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. While visual systems have repeatedly undergone degeneration or loss, examples of innovation are rare and poorly understood. Mechanistic insight into such gains requires tractable systems in which novel visual functions have evolved. Here we leverage the four-eyed fish Anableps anableps to uncover the cellular and molecular basis of adaptation to simultaneous aerial and aquatic light. Combining single-nucleus and spatial transcriptomics, we show that the Anableps ventral retina has been developmentally and functionally reconfigured for aerial vision. In fully aquatic teleosts such as zebrafish, ventral photoreceptors are tuned to red-shifted, downwelling light. In contrast, we show that Anableps exhibits an expanded and diversified ventral cone population encompassing ultraviolet-, blue-, green-, and red-sensitive types, enabling broad-spectrum, high-intensity vision from above water. To estimate Anableps opsin spectral properties, we employed OPTICS, a machine-learning model of opsin spectral tuning, which predicts that the ventrally expressed opn1mw2 opsin is red-shifted relative to typical green-sensitive pigments. Analysis of dorsal-ventral patterning markers reveals an overrepresentation of ventral retinal cell types. Consistent with this, comparative spatial transcriptomic profiling of developing and adult Anableps and the killifish (Fundulus heteroclitus) indicates a dorsal displacement of the dorsal-ventral boundary in Anableps, resulting in a proportionally larger ventral retinal domain. Collectively, our results reveal that Anableps achieved its unique dual visual capacity by developmentally expanding and functionally reprogramming the ventral retina for aerial light detection, an evolutionary innovation that redefines how retinal domains can adapt to radically different visual environments.