Accounting for only 8% of Earth's land cover, freshwater wetlands remain the foremost contributors to global methane emissions. Yet the microorganisms and processes underlying methane emissions from wetland soils remain poorly understood. Over a five-year period, we surveyed the microbial membership and in situ methane measurements from over 700 samples in one of the most prolific methane-emitting wetlands in the United States. We constructed a catalog of 2,502 metagenome-assembled genomes (MAGs), with more than half of the 70 bacterial and archaeal phyla sampled containing novel lineages. Integration of these data with 133 soil metatranscriptomes provided a genome-resolved view of the biogeochemical specialization and versatility expressed over wetland soil spatial and temporal gradients. Centimeter-scale depth differences best explained patterns of microbial community structure and transcribed functionalities, even more than land cover or temporal information. Moreover, while extended flooding restructured soil redox, this perturbation failed to reconfigure the transcriptional profiles of methane-cycling microorganisms, contrasting with theoretically expected responses to hydrological perturbations. Co-expression analyses, coupled with depth-resolved methane measurements, revealed the metabolisms and trophic structures most predictive of methane hotspots. Mapping the spatiotemporal transcriptional patterns on this compendium of biogeochemically classified soil-derived genomes begins to untangle the microbial carbon, energy, and nutrient processing contributing to wetland methane production.IMPORTANCESoil microbial ecology is increasingly recognized as essential to climate mitigation, but realizing its full potential requires shifting from static genome inventories to dynamic assessments of microbial activity. This study shows that methane-cycling microbes exhibit stable, depth-stratified expression patterns, even in response to major redox and flooding shifts, undermining assumptions that water-table manipulations common in wetland management can alone reduce methanogenesis. Instead, methane cycling is shaped by spatially organized, transcriptionally active networks involving not only methanogens but also methanotrophs, fermenters, and iron reducers. These findings expose the limitations of genome-only models and highlight the need for soil diagnostics that capture in situ activity. Together, we provide a foundation for developing activity-based microbiome tools, embedding microbial functions into Earth system models, and designing interventions that move beyond "single-lever" strategies and instead work with the structure and dynamics of microbial communities as complex, layered systems.

Environmental DNA (eDNA) approaches to monitoring biodiversity in terrestrial environments have largely focused on sampling water bodies, potentially limiting the geographic and taxonomic scope of eDNA investigations. We assessed the performance of two strictly terrestrial eDNA sampling approaches to detect arboreal mammals, a guild with many threatened and poorly studied taxa worldwide, within two central New Jersey (USA) woodlands. We evaluated species detected with metabarcoding using two eDNA collection methods (tree bark vs. soil sampling), and compared the performance of two detection methods (qPCR vs. metabarcoding) within a single species. Our survey, which included 94 sampling events at 21 trees, detected 16 species of mammals, representing over 60% of the diversity expected in the area. More DNA was found for the 8 arboreal versus 8 non-arboreal species detected (mean: 2466 vs. 289 reads/sample). Soil samples revealed a generally similar composition, but a lower diversity, of mammal species. Detection rates for big brown bat were 3.4 × higher for qPCR over metabarcoding, illustrating the enhanced sensitivity of single-species approaches. Our results suggest that sampling eDNA from on and around trees could serve as a useful new monitoring tool for cryptic arboreal mammal communities globally.

Terrestrial arthropods are a diverse taxonomic group of significant ecological and economic importance. Our ability to understand the diversity that comprises this group is hampered by the variety of sampling techniques and high level of taxonomic expertise required to identify individual species. DNA metabarcoding approaches have potential to overcome these challenges but have been mainly limited to studies where DNA is directly extracted from trapped individuals. We posit that collection of environmental DNA (eDNA) deposited on vegetation surfaces could provide an alternative method of conducting metabarcoding-based arthropod inventories. In this study, we illustrate the promise of characterizing arthropod biodiversity based on eDNA collected from terrestrial plant surfaces. We collected 40 paired samples using two novel eDNA sampling techniques-tree bark and foliage sampling-in a New Jersey, USA, pine barrens forest. Metabarcoding using two primer sets revealed significantly higher taxonomic richness for the 16S versus COI primer set (1077 vs. 650 molecular operational taxonomic units; MOTUs), as well as higher richness and diversity in foliage versus bark samples. Accumulation curves suggest that our samples captured about half of the available MOTU-level diversity. Matching to reference databases revealed 28 arthropod orders, 181 families, 353 genera, and 292 species. Despite having lower MOTU-level richness, the COI primer set revealed more taxa that were identified to species (197 vs. 115) and genus (227 vs. 173) thanks to a more complete reference database. The two primer sets and sampling substrates showed distinct community compositions that differed in important ecological traits (feeding guild, body size), demonstrating the utility of a multi-faceted sampling and analytical approach. Our study highlights the value of exploiting eDNA left on plant surfaces via metabarcoding for contributing to rapid arthropod inventories, and thus realizing a range of ecological research and management goals.

