Comparative pangenome analysis of methanogenic archaea from diverse ecosystems reveals potential targets for methane mitigation in rumen microbiome

Rumen methanogenesis is a major biological contributor to methane emissions in ruminants, yet the extent to which functional markers align with taxonomic relationships and how genome content varies across habitats, remains poorly resolved. In this study, we integrated broad phylogenetic frameworks with pangenome-resolved analysis to characterize methanogenic archaea from diverse ecosystems, including seawater, freshwater, sewage, rumen, human gut, soil, and cockroach sources. By combining these insights with pangenome reconstruction and KEGG-based pathway mapping of methanogenesis, we reveal key evolutionary and functional patterns. Notably, phylogenies based on 16S rRNA and <italic>mcrA</italic> genes showed limited concordance: only two clades exhibited overlap between trees, with most clustering patterns lacking environmental specificity. This discrepancy reflects the deep conservation of 16S rRNA compared with the evolutionary plasticity of <italic>mcr</italic> genes, shaped by lateral gene transfer, gene loss, and pathway modularity. The pangenome comprised of 8,695 orthogroups across 71 genomes, with core and soft-core genes enriched in translation, amino acid metabolism, and coenzyme biosynthesis, while the shell contained many poorly annotated orthogroups, highlighting annotation gaps in archaeal genomes. KEGG analysis revealed habitat-specific signatures: rumen methanogens were notably depleted in genes of the acetyl-CoA pathway, whereas human gut methanogens lacked key cofactor biosynthesis modules, including those for coenzymes M, B, F₄₂₀, and methanofuran. From rumen-derived shotgun metagenomes, we identified 53 methane-producing, 4 canonical methanogenic, 10 potential competitor, and 1 methanotrophic metagenome-assembled genomes (MAGs) based on functional gene content. Competitor candidates included nitrate-reducing and Wood–Ljungdahl pathway (WLP)-utilizing acetogens, suggesting hydrogen redirection under high-hydrogen or inhibitor conditions. These findings support a functional marker strategy that integrates 16S rRNA with pathway-specific genes and a pangenome framework to enhance ecological interpretations of methanogens and to prioritize potential targets for methane mitigation in ruminants.