", "given_name": "Danielle", "orcid": "0000-0002-3785-032X", "clpid": "Goudeau-Danielle" }, { "family_name": "Woyke", "given_name": "Tanja", "orcid": "0000-0002-9485-5637", "clpid": "Woyke-Tanja" }, { "family_name": "Hatzenpichler", "given_name": "Roland", "orcid": "0000-0002-5489-3444", "clpid": "Hatzenpichler-Roland" }, { "family_name": "Chadwick", "given_name": "Grayson L.", "orcid": "0000-0003-0700-9350", "clpid": "Chadwick-Grayson-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we explore the metabolic adaptation of four syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a and Seep-SRB1g) from a phylogenomics perspective, tracing the evolution of conserved proteins in the syntrophic SRB clades, and comparing the genomes of syntrophic SRB to their nearest evolutionary neighbors in the phylum Desulfobacterota. We note several examples of gain, loss or biochemical adaptation of proteins within pathways involved in extracellular electron transfer, electron transport chain, nutrient sharing, biofilm formation and cell adhesion. We demonstrate that the metabolic adaptations in each of these syntrophic clades are unique, suggesting that they have independently evolved, converging to a syntrophic partnership with ANME. Within the clades we also investigated the specialization of different syntrophic SRB species to partnerships with different ANME clades, using metagenomic sequences obtained from ANME and SRB partners in individual consortia after fluorescent-sorting of cell aggregates from anaerobic sediments. In one instance of metabolic adaptation to different partnerships, we show that Seep-SRB1a partners of ANME-2c appear to lack nutritional auxotrophies, while the related Seep-SRB1a partners of a different methanotrophic archaeal lineage, ANME-2a, are missing the cobalamin synthesis pathway, suggesting that the Seep-SRB1a partners of ANME-2a may have a nutritional dependence on its partner. Together, our paired genomic analysis of AOM consortia highlights the specific adaptation and diversification of syntrophic SRB clades linked to their associated ANME lineages.", "doi": "10.1101/2022.11.23.517749", "publication_date": "2022-11-24" }, { "id": "authors:bzbxb-4t885", "collection": "authors", "collection_id": "bzbxb-4t885", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220808-886882000", "type": "monograph", "title": "Evolutionary Diversification of Methanotrophic Ca. Methanophagales (ANME-1) and Their Expansive Virome", "author": [ { "family_name": "Laso-P\u00e9rez", "given_name": "Rafael", "orcid": "0000-0002-6912-7865", "clpid": "Laso-P\u00e9rez-Rafael" }, { "family_name": "Wu", "given_name": "Fabai", "orcid": "0000-0001-5812-5621", "clpid": "Wu-Fabai" }, { "family_name": "Cr\u00e9mi\u00e8re", "given_name": "Antoine", "orcid": "0000-0001-7382-2097", "clpid": "Cr\u00e9mi\u00e8re-Antoine" }, { "family_name": "Speth", "given_name": "Daan R.", "orcid": "0000-0002-2361-5935", "clpid": "Speth-Daan-R" }, { "family_name": "Magyar", "given_name": "John S.", "orcid": "0000-0002-3586-8286", "clpid": "Magyar-John-S" }, { "family_name": "Krupovic", "given_name": "Mart", "orcid": "0000-0001-5486-0098", "clpid": "Krupovic-Mart" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "'Candidatus Methanophagales' (ANME-1) is a major order-level clade of archaea responsible for methane removal in deep-sea sediments through anaerobic oxidation of methane. Yet the extent of their diversity and factors which drive their dynamics and evolution remain poorly understood. Here, by sampling hydrothermal rocks and sediments, we expand their phylogenetic diversity and characterize a new deep-branching, thermophilic ANME-1 family, 'Candidatus Methanoxibalbaceae' (ANME-1c). They are phylogenetically closest to the short-chain-alkane oxidizers 'Candidatus Syntrophoarchaeales' and 'Candidatus Alkanophagales', and encode ancestral features including a methyl coenzyme M reductase chaperone McrD and a hydrogenase complex. Global phylogeny and near-complete genomes clarified that the debated hydrogen metabolism within ANME-1 is an ancient trait that was vertically inherited but differentially lost during lineage diversification. Our expanded genomic and metagenomic sampling allowed the discovery of viruses constituting 3 new orders and 16 new families that so far are exclusive to ANME-1 hosts. These viruses represent 4 major archaeal virus assemblages, characterized by tailless icosahedral, head-tailed, rod-shaped, and spindle-shaped virions, but display unique structural and replicative signatures. Exemplified by the analyses of thymidylate synthases that unveiled a virus-mediated ancestral process of host gene displacement, this expansive ANME-1 virome carries a large gene repertoire that can influence their hosts across