Reptiles are increasingly of conservation concern due to their susceptibility to habitat loss, emerging disease, and harvest in the wildlife trade. However, reptile populations are often difficult to monitor given the frequency of crypsis in their life history. This difficulty has left uncertain the conservation status of many species and the efficacy of conservation actions unknown. Environmental DNA (eDNA) surveys consistently elevate the detection rate of species they are designed to monitor, and while their use is promising for terrestrial reptile conservation, successes in developing such surveys have been sparse. We tested the degree to which inclusion of surface and soil eDNA sampling into conventional artificial-cover methods elevates the detection probability of a small, cryptic terrestrial lizard, Scincella lateralis. The eDNA sampling of cover object surfaces with paint rollers elevated per sample detection probabilities for this species 4-16 times compared with visual surveys alone. We readily detected S. lateralis eDNA under cover objects up to 2 weeks after the last visual detection, and at some cover objects where no S. lateralis were visually observed in prior months. With sufficient sampling intensity, eDNA testing of soil under cover objects produced comparable per sample detection probabilities as roller surface methods. Our results suggest that combining eDNA and cover object methods can considerably increase the detection power of reptile monitoring programs, allowing more accurate estimates of population size, detection of temporal and spatial changes in habitat use, and tracking success of restoration efforts. Further research into the deposition and decay rates of reptile eDNA under cover objects, as well as tailored protocols for different species and habitats, is needed to bring the technique into widespread use.

Terrestrial arthropods are abundant and diverse with outsized ecological and economic importance. Our ability to monitor this diversity is hampered by the variety of sampling techniques and taxonomic expertise required to catalog the species in an area. DNA metabarcoding approaches show promise but have mainly been limited to trapping studies where DNA is extracted from captured individuals. Here we illustrate the promise of terrestrial plant surfaces as reservoirs of environmental DNA (eDNA) that is rich in arthropod biodiversity information. We posit that collection of surface eDNA will enable easier and more rapid arthropod inventories. We collected 40 paired samples using two novel terrestrial surface eDNA sampling techniques – ‘roller’ tree bark and ‘spray’ foliage sampling – in a New Jersey, USA pine barrens forest. Metabarcoding using two primer sets (COI and 16S) revealed the presence of 177 arthropod families (from 21 orders), representing 80% of the family-level diversity expected in the area based on accumulation curves. Spray samples revealed more families than roller (148 vs. 126), while the two methods showed distinct, though overlapping, community composition. The two primer sets revealed similar alpha diversity, although they also captured different taxonomic subsets. A more limited comparison of roller and spray sampling with traditional aquatic and soil eDNA samples revealed a greater family diversity in surface samples, especially compared with soil. Our study highlights the value of eDNA metabarcoding surveys for achieving the elusive goal of rapid, cost-effective arthropod inventories, and thus realizing a range of ecological research and management goals.

Environmental genomics is a rapidly advancing field that promises to revolutionise the way in which industry conducts biodiversity monitoring. The International Association of Oil and Gas Producers Environmental Genomics Joint Industry Program (JIP) was formed in June 2019 with the aim of facilitating the development and uptake of environmental genomics within the oil and gas industry. Towards this goal, a white paper was produced that summarises the state‐of‐the‐art in environmental genomics research, and the opportunities and limitations of applying environmental genomics within industry. The white paper included a comprehensive literature review, and importantly, involved consultation with professionals from academic, regulatory and industry backgrounds from across the globe that had expertise in environmental genomics applications. While this consultation revealed a consensus that the application of environmental genomics has advanced greatly in a brief period, with demonstrable benefits, there was an acknowledgement that key aspects are still lacking that would allow confident application of genomics approaches within industry. Through the review and consultation process, a range of knowledge gaps and areas requiring further development were identified. To elucidate which of these areas were most critical to the successful application of environmental genomics within industry, the JIP is drafting guidance that describes sampling design considerations, minimum standards for laboratory analyses and approaches to genomics data interpretation. Through the drafting of guidance, the JIP hopes to determine which gaps are most critical, enabling these to be prioritised for targeted research. The guidance will then be updated regularly to capture the latest research outcomes.

