In the genetic sex-determining system, the sex-determining genes on the sex chromosomes, which bifurcate for female and male development, have been diversified even among closely-related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. We previously isolated a W-linked sex-determining gene dm-W in the frog Xenopus laevis, and found that three neofunctionalization-type sex-determining genes, dm-W, the medaka fish dmy, and mammalian Sry, have a higher substitution rate in their DNA-binding domain-coding regions than their prototype genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of the two sex determining-genes, dmy and dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, a common amino acid substitution from serine (S) to threonine (T) was found at position 15 on the DNA-binding DM domains of both ancestral DMY and DM-W. We confirmed that the parallel S15T substitutions on the two DNA-binding domains are under positive selection. Both two types of DNA-protein binding experiments, an electrophoretic mobility shift assay and bio-layer interferometry, demonstrated that these S15T substitutions could strengthen the DNA-binding abilities. We also revealed that the S15T substitutions enhanced the transcriptional regulation function in a luciferase reporter assay in co-transfected 293T cells. These findings suggest that the parallel S15T substitutions after dmrt1 duplication may have contributed to the establishment of both dmy and dm-W in the ancestors of the<br />
medaka fish and the frog X. laevis as sex-determining genes.