Freshwater temperate wetlands represent the largest natural source of methane emitted to the atmosphere, yet we know little about the factors impacting emission in these habitats. Here, we identify the biogeochemical and microbial genomic determinants impacting methane cycling, and the scale at which they operate along temperate freshwater wetland gradients.
Mud ecosites lacking above-ground vegetation or standing water produced significantly more methane in colder seasons, while summer plant primary productivity had the greatest methane emission.Consistently, the greatest in situ methane and methanogenic potential was associated with surface samples with high dissolved oxygen concentrations, and not the deeper anoxic samples. 16S rRNA gene analyses and shotgun metagenomics revealed Methanosaeta spp. were dominant in high methane emitting surface samples. Two reconstructed surface Methanosaeta genomes encode multiple, different oxygen tolerance mechanisms. We posit that recalictrant carbon constrains methanogenic activity, and we observed statistically different high-molecular-weight carbon profiles and a greater concentration of methanogenic substrates in methane-rich pore waters from surface samples. Although methanotrophs were broadly distributed across ecosites and depths, the highest relative abundances were detected in surface samples, and thus strongly correlated to in situ methane and oxygen concentrations. Near-complete reconstructed Methylobacter genomes revealed redox tolerance as an explanation for site-wide prevalence, with the potential for methane oxidation coupled to oxygen and nitrate. Despite the high richness and broad phylogenetic diversity of wetland soils, the majority of methane cycling across time, season, and depth was limited to a handful of taxa active primarily in shallow, rather than deep, samples. Together, our findings show that carbon quality and the abundance of specific microbial genera, not depth or dissolved oxygen concentrations, are key predictors of ecosystem-scale methane emissions.