A Splicing Mutation in mitfa is Involved in the Depigmentation of Cavefish Triplophysa rosa.

Regression traits such as pigmentation loss in cave-dwelling species offer powerful models for understanding evolutionary mechanisms under extreme environments. In this study, we investigated the genetic and evolutionary mechanisms underlying pigmentation loss in the cavefish Triplophysa rosa, a depigmented, eyeless species endemic to subterranean habitats. Compared with its surface-dwelling relative T. stenura, T. rosa exhibited significantly reduced expression of melanogenesis genes, indicating transcriptional repression of pigmentation pathways. Further analysis revealed a novel splicing site mutation in melanocyte inducing transcription factor a (mitfa), which results in a 63-nt deletion and loss of 21 amino acids in the activation domain. Functional rescue assays in zebrafish confirmed that the loss of 21 amino acids in Mitfa severely compromises melanin synthesis. Additionally, a premature stop codon in tyrosinase-related protein 1a (tyrp1a) was detected, which may also contribute to the depigmented phenotype. Evolutionary analyses indicated that pigmentation-specific genes in the T. rosa lineage are under relaxed purifying selection, consistent with weakened selective constraints on pigmentation in cave environments. Collectively, our findings indicate that a splice-site mutation in mitfa, acting against a background of relaxed selection on pigmentation genes, contributes to pigmentation loss in T. rosa, offering integrated proximate (molecular) and evolutionary insights into the troglomorphic traits in cavefish.