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Methanogenic Thermoplasmata of the novel order Methanomassiliicoccales were recently discovered in human and animal gastro-intestinal tracts (GITs). However, their distribution in other methanogenic environments has not been addressed systematically. Here, we surveyed Methanomassiliicoccales presence in wetland soils, a globally important source of methane emissions to the atmosphere, and in the GITs of different animals by PCR targeting their 16S rRNA and methyl:coenzyme M reductase (α-subunit) genes. We detected Methanomassiliicoccales in all 16 peat soils investigated, indicating their wide distribution in these habitats. Additionally, we detected their genes in various animal faeces. Methanomassiliicoccales were subdivided in two broad phylogenetic clades designated 'environmental' and 'GIT' clades based on differential, although non-exclusive, habitat preferences of their members. A well-supported cluster within the environmental clade comprised more than 80% of all wetland 16S rRNA gene sequences. Metagenome assembly from bovine rumen fluid enrichments resulted in two almost complete genomes of both Methanomassiliicoccales clades. Comparative genomics revealed that members of the environmental clade contain larger genomes and a higher number of genes encoding anti-oxidative enzymes than animal GIT clade representatives. This study highlights the wide distribution of Methanomassiliicoccales in wetlands, which suggests that they contribute to methane emissions from these climate-relevant ecosystems.

Phylogenetic and genomic analysis ofMethanomassiliicoccalesin wetlands and animal intestinal tracts reveals clade-specific habitat preferences