Salinity-driven niche differentiation within the aquatic Luna-1 subcluster

Salinity imposes a major barrier to microbial dispersal and colonization due to the requirement for osmoadaptations to maintain cell turgor and protein stability. Estuaries may facilitate infrequent evolutionary transitions between freshwater and marine habitats, which are characterized by differences in both salinity and resource availability. Here we illustrate niche differentiation of the <i>Actinomycetota</i> Luna-1 subcluster sister lineages within an estuarine system: freshwater-adapted <i>Rhodoluna</i> and saltwater-adapted <i>Aquiluna</i>. Comparative genomic and transcriptomic analyses highlighted key differences in osmoregulation, photoheterotrophy, and nutrient acquisition. Both genera are differentiated by mechanisms for osmoregulation, phosphate and iron uptake, and carbohydrate utilization, and by their rhodopsin preference (actinorhodopsin or heliorhodopsin). To clarify which traits are habitat versus lineage specific, we investigated the global distribution of Luna-1 subcluster taxa. The two constituent genera are both more commonly known from freshwater sources, although there are reports of <i>Aquiluna</i> isolated from saltwater. Results here confirm that <i>Rhodoluna</i> is almost exclusively freshwater-derived. <i>Aquiluna</i> instead comprises distinct clades of predominantly freshwater- or saltwater-derived taxa, with approximately half of <i>Aquiluna</i> representing slight halophiles from brackish and marine waters. Consistent with observations from the estuary, traits associated with osmoregulation and photoheterotrophy (rhodopsin preference and carbohydrate utilization) differentiated saltwater <i>Aquiluna</i> and freshwater members of the global dataset (both <i>Aquiluna</i> and <i>Rhodoluna</i>), and are therefore likely to be habitat rather than lineage-specific traits. Together, findings demonstrate various genomic characteristics enabling habitat-based niche differentiation between and within lineages of the Luna-1 subcluster, providing insights into microbial adaptation across salinity gradients.