Sampling environmental DNA from trees and soil to detect cryptic arboreal mammals.

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.

Combining surface and soil environmental DNA with artificial cover objects to improve terrestrial reptile survey detection.

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.

El interés por la conservación de los reptiles es cada vez mayor debido a su susceptibilidad ante la pérdida del hábitat, enfermedades emergentes y la captura para el mercado de fauna. Sin embargo, las poblaciones de reptiles son difíciles de monitorear por lo frecuente que es la cripsis en sus historias de vida. Esta dificultad deja incierto el estado de conservación de muchas especies y desconocida la eficacia de las acciones de conservación. Los censos de ADN ambiental (DNAa) elevan sistemáticamente la tasa de detección de las especies que monitorean, y aunque su uso es prometedor para la conservación de los reptiles terrestres, han sido escasos los éxitos en el desarrollo de dichos censos. Analizamos el grado al que la inclusión del muestreo de DNAa superficial y del suelo a los métodos convencionales de cobertura artificial eleva la probabilidad de detección de una pequeña lagartija terrestre críptica: Scincella lateralis. El muestreo de DNAa de las superficies con cobertura de objetos con rodillos de pintura elevó las probabilidades de detección por muestra para esta especie 4-16 veces más que los censos visuales. Detectamos fácilmente el DNAa de S. lateralis bajo los objetos de cubierta hasta dos semanas después de la última detección visual y en algunos objetos de cubierta en donde no se había observado en los meses previos a S. lateralis. Con suficiente intensidad de muestreo, el análisis de DNAa del suelo bajo objetos de cubierta produjo probabilidades de detección por muestra comparables como métodos de rodillo superficial. Nuestros resultados sugieren que la combinación del DNAa y los métodos de objetos de cobertura puede incrementar considerablemente el poder de detección de los programas de monitoreo de reptiles, lo que permite estimaciones más precisas del tamaño poblacional, detección de los cambios espaciales y temporales en el uso de hábitat y el éxito de rastreo de los esfuerzos de restauración. Además, se necesita la investigación sobre las tasas de depósito y descomposición del DNAa de reptiles bajo objetos de cubierta, así como los protocolos hechos para diferentes especies y hábitats, para que la técnica entre al uso difundido.

Methane and nitrous oxide porewater concentrations and surface fluxes of a regulated river.

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.

Uncovering the Diversity and Activity of Methylotrophic Methanogens in Freshwater Wetland Soils.

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.

Members of the Genus Methylobacter Are Inferred To Account for the Majority of Aerobic Methane Oxidation in Oxic Soils from a Freshwater Wetland.

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.

Methanogenesis in oxygenated soils is a substantial fraction of wetland methane emissions.

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.

High-resolution sequencing reveals unexplored archaeal diversity in freshwater wetland soils.

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.