Evolutionary conservation of the dmrt gene family and sex-biased dmrt2a expression during pseudo-male testis differentiation in flatfish

The dmrt (doublesex and mab-3 related transcription factor) gene family is pivotal in sex determination, gonadal differentiation, and developmental patterning in vertebrates, including flatfish. To further understand the evolution and potential functions of dmrt in flatfish, we conducted a genome-wide characterization of dmrt genes in the Chinese tongue sole ( Cynoglossus semilaevis ). Six genes— dmrt1 , dmrt2a , dmrt2b , dmrt3 , dmrt4 , and dmrt5 —were identified in the species, all containing a conserved DM (doublesex and mab-3) DNA-binding domain. Phylogenetic and synteny analyses revealed a well-conserved dmrt1/2/3 genomic cluster, suggesting strong evolutionary constraints and functional interdependence within teleosts. Tissue-specific transcriptome profiling demonstrated distinct sex-biased expression patterns among dmrt genes. Strikingly, dmrt2a exhibited significant upregulation in pseudo-males compared to females, providing evidence of its potential involvement in pseudo-male testis formation. Proteomic and transcriptomic analyses further indicated that dmrt2a may act upstream of key signaling cascades, including transforming growth factor-beta (TGF-β), mitogen-activated protein kinase (MAPK), and Wnt pathways, while repressing ribosome and steroid biosynthesis. These results suggest that dmrt2a may contribute to testicular differentiation by coordinating chromatin remodeling and signaling integration. Overall, this study provides new insight into the evolution and potential functions of the dmrt gene family in C. semilaevis and identifies dmrt2a as a candidate regulator involved in pseudo-male differentiation and sex reversal in flatfish.