different timescales. Our study thus puts forth an emerging evolutionary continuum between anaerobic methane and short-chain-alkane oxidizers and opens doors for exploring the impacts of viruses on the dynamics and evolution of the anaerobic methane-driven ecosystems.", "doi": "10.1101/2022.07.04.498658", "publication_date": "2022-07-06" }, { "id": "authors:ddbgb-8jb47", "collection": "authors", "collection_id": "ddbgb-8jb47", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210804-220359405", "type": "monograph", "title": "Microbial community of recently discovered Auka vent field sheds light on vent biogeography and evolutionary history of thermophily", "author": [ { "family_name": "Speth", "given_name": "Daan R.", "orcid": "0000-0002-2361-5935", "clpid": "Speth-Daan-R" }, { "family_name": "Yu", "given_name": "Feiqiao B.", "orcid": "0000-0003-3416-3046", "clpid": "Yu-Feiqiao-B" }, { "family_name": "Connon", "given_name": "Stephanie A.", "clpid": "Connon-Stephanie-A" }, { "family_name": "Lim", "given_name": "Sujung", "orcid": "0000-0001-6040-729X", "clpid": "Lim-Sujung" }, { "family_name": "Magyar", "given_name": "John S.", "orcid": "0000-0002-3586-8286", "clpid": "Magyar-John-S" }, { "family_name": "Pe\u00f1a", "given_name": "Manet E.", "orcid": "0000-0002-5835-0455", "clpid": "Pe\u00f1a-Manet-E" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-Stephen-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physico-chemical data and 16S rRNA amplicons to characterize the diversity, temperature optima, and biogeographic distribution of sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California, the deepest known hydrothermal vent field in the Pacific Ocean. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 1/3 of the currently known phylum diversity, showing the microbial community in Auka hydrothermal sediments is highly diverse. Large scale 16S rRNA amplicon screening of 227 sediment samples across the vent field indicates that the MAGs are largely representative of the microbial community. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota (Tharpobacteria) suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison of the average nucleotide identity of the Auka MAGs with MAGs from the Guaymas Basin vent field, found 400 km to the Northwest, revealed a remarkable 20% species-level overlap between vent sites, suggestive of long-distance species transfer and sediment colonization. An adapted version of a recently developed model for predicting optimal growth temperature to the Auka and Guaymas MAGs indicates several of these uncultured microorganisms could grow at temperatures exceeding the currently known upper limit of life. Extending this analysis to reference data shows that thermophily is a trait that has evolved frequently among Bacteria and Archaea. Combined, our results show that Auka vent field offers new perspectives on our understanding of hydrothermal vent microbiology.", "doi": "10.1101/2021.08.02.454472", "publication_date": "2021-08-03" }, { "id": "authors:cyt72-4zt71", "collection": "authors", "collection_id": "cyt72-4zt71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191004-102107177", "type": "monograph", "title": "ASM-Clust: classifying functionally diverse protein families using alignment score matrices", "author": [ { "family_name": "Speth", "given_name": "Daan R.", "orcid": "0000-0002-2361-5935", "clpid": "Speth-D-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "abstract": "Rapid advances in sequencing technology have resulted in the availability of genomes from organisms across the tree of life. Accurately interpreting the function of proteins in these genomes is a major challenge, as annotation transfer based on homology frequently results in misannotation and error propagation. This challenge is especially pressing for organisms whose genomes are directly obtained from environmental samples, as interpretation of their physiology and ecology is often based solely on the genome sequence. For complex protein (super)families containing a large number of sequences, classification can be used to determine whether annotation transfer is appropriate, or whether experimental evidence for function is lacking. Here we present a novel computational approach for de novo classification of large protein (super)families, based on clustering an alignment score matrix obtained by aligning all sequences in the family to a small subset of the data. We evaluate our approach on the enolase family in the Structure Function Linkage Database.", "doi": "10.1101/792739", "publication_date": "2019-10-03" }, { "id": "authors:h37x3-kbc45", "collection": "authors", "collection_id": "h37x3-kbc45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190401-084306692", "type": "monograph", "title": "FIND: