Dietrich, Carsten

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

Summary The bacterial tree contains many deep‐rooting clades without any cultured representatives. One such clade is ‘ E ndomicrobia’, a class‐level lineage in the phylum E lusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free‐living member of this clade from sterile‐filtered gut homogenate of defaunated (starch‐fed) R eticulitermes santonensis . Strain R sa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO 2 . Ultrastructural analysis revealed a G ram‐negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of G roup IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and 15 N 2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to ‘ C andidatus   E ndomicrobium trichonymphae’ and other obligate endosymbionts, we propose the name ‘ E ndomicrobium proavitum ’ gen. nov., sp. nov. for the first isolate of this lineage and the name ‘ E ndomicrobia ’ class. nov. for the entire clade.