DNA methylation in cavefishGore et al 2 Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachón blind cave morph of the Mexican tetra Astyanax mexicanus 1-6 , no inactivating mutations have been found in any of these genes 2,3,7-10 . Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus.By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development.The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.Subterranean animals offer an excellent opportunity to study morphological, molecular and physiological changes that allow organisms to adapt to unique environments. Loss of eyes is one of the most common morphological features of cave-adapted animals, including many fish species. Blind cave fish (CF) morphs of Astyanax mexicanus evolved from surface fish (SF) during a few million years of isolation in dark Mexican caves 11 , with recent studies suggesting that regression of eyes evolved as part of a strategy to conserve energy in fish adapted to dark and nutrient deficient caves 12 .Although a number of studies have examined molecular mechanisms underlying eye loss DNA methylation in cavefish Gore et al 3 in Pachón cave-derived Astyanax mexicanus CF, recent sequencing of the Pachón CF genome and other studies revealed no inactivating null mutations in essential eye development genes 2,3,7-9 . In contrast, genome sequencing of another subterranean animal, the naked mole rat Heterocephalus glaber, showed combined functional loss of more than a dozen key eye genes due to inactivating mutations 13 . These findings suggest the possibility that epigenetic rather than genetic changes may mediate eye loss in Pachón cave fish. To test this possibility, we used CF and SF morphs of Astyanax mexicanus as well as wild type and DNA methylation and demethylation-deficient zebrafish Danio rerio to examine whether DNA methylation regulates eye formation, and whether eye loss in Pachón cave fish evolved at least in part through hypermethylation of key eye genes.At 36 hpf Astyanax mexicanus CF and SF embryos are superficially indistinguishable with properly formed lenses and optic cups in both morphs (Fig. 1a,b). By five days of development, however, degeneration of eye tissue is clearly evident (Fig. 1c,d), and by adulthood CF eyes ar...