New isolated from reveals two cosmopolitan subfamilies of single-stranded DNA phages infecting marine and terrestrial Alphaproteobacteria.

The 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 . We have isolated 'Ascunsovirus oldenburgi' ICBM5, a novel phage distantly related to known . It infects 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 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 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 tree, suggesting that much of the ssDNA phage diversity remains in the dark.