Regulation of the amh gene promoter activity by SF-1 in sex-reversing Cheilinus undulatus and its adaptive evolution

The humphead wrasse (Cheilinus undulatus) is a protogynous hermaphroditic fish and a top predator in coral reef ecosystems. However, overfishing has led to a significant decline in its population, making it challenging to study its adaptive evolution and sex reversal mechanisms. In this study, comparative genomics analyses revealed adaptive evolutionary signals associated with feeding capacity, digestive function, and color pattern transformation in C. undulatus. Notably, positive selection signals were detected in the key masculinization pathway gene amh in C. undulatus, Amphiprion percula (a protandrous hermaphrodite), and Oryzias latipes (an XY sex-determination species). Furthermore, a positive correlation was observed between body size and transposon content in C. undulatus and Labrus bergylta. A total of 75 nuclear receptors (NRs) were identified in C. undulatus, with all family members containing both the zf-C4 (PF00105) and Hormone_recep (PF00104) domains, except for the three NR0 members, which possessed only the Hormone_recep (PF00104) domain. Additionally, a 184 bp fragment of exon 1 and a 2229 bp upstream sequence of the amh gene were cloned, revealing three predicted SF-1 transcription factor binding sites. Dual-luciferase reporter assays demonstrated that the recombinant amh promoter construct (pGL4.10-amh) exhibited basal promoter activity, and SF-1 significantly enhanced this activity. Site-directed mutagenesis further confirmed that the SF-1 regulatory site is located between -1413 bp and -1403 bp upstream of the amh gene, indicating SF-1’s direct role in amh regulation and its potential involvement in the sex reversal process of C. undulatus. Overall, this study provides molecular evidence of the sex reversal mechanism and signals of adaptive evolution in C. undulatus, enhancing the understanding of this endangered species.