AbstractEvolutionary transitions between fresh and salt water happen infrequently among bacterioplankton. Within the ubiquitous and highly abundant heterotrophic Alphaproteobacteria order Pelagibacterales (SAR11), most members live in marine habitats, but the LD12 subclade has evolved as a unique freshwater lineage. LD12 cells occur as some of the most dominant freshwater bacterioplankton, yet this group has remained elusive to cultivation, hampering a more thorough understanding of its biology. Here, we report the first successful isolation of an LD12 representative, strain LSUCC0530, using high-throughput dilution-to-extinction cultivation methods, and its complete genome sequence. Growth experiments corroborate ecological data suggesting active populations of LD12 in brackish water up to salinities of ~5. LSUCC0530 has the smallest closed genome thus far reported for a SAR11 strain (1.16 Mbp). The genome affirms many previous metabolic predictions from cultivation-independent analyses, like a complete Embden–Meyerhof–Parnas glycolysis pathway, but also provides novel insights, such as the first isocitrate dehydrogenase in LD12, a likely homologous recombination of malate synthase from outside of the SAR11 clade, and analogous substitutions of ion transporters with others that occur throughout the rest of the SAR11 clade. Growth data support metagenomic recruitment results suggesting temperature-based ecotype diversification within LD12. Key gene losses for osmolyte uptake provide a succinct hypothesis for the evolutionary transition of LD12 from salt to freshwater. For strain LSUCC0530, we propose the provisional nomenclature Candidatus fonsibacter ubiquis.