Visual pigments of basal lineages of bony fishes support independent ecological shifts from a shallow marine to a freshwater niche.

Bony fishes (Osteichthyes) occupy a diverse range of aquatic habitats, yet the ecological transitions underlying their early evolution remain debated. Extant "living fossil" lineages-such as lungfishes and basal ray-finned fishes-are primarily restricted to benthic freshwater habitats, raising questions about the ancestral ecology of bony fishes. To investigate this, we reconstructed and expressed visual pigments from both extant and inferred ancestral taxa in vitro, enabling characterization of their spectral sensitivities. The results reveal that the ancestral visual phenotype is most consistent with adaptation to shallow-water light conditions. Furthermore, parallel shifts in the spectral tuning of visual pigments across both lobe-finned and ray-finned fish lineages were observed, with consistent patterns of shorter wavelength tuning in middle/long-wavelength-sensitive pigments, paired with longer wavelength shifts in others. The shifts of spectral tuning support an ecological transition from marine to freshwater habitats. Additionally, changes in rhodopsin retinal release rates and signatures of positive selection on opsin genes further point to independent visual adaptations to freshwater environments in both lineages. These findings suggest that early bony fish evolution involved ecological expansion from shallow marine habitats into deeper or more turbid freshwater environments, as reflected in parallel adaptations of visual systems to benthic photic conditions.