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 Actinomycetota Luna-1 subcluster sister lineages within an estuarine system: freshwater-adapted Rhodoluna and saltwater-adapted Aquiluna. 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 Aquiluna isolated from saltwater. Results here confirm that Rhodoluna is almost exclusively freshwater-derived. Aquiluna instead comprises distinct clades of predominantly freshwater- or saltwater-derived taxa, with approximately half of Aquiluna 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 Aquiluna and freshwater members of the global dataset (both Aquiluna and Rhodoluna), 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.