ABSTRACT 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. IMPORTANCE Soil 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.
Borton et al. (Mon,) studied this question.