Summary Recent discoveries of mcr and mcr‐like genes in genomes from diverse archaeal lineages suggest that methane metabolism is an ancient pathway with a complicated evolutionary history. One conventional view is that methanogenesis is an ancestral metabolism of the class Thermoplasmata. Through comparative genomic analysis of 12 Thermoplasmata metagenome‐assembled genomes (MAGs) basal to the Methanomassiliicoccales, we show that these microorganisms do not encode the genes required for methanogenesis. Further analysis of 770 Ca. Thermoplasmatota genomes/MAGs found no evidence of mcrA homologues outside of the Methanomassiliicoccales. Together, these results suggest that methanogenesis was laterally acquired by an ancestor of the Methanomassiliicoccales. The 12 analysed MAGs include representatives from four orders basal to the Methanomassiliicoccales, including a high‐quality MAG that likely represents a new order, Ca. Lunaplasma lacustris ord. nov. sp. nov. These MAGs are predicted to use diverse energy conservation pathways, including heterotrophy, sulfur and hydrogen metabolism, denitrification, and fermentation. Two lineages are widespread among anoxic, sedimentary environments, whereas Ca. Lunaplasma lacustris has thus far only been detected in alpine caves and subarctic lake sediments. These findings advance our understanding of the metabolic potential, ecology, and global distribution of the Thermoplasmata and provide insight into the evolutionary history of methanogenesis within the Ca. Thermoplasmatota.

Viromics is becoming an increasingly popular method for characterizing soil viral communities. DNase treatment of the viral size fraction prior to DNA extraction is meant to reduce contaminating free DNA and is a common step within viromics protocols to ensure that sequences are of viral origin.

0Recent discoveries of mcr and mcr-like complexes in genomes from diverse archaeal 2 1 lineages suggest that methane (and more broadly alkane) metabolism is an ancient pathway with 2 2 complicated evolutionary histories. The conventional view is that methanogenesis is an ancestral 2 3 metabolism of the archaeal class Thermoplasmata. Through comparative genomic analysis of 12 2 4Thermoplasmata metagenome-assembled genomes (MAGs), we show that these microorganisms 2 5do not encode the genes required for methanogenesis, which suggests that this metabolism may 2 6 have been laterally acquired by an ancestor of the order Methanomassiliicoccales. These MAGs 2 7 include representatives from four orders basal to the Methanomassiliicoccales, including a high-2 8 quality MAG (95% complete) that likely represents a new order, Ca. Lunaplasma lacustris ord. 2 9 nov. sp. nov. These MAGs are predicted to use diverse energy conservation pathways, such as 3 0 heterotrophy, sulfur and hydrogen metabolism, denitrification, and fermentation. Two of these 3 1 lineages are globally widespread among anoxic, sedimentary environments, with the exception 3 2 of Ca. Lunaplasma lacustris, which has thus far only been detected in alpine caves and subarctic 3 3 lake sediments. These findings advance our understanding of the metabolic potential, ecology, 3 4 and global distribution of the Thermoplasmata and provide new insights into the evolutionary 3 5 history of methanogenesis within the Thermoplasmata. 3 6 3 7

The small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms. Viral size-fraction metagenomics (viromics) protocols facilitate enrichment of viral DNA from environmental samples, and these protocols typically include a DNase treatment of the post-0.2 μm viromic fraction to remove contaminating free DNA prior to virion lysis. However, DNase may also remove desirable viral genomic DNA (e.g., contained in virions compromised due to frozen storage or laboratory processing), suggesting that DNase-untreated viromes might be useful in some cases. In order to understand how virome preparation with and without DNase treatment influences the resultant data, here we compared 15 soil viromes (7 DNase-treated, 8 untreated) from 8 samples collected from agricultural fields prior to tomato planting. DNase-treated viromes yielded significantly more assembled viral contigs, contained significantly less non-viral microbial DNA, and recovered more viral populations (vOTUs) through read mapping. However, DNase-treated and untreated viromes were statistically indistinguishable, in terms of ecological patterns across viral communities. Although results suggest that DNase treatment is preferable where possible, in comparison to previously reported total metagenomes from the same samples, both DNase-treated and untreated viromes were significantly enriched in viral signatures by all metrics compared, including a ~225 times greater proportion of viral reads in untreated viromes compared to total metagenomes. Thus, even without DNase treatment, viromics was preferable to total metagenomics for capturing viral diversity in these soils, suggesting that preparation of DNase-untreated viromes can be worthwhile when DNase treatment is not possible