Identifying Functionally and Structurally Important Features in Protein Sequences with Deep Neural Networks", "author": [ { "family_name": "Murali", "given_name": "Ranjani", "clpid": "Murali-R" }, { "family_name": "Hemp", "given_name": "James", "orcid": "0000-0001-7193-0553", "clpid": "Hemp-J" }, { "family_name": "Orphan", "given_name": "Victoria", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Bisk", "given_name": "Yonatan", "clpid": "Bisk-Y" } ], "abstract": "The ability to correctly predict the functional role of proteins from their amino acid sequences would significantly advance biological studies at the molecular level by improving our ability to understand the biochemical capability of biological organisms from their genomic sequence. Existing methods that are geared towards protein function prediction or annotation mostly use alignment-based approaches and probabilistic models such as Hidden-Markov Models. In this work we introduce a deep learning architecture (Function Identification with Neural Descriptions or FIND) which performs protein annotation from primary sequence. The accuracy of our methods matches state of the art techniques, such as protein classifiers based on Hidden Markov Models. Further, our approach allows for model introspection via a neural attention mechanism, which weights parts of the amino acid sequence proportionally to their relevance for functional assignment. In this way, the attention weights automatically uncover structurally and functionally relevant features of the classified protein and find novel functional motifs in previously uncharacterized proteins. While this model is applicable to any database of proteins, we chose to apply this model to superfamilies of homologous proteins, with the aim of extracting features inherent to divergent protein families within a larger superfamily. This provided insight into the functional diversification of an enzyme superfamily and its adaptation to different physiological contexts. We tested our approach on three families (nitrogenases, cytochrome bd-type oxygen reductases and heme-copper oxygen reductases) and present a detailed analysis of the sequence characteristics identified in previously characterized proteins in the heme-copper oxygen reductase (HCO) superfamily. These are correlated with their catalytic relevance and evolutionary history. FIND was then applied to discover features in previously uncharacterized members of the HCO superfamily, providing insight into their unique sequence features. This modeling approach demonstrates the power of neural networks to recognize patterns in large datasets and can be utilized to discover biochemically and structurally important features in proteins from their amino acid sequences.", "doi": "10.1101/592808", "publication_date": "2019-03-30" }, { "id": "authors:3gcg3-khn91", "collection": "authors", "collection_id": "3gcg3-khn91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180426-110252898", "type": "monograph", "title": "Methane on Mars and Habitability: Challenges and Responses", "author": [ { "family_name": "Yung", "given_name": "Yuk L.", "orcid": "0000-0002-4263-2562", "clpid": "Yung-Y-L" }, { "family_name": "Chen", "given_name": "Pin", "clpid": "Chen-Pin" }, { "family_name": "Nealson", "given_name": "Kenneth", "orcid": "0000-0001-5189-3732", "clpid": "Nealson-K-H" }, { "family_name": "Atreya", "given_name": "Sushil", "orcid": "0000-0002-1972-1815", "clpid": "Atreya-S-K" }, { "family_name": "Beckett", "given_name": "Patrick", "clpid": "Beckett-P" }, { "family_name": "Blank", "given_name": "Jennifer", "clpid": "Blank-J-G" }, { "family_name": "Ehlmann", "given_name": "Bethany", "orcid": "0000-0002-2745-3240", "clpid": "Ehlmann-B-L" }, { "family_name": "Eiler", "given_name": "John", "clpid": "Eiler-J-M" }, { "family_name": "Etiope", "given_name": "Giuseppe", "clpid": "Etiope-G" }, { "family_name": "Ferry", "given_name": "James G.", "clpid": "Ferry-J-G" }, { "family_name": "Forget", "given_name": "Francois", "clpid": "Forget-F" }, { "family_name": "Gao", "given_name": "Peter", "orcid": "0000-0002-8518-9601", "clpid": "Gao-Peter" }, { "family_name": "Hu", "given_name": "Renyu", "orcid": "0000-0003-2215-8485", "clpid": "Hu-Renyu" }, { "family_name": "Kleinb\u00f6hl", "given_name": "Armin", "clpid": "Kleinb\u00f6hl-A" }, { "family_name": "Klusman", "given_name": "Ronald", "clpid": "Klusman-R" }, { "family_name": "Lef\u00e8vre", "given_name": "Franck", "clpid": "Lef\u00e8vre-F" }, { "family_name": "Miller", "given_name": "Charles", "orcid": "0000-0002-9380-4838", "clpid": "Miller-C-E" }, { "family_name": "Mischna", "given_name": "Michael", "orcid": "0000-0002-8022-5319", "clpid": "Mischna-M-A" }, { "family_name": "Mumma", "given_name": "Michael", "clpid": "Mumma-M-J" }, { "family_name": "Newman", "given_name": "Sally", "orcid": "0000-0003-0710-995X", "clpid": "Newman-S" }, { "family_name": "Oehler", "given_name": "Dorothy", "clpid": "Oehler-D-Z" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Oremland", "given_name": "Ronald", "clpid": "Oremland-R-S" }, { "family_name": "Orphan", "given_name": "Victoria", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Popa", "given_name": "Radu", "clpid": "Popa-R" }, { "family_name": "Russell", "given_name": "Michael", "clpid": "Russell-M-J" }, { "family_name": "Shen", "given_name": "Linhan", "orcid": "0000-0003-3871-655X", "clpid": "Shen-Linhan" }, { "family_name": "Sherwood