Functional Conservation Amid Extreme Divergence in Freshwater SAR11 Hypervariable Region 2

The Ca. Pelagibacterales order (SAR11) is a ubiquitous bacterioplankton in aquatic ecosystems. The genomes of SAR11 bacteria contain hypervariable region 2 (HVR2), the largest HVR with extreme diversity. Previous studies collectively indicated the enrichment of cell-surface modification genes on HVR2, which have been hypothesized to facilitate phage evasion. However, characterisation of HVR2 has primarily targeted marine clades and been limited for freshwater SAR11 (aka. Fontibacterium). Here, we employed single-cell genomics and recovered 176 Fontibacterium HVR2 sequences from Lake Biwa (Japan) and Lake Zurich (Switzerland), across multiple sampling years for Lake Biwa. Comparative sequence analyses revealed that freshwater HVR2 showed high intra-population diversity and appears to evolve independently of the rest of the genome, mirroring patterns observed in marine counterparts. Nevertheless, highly conserved HVR2 sequence pairs were observed for distinct Fontibacterium species from the two intercontinental habitats, suggesting horizontal transfers of HVR2 with conserved functional configurations across divergent lineages and large spatial scales. Further comparison with 145 marine HVR2 sequences revealed HVR2 sequences displaying synteny between Fontibacterium and marine SAR11, and 209 gene families conserved across habitats, including three near-universally conserved families. All examined freshwater HVR2s contained initiator methionine tRNA genes concentrated near the boundaries of the 5S region, suggesting a tDNA-anchored integration mechanism. These results suggest the functional equivalence of HVR2 across marine and freshwater SAR11 clades, and the existence of a shared pool of HVR2 sequences that evolve reticulately through mutual genetic exchange, independent of the vertical inheritance of the rest of the genome.