摘要:SummaryAnimal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medakadmyand the clawed frogdm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization genedmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment ofdmyanddm-Was sex-determining genes.Graphical AbstractDisplay OmittedHighlights•We detected parallel amino acid substitutions in two sex-determining genes,dmyanddm-W•Both the substitutions fromdmrt1duplication are under positive selection•These substitutions enhanced their DNA-binding activity as transcription factors•These substitutions might have contributed to the establishment ofdmyanddm-WBiological Sciences; Genetics; Evolutionary Biology