Long-term hybridization in a karst window reveals the genetic basis of eye loss in cavefish.

Eye loss is a hallmark trait of animals inhabiting perpetual darkness, such as caves. The Mexican tetra ( provides an unparalleled model for studying the genetic basis of eye loss. There are two interfertile morphs of the Mexican tetra, sighted surface fish and multiple independently evolved eyeless, blind cavefish populations. Despite decades of research on eye loss in this species, our understanding of the precise genetic basis remains sparse. Here, we focused on the unique Caballo Moro cave, where there is a karst window collapse that introduced sunlight and coexistence of both eyed and eyeless cavefish of similar genetic background. This unique genetic mosaic allowed us to pinpoint coding mutations in Connexin 50 (Cx50), also known as gap junction protein alpha 8b (Gja8b), as critical in the genetic basis of eye loss. CRISPR based knockouts of Cx50 in surface fish result in small or absent eyes as young as 48 hours post-fertilization. Further, we identified similar mutations in Cx50 that alter predicted protein structure among other cave-dwelling fish and even subterranean mammals, indicating a conserved evolutionary mechanism of Cx50 mutations. We introduced a mutation (Cx50-S89K) in mice, which resulted in cataracts, smaller eyes, and smaller lenses. Mutations in Cx50 mimic those identified in human congenital cataracts. We additionally leveraged phenotypic variation in a hybrid cavefish population to demonstrate that eyes provide fish with a metabolic advantage, providing a mechanism by which loss of eyes could be favored by selection. This unique hybridization event allowed us to identify novel alleles that contribute to the convergent evolution of eye loss, providing profound insights into the genetic underpinnings of one of nature's most fascinating adaptive traits.