Offshore oil and gas platforms have a finite life of production operations. Once production ceases, decommissioning options for the platform are evaluated. The role that a platform’s jacket plays as invertebrate habitat may inform the decommissioning decision. Worldwide, few scientific surveys specific to marine invertebrates have been conducted at platforms to collect data needed for biological assessments; however, recent research has examined the biological community utilizing historical industry-collected Remotely Operated Video (ROV) imagery that was not specifically collected for such purposes. To address concerns of bias in using video footage from industry structural surveys for biological assessments, we compared estimates of invertebrate density and species richness beneath a California oil platform determined using data from three sources: video from an industry ROV “structural” survey, video from a scientific ROV “biological” survey using the same vehicle, and photoquadrat still imagery from a scientific scuba diver survey. All surveys were conducted along the same jacket crossbeams. We found that the epibenthic assemblages characterized by the two ROV methods were similar. More species were observed in the diver photoquadrats than in either of the two kinds of ROV surveys, because smaller and more cryptic species could be resolved. We cannot extrapolate from this preliminary, localized study to all platforms worldwide; however, our results indicate routine platform structural integrity surveys are as valuable as dedicated scientific biological surveys for macroinvertebrates and represent an invaluable source for opportunistic marine community assessments.

The International Association of Oil and Gas Producers (IOGP) Environmental Genomics Joint Industry Program (JIP) was formed in June 2019. The aim of the JIP is to facilitate the development of guidelines for the application of environmental genomics to support environmental management activities in the oil and gas industry. Towards this goal, a white paper summarizing the state-of-the-art in environmental genomics research and how it may be used to advance technology development opportunities for the oil and gas industry was drafted. More specifically, a series of applications and focus areas of primary interest to oil and gas companies were covered including: baseline assessments; detection of key species; rapid assessment of invasive species; population status and dynamics; monitoring of environmental effects of oil and gas activities; remediation and restoration; sampling design; data analysis and interpretation; community representation; species abundance, distribution and viability; and real-time on-site measurement and analysis. baseline assessments; detection of key species; rapid assessment of invasive species; population status and dynamics; monitoring of environmental effects of oil and gas activities; remediation and restoration; sampling design; data analysis and interpretation; community representation; species abundance, distribution and viability; and real-time on-site measurement and analysis. In addition to the literature review, consultation of professionals from academic, regulatory, and industrial backgrounds with expertise on these topics was conducted. While there was a consensus that the application of environmental genomics has advanced greatly in a short period of time with demonstrable benefit potential, there was acknowledgement that key aspects of best management practices are still lacking. Furthermore, while the majority of regulators interviewed were aware to varying degrees of the methodological limitations which restrict the present use of environmental genomics in regulatory affairs, it transpired that there is considerable appetite and capacity amongst the regulatory community to engage in collaborative research initiatives with the oil and gas industry and academia. Through these academic, regulatory, and industrial consultation, specific environmental genomics study areas and applications requiring further development and refinement were identified. These include: methodological standardization, persistence and dispersal of eDNA; eDNA data integration with various other data types; improvement of reference databases; and refinement of molecular indices. methodological standardization, persistence and dispersal of eDNA; eDNA data integration with various other data types; improvement of reference databases; and refinement of molecular indices. Based on the above and considering the most efficient path to greater regulatory uptake for environmental genomic approaches for the oil and gas industry, the JIP’s recommendation is to pursue a Common-Garden Experiment. Such experiment should seek the involvement and ultimately endorsement from the Regulators marking the path towards wider regulatory acceptance and uptake of eDNA-based approaches.

