Diversification in ANME-1 archaea is associated with the presence of highly variable genomic hotspots.

Anaerobic methanotrophic (ANME) archaea have been primarily documented by metagenomic analysis of environmental samples. The mechanisms that drive their diversification and speciation are poorly understood. Here we analyse the phylogenomic diversity at the species and strain levels of clade ANME-1 from deep-sea cold seeps, as a model system with a well-studied phylogenetic framework. We reconstruct high-quality circular metagenomic-assembled genomes (cMAGs) and identify highly variable genomic hotspots that distinguish them. Genomic differentiation and diversification in ANME-1 is associated with genes involved in prokaryotic defense systems, transport mechanisms and methane metabolism. In addition, heterologous expression of ANME-1 hicAB operons supports their proposed role as toxin/antitoxin systems, possibly involved in mediating responses to environmental stresses.