Lollar", "given_name": "Barbara", "clpid": "Sherwood-Lollar-B" }, { "family_name": "Stamenkovi\u0107", "given_name": "Vlada", "orcid": "0000-0003-2416-3683", "clpid": "Stamenkovi\u0107-V" }, { "family_name": "Staehle", "given_name": "Robert", "clpid": "Staehle-R-L" }, { "family_name": "Stolper", "given_name": "Daniel", "orcid": "0000-0003-3299-3177", "clpid": "Stolper-D-A" }, { "family_name": "Templeton", "given_name": "Alexis", "clpid": "Templeton-A" }, { "family_name": "Vandaele", "given_name": "Ann C.", "clpid": "Vandaele-A-C" }, { "family_name": "Viscardy", "given_name": "S\u00e9bastien", "clpid": "Viscardy-S" }, { "family_name": "Webster", "given_name": "Chris", "clpid": "Webster-C-R" }, { "family_name": "Wennberg", "given_name": "Paul O.", "orcid": "0000-0002-6126-3854", "clpid": "Wennberg-P-O" }, { "family_name": "Wong", "given_name": "Michael", "clpid": "Wong-Michael-L" }, { "family_name": "Worden", "given_name": "John", "orcid": "0000-0003-0257-9549", "clpid": "Worden-J-R" } ], "abstract": "Recent measurements of methane (CH_4) by the Mars Science Laboratory (MSL) now confront us with robust data that demand interpretation. Thus far, the MSL data have revealed a baseline level of CH_4 (\u223c0.4 parts per billion by volume [ppbv]), with seasonal variations, as well as greatly enhanced spikes of CH_4 with peak abundances of \u223c7\u2009ppbv. What do these CH_4 revelations with drastically different abundances and temporal signatures represent in terms of interior geochemical processes, or is martian CH_4 a biosignature? Discerning how CH_4 generation occurs on Mars may shed light on the potential habitability of Mars. There is no evidence of life on the surface of Mars today, but microbes might reside beneath the surface. In this case, the carbon flux represented by CH_4 would serve as a link between a putative subterranean biosphere on Mars and what we can measure above the surface. Alternatively, CH_4 records modern geochemical activity. Here we ask the fundamental question: how active is Mars, geochemically and/or biologically? In this article, we examine geological, geochemical, and biogeochemical processes related to our overarching question. The martian atmosphere and surface are an overwhelmingly oxidizing environment, and life requires pairing of electron donors and electron acceptors, that is, redox gradients, as an essential source of energy. Therefore, a fundamental and critical question regarding the possibility of life on Mars is, \"Where can we find redox gradients as energy sources for life on Mars?\" Hence, regardless of the pathway that generates CH_4 on Mars, the presence of CH_4, a reduced species in an oxidant-rich environment, suggests the possibility of redox gradients supporting life and habitability on Mars. Recent missions such as ExoMars Trace Gas Orbiter may provide mapping of the global distribution of CH_4. To discriminate between abiotic and biotic sources of CH_4 on Mars, future studies should use a series of diagnostic geochemical analyses, preferably performed below the ground or at the ground/atmosphere interface, including measurements of CH_4 isotopes, methane/ethane ratios, H_2 gas concentration, and species such as acetic acid. Advances in the fields of Mars exploration and instrumentation will be driven, augmented, and supported by an improved understanding of atmospheric chemistry and dynamics, deep subsurface biogeochemistry, astrobiology, planetary geology, and geophysics. Future Mars exploration programs will have to expand the integration of complementary areas of expertise to generate synergistic and innovative ideas to realize breakthroughs in advancing our understanding of the potential of life and habitable conditions having existed on Mars. In this spirit, we conducted a set of interdisciplinary workshops. From this series has emerged a vision of technological, theoretical, and methodological innovations to explore the martian subsurface and to enhance spatial tracking of key volatiles, such as CH_4.", "doi": "10.7907/Z990220K", "publisher": "Astrobiology", "publication_date": "2018-01" } ]