Greenhouse gas (GHG) emissions from rivers are a critical missing component of current global GHG models. Their exclusion is mainly due to a lack of in-situ measurements and a poor understanding of the spatiotemporal dynamics of GHG production and emissions, which prevents optimal model parametrization. We combined simultaneous observations of porewater concentrations along different beach positions and depths, and surface fluxes of methane and nitrous oxide at a plot scale in a large regulated river during three water stages: rising, falling, and low. Our goal was to gain insights into the interactions between hydrological exchanges and GHG emissions and elucidate possible hypotheses that could guide future research on the mechanisms of GHG production, consumption, and transport in the hyporheic zone (HZ). Results indicate that the site functioned as a net source of methane. Surface fluxes of methane during river water stages at three beach positions (shallow, intermediate and deep) correlated with porewater concentrations of methane. However, fluxes were significantly higher in the intermediate position during the low water stage, suggesting that low residence time increased methane emissions. Vertical profiles of methane peaked at different depths, indicating an influence of the magnitude and direction of the hyporheic mixing during the different river water stages on methane production and consumption. The site acted as either a sink or a source of nitrous oxide depending on the elevation of the water column. Nitrous oxide porewater concentrations peaked at the upper layers of the sediment throughout the different water stages. River hydrological stages significantly influenced porewater concentrations and fluxes of GHG, probably by influencing heterotrophic respiration (production and consumption processes) and transport to and from the HZ. Our results highlight the importance of including dynamic hydrological exchanges when studying and modeling GHG production and consumption in the HZ of large rivers.

ABSTRACT Wetland soils are one of the largest natural contributors to the emission of methane, a potent greenhouse gas. Currently, microbial contributions to methane emissions from these systems emphasize the roles of acetoclastic and hydrogenotrophic methanogens, while less frequently considering methyl-group substrates (e.g., methanol and methylamines). Here, we integrated laboratory and field experiments to explore the potential for methylotrophic methanogenesis in Old Woman Creek (OWC), a temperate freshwater wetland located in Ohio, USA. We first demonstrated the capacity for methylotrophic methanogenesis in these soils using laboratory soil microcosms amended with trimethylamine. However, subsequent field porewater nuclear magnetic resonance (NMR) analyses to identify methanogenic substrates failed to detect evidence for methylamine compounds in soil porewaters, instead noting the presence of the methylotrophic substrate methanol. Accordingly, our wetland soil-derived metatranscriptomic data indicated that methanol utilization by the Methanomassiliicoccaceae was the likely source of methylotrophic methanogenesis. Methanomassiliicoccaceae relative contributions to mcrA transcripts nearly doubled with depth, accounting for up to 8% of the mcrA transcripts in 25-cm-deep soils. Longitudinal 16S rRNA amplicon and mcrA gene surveys demonstrated that Methanomassiliicoccaceae were stably present over 2 years across lateral and depth gradients in this wetland. Meta-analysis of 16S rRNA sequences similar (>99%) to OWC Methanomassiliicoccaceae in public databases revealed a global distribution, with a high representation in terrestrial soils and sediments. Together, our results demonstrate that methylotrophic methanogenesis likely contributes to methane flux from climatically relevant wetland soils. IMPORTANCE Understanding the sources and controls on microbial methane production from wetland soils is critical to global methane emission predictions, particularly in light of changing climatic conditions. Current biogeochemical models of methanogenesis consider only acetoclastic and hydrogenotrophic sources and exclude methylotrophic methanogenesis, potentially underestimating microbial contributions to methane flux. Our multi-omic results demonstrated that methylotrophic methanogens of the family Methanomassiliicoccaceae were present and active in a freshwater wetland, with metatranscripts indicating that methanol, not methylamines, was the likely substrate under the conditions measured here. However, laboratory experiments indicated the potential for other methanogens to become enriched in response to trimethylamine, revealing the reservoir of methylotrophic methanogenesis potential residing in these soils. Collectively, our approach used coupled field and laboratory investigations to illuminate metabolisms influencing the terrestrial microbial methane cycle, thereby offering direction for increased realism in predictive process-oriented models of methane flux in wetland soils.

