Group II Mcr-encoding archaea exhibit methane-cycling potential in geothermal springs

Methane is a potent greenhouse gas with profound implications for climate change, and its global emissions are largely driven by biological methanogenesis—a process long thought to be restricted to Euryarchaeota. However, emerging evidence indicates that Thermoproteota (formerly the TACK superphylum) also carry this capability. Through metagenomic sequencing of 157 terrestrial geothermal springs in Tengchong, China, we here reconstructed 104 methyl-coenzyme M reductase (Mcr)-containing metagenome-assembled genomes (MAGs). Among these, 90 were identified as MAGs encoding Group II Mcr proteins, which predominated in relative abundance over euryarchaeotal MAGs (Group I Mcr lineages) in most geothermal spring samples. Methanosuratincolia was the most numerous lineage among the recovered MAGs, and a typical operon structure associated with methyl catabolism was delineated across this group. Physicochemical factors, particularly pH and temperature, were associated with the distribution of Mcr-containing archaea, with metatranscriptomics revealing high transcriptional activity of genes associated with methane metabolism in thermal (> 70 °C) and alkaline (pH > 9) environments. We identified three Bathyarchaeia MAGs encoding pathways potentially linked to hydrogenotrophic methanogenesis and heterotrophic metabolism. Evolutionary analyses suggest a possible horizontal gene transfer event between Mcr-containing Bathyarchaeia and Methanonezhaarchaeia, although the direction of transfer cannot be resolved. Phylogenetic reconstruction of methanogenesis-associated proteins is consistent with methylotrophic methanogenesis being ancestral among currently sampled Mcr-containing Thermoproteota, followed by lineage-specific acquisition, loss and modification of hydrogenotrophic components. Our study expands the known diversity of Mcr-containing archaea, offering new insights into their biogeography, ecological functions and evolutionary origins of underdescribed lineages.