Environmental gradients and methanogen guild composition underlie global variation in methanogenic pathway potential across soil and sediment microbiomes

Methanogenesis is a central microbial process in anaerobic soil and sediment carbon turnover and contributes to the biological potential for methane production. It is mediated by diverse archaeal lineages that use distinct substrate-entry pathways, yet how methanogenic pathway potential is organized across contrasting environmental microbiomes remains insufficiently understood. We analyzed 904 metagenomes spanning seven ecosystem types sampled from soil and sediment environments to characterize global patterns of methanogenic pathway potential, environmental associations, and methanogen guild composition. Across ecosystems, methanogenic pathway potential showed a recurring pattern of pathway contributions, with acetoclastic methanogenesis accounted for the largest proportion, followed by hydrogenotrophic and methylotrophic pathways. However, the relative contributions of these pathways varied significantly among ecosystems and were associated with soil organic carbon, pH, and temperature-related variables. Metatranscriptomic data from wetland soils, peatlands and paddy soils provided ecosystem-level evidence that pathway expression patterns were broadly consistent with metagenomic pathway profiles while also revealing differences between genomic potential and transcriptional activity. To link pathway-level functional variation to microbial populations, we reconstructed 110 high-quality methanogen metagenome-assembled genomes and assigned them to acetoclastic, hydrogenotrophic, methylotrophic, and multi-pathway guilds. Ecosystem-specific differences in pathway composition were associated with methanogen guild composition, while similar guild composition patterns could be maintained by distinct taxonomic lineages, indicating lineage composition variation within guilds. Methanogen taxa also differed in their associations with pathway composition, with taxa assigned to single pathway guilds showing stronger associations with pathway differentiation than multi-pathway taxa. These findings suggest that methanogenic pathway potential across soil and sediment microbiomes is associated with environmental gradients, methanogen functional guild composition and lineage composition variation within methanogenic guilds. By linking pathway-level functional potential to methanogen community composition, this study provides a genome-resolved basis for understanding methanogenic pathway variation in anaerobic environmental microbiomes.