Microbial carbon degradation and methanogenesis in wetland soils generate a large proportion of atmospheric methane, a highly potent greenhouse gas. Despite their potential to mitigate greenhouse gas emissions, knowledge about methane-consuming methanotrophs is often limited to lower-resolution single-gene surveys that fail to capture the taxonomic and metabolic diversity of these microorganisms in soils. Here our objective was to use genome-enabled approaches to investigate methanotroph membership, distribution, and in situ activity across spatial and seasonal gradients in a freshwater wetland near Lake Erie. 16S rRNA gene analyses demonstrated that members of the methanotrophic Methylococcales were dominant, with the dominance largely driven by the relative abundance of four taxa, and enriched in oxic surface soils. Three methanotroph genomes from assembled soil metagenomes were assigned to the genus Methylobacter and represented the most abundant methanotrophs across the wetland. Paired metatranscriptomes confirmed that these Old Woman Creek (OWC) Methylobacter members accounted for nearly all the aerobic methanotrophic activity across two seasons. In addition to having the capacity to couple methane oxidation to aerobic respiration, these new genomes encoded denitrification potential that may sustain energy generation in soils with lower dissolved oxygen concentrations. We further show that Methylobacter members that were closely related to the OWC members were present in many other high-methane-emitting freshwater and soil sites, suggesting that this lineage could participate in methane consumption in analogous ecosystems. This work contributes to the growing body of research suggesting that Methylobacter may represent critical mediators of methane fluxes in freshwater saturated sediments and soils worldwide. IMPORTANCE Here we used soil metagenomics and metatranscriptomics to uncover novel members within the genus Methylobacter. We denote these closely related genomes as members of the lineage OWC Methylobacter. Despite the incredibly high microbial diversity in soils, here we present findings that unexpectedly showed that methane cycling was primarily mediated by a single genus for both methane production ("Candidatus Methanothrix paradoxum") and methane consumption (OWC Methylobacter). Metatranscriptomic analyses revealed that decreased methanotrophic activity rather than increased methanogenic activity possibly contributed to the greater methane emissions that we had previously observed in summer months, findings important for biogeochemical methane models. Although members of this Methylococcales order have been cultivated for decades, multi-omic approaches continue to illuminate the methanotroph phylogenetic and metabolic diversity harbored in terrestrial and marine ecosystems.

The current paradigm, widely incorporated in soil biogeochemical models, is that microbial methanogenesis can only occur in anoxic habitats. In contrast, here we show clear geochemical and biological evidence for methane production in well-oxygenated soils of a freshwater wetland. A comparison of oxic to anoxic soils reveal up to ten times greater methane production and nine times more methanogenesis activity in oxygenated soils. Metagenomic and metatranscriptomic sequencing recover the first near-complete genomes for a novel methanogen species, and show acetoclastic production from this organism was the dominant methanogenesis pathway in oxygenated soils. This organism, Candidatus Methanothrix paradoxum, is prevalent across methane emitting ecosystems, suggesting a global significance. Moreover, in this wetland, we estimate that up to 80% of methane fluxes could be attributed to methanogenesis in oxygenated soils. Together, our findings challenge a widely held assumption about methanogenesis, with significant ramifications for global methane estimates and Earth system modeling.

Summary Despite being key contributors to biogeochemical processes, archaea are frequently outnumbered by bacteria, and consequently are underrepresented in combined molecular surveys. Here, we demonstrate an approach to concurrently survey the archaea alongside the bacteria with high‐resolution 16S rRNA gene sequencing, linking these community data to geochemical parameters. We applied this integrated analysis to hydric soils sampled across a model methane‐emitting freshwater wetland. Geochemical profiles, archaeal communities, and bacterial communities were independently correlated with soil depth and water cover. Centimeters of soil depth and corresponding geochemical shifts consistently affected microbial community structure more than hundreds of meters of lateral distance. Methanogens with diverse metabolisms were detected across the wetland, but displayed surprising OTU‐level partitioning by depth. Candidatus Methanoperedens spp. archaea thought to perform anaerobic oxidation of methane linked to iron reduction were abundant. Domain‐specific sequencing also revealed unexpectedly diverse non‐methane‐cycling archaeal members. OTUs within the underexplored Woesearchaeota and Bathyarchaeota were prevalent across the wetland, with subgroups and individual OTUs exhibiting distinct occupancy and abundance distributions aligned with environmental gradients. This study adds to our understanding of ecological range for key archaeal taxa in a model freshwater wetland, and links these taxa and individual OTUs to hypotheses about processes governing biogeochemical cycling.