Despite their large number, viruses present in the environment remain largely unknown. Metagenomic approaches, targeting viruses specifically or not, have allowed us a better understanding of the composition of natural viral communities, with Caudoviricetes, Microviridae, Cressdnaviricota or Phycodnaviridae being the most frequently found viral groups. Metagenomes are gradually revealing the extent of the diversity of these groups and their structure, highlighting the large number of species, genera and even viral families, most of which being seen for the first time. Within these groups, the gene content, infected hosts and inhabited ecosystems are often consistent with the evolutionary history traced with marker genes. Thus, the diversity of viruses and their genes is more a reflection of their ancient origin and long coevolution with their hosts than of their ability to mutate rapidly.

The Microviridae family represents one of the major clades of single-stranded DNA (ssDNA) phages. Their cultivated members are lytic and infect Proteobacteria, Bacteroidetes, and Chlamydiae. Prophages have been predicted in the genomes from Bacteroidales, Hyphomicrobiales, and Enterobacteriaceae and cluster within the 'Alpavirinae', 'Amoyvirinae', and Gokushovirinae. We have isolated 'Ascunsovirus oldenburgi' ICBM5, a novel phage distantly related to known Microviridae. It infects Sulfitobacter dubius SH24-1b and uses both a lytic and a carrier-state life strategy. Using ICBM5 proteins as a query, we uncovered in publicly available resources sixty-five new Microviridae prophages and episomes in bacterial genomes and retrieved forty-seven environmental viral genomes (EVGs) from various viromes. Genome clustering based on protein content and phylogenetic analysis showed that ICBM5, together with Rhizobium phages, new prophages, episomes, and EVGs cluster within two new phylogenetic clades, here tentatively assigned the rank of subfamily and named 'Tainavirinae' and 'Occultatumvirinae'. They both infect Rhodobacterales. Occultatumviruses also infect Hyphomicrobiales, including nitrogen-fixing endosymbionts from cosmopolitan legumes. A biogeographical assessment showed that tainaviruses and occultatumviruses are spread worldwide, in terrestrial and marine environments. The new phage isolated here sheds light onto new and diverse branches of the Microviridae tree, suggesting that much of the ssDNA phage diversity remains in the dark.