Deep-branching Chloroflexota lineages illuminate the eco-evolutionary foundation of cross-ecosystem colonization.

Bacteria span Earth's ecosystems, coupling ecological versatility with genome-architectural reconfiguration across shifting physicochemical conditions. Yet the genomic routes by which free-living lineages cross ecosystem boundaries, and the consequences for genome architecture, remain poorly understood. Here, we use comparative and evolutionary genomics to investigate a soil-to-sediment-to-freshwater transition in Limnocylindria, an abundant clade within the Chloroflexota phylum. Two sister families show contrasting strategies. CSP1-4 expands genomes through niche-specific gene acquisition, whereas Limnocylindraceae undergoes genome reduction and metabolic simplification-revealing alternative evolutionary routes to similar ecological outcomes. In Limnocylindraceae, the loss of key DNA glycosylases coincides with degradation of base excision repair and is consistent with a hypermutator state that may have accelerated genomic erosion during freshwater specialization, potentially facilitating ecological expansion. This reductive genome trajectory is associated with a freshwater-adapted lineage with unexpectedly high GC content, challenging canonical links between base composition and genome size. While mutational processes appear to dominate genome restructuring, proteome-level patterns suggest selection favoring carbon- and nitrogen-efficient amino acid usage, implying that adaptive refinement can emerge alongside primarily non-adaptive dynamics. Overall, our findings are consistent with mutation-driven genome reduction and proteome optimization acting in concert to support cross-ecosystem boundary crossing and freshwater specialization in a free-living Chloroflexota lineage.