Evolutionary remodeling of the ventral retina enables aerial vision in the four-eyed fish Anableps anableps

The emergence of evolutionary novelties remains a central challenge in biology, particularly for complex organs such as the vertebrate eye. Here, we use the four-eyed fish Anableps anableps to investigate adaptation to simultaneous aerial and aquatic vision. Single-nucleus and spatial transcriptomics reveal that the ventral retina, which processes aerial light, is developmentally expanded and exhibits region-specific opsin expression. Ventral PR1 cones express opn1mw2 rather than the longer-wavelength-sensitive opn1lw paralogs expressed dorsally. Machine-learning predictions indicate that Opn1mw2 is red-shifted relative to Opn1mw1 but remains blue-shifted relative to dorsal Opn1lw pigments. Dorsal-ventral patterning markers further reveal an overrepresentation of ventral retinal identities. Comparative spatial profiling of developing and adult Anableps and killifish indicates dorsal displacement of the retinal boundary, producing a proportionally larger ventral domain. Thus, Anableps evolved aerial vision through developmental expansion of the ventral retina and regional reconfiguration of opsin expression within conserved cone subtypes.