Lineage-Specific Gene Expansion and Atypical Expression Pattern of Feminizing Gene Transformer in Stag Beetles

The molecular cascade of sex determination in insects exhibits remarkable diversity in its upstream factors while conserving the downstream doublesex (dsx) gene across various taxa. dsx encodes a transcription factor that undergoes sex-specific splicing, playing a central role in sex determination. This sex-specific splicing of dsx is generally regulated by the Transformer (Tra) protein. Functional Tra is exclusively expressed in females, governing the female-specific splicing of dsx and inducing feminization. Although the tra gene is widely conserved across various insect species, with the genome typically containing a single copy of the tra gene, it is lost or duplicated in specific taxa, resulting in lineage-specific variations in numbers. Given the flexibility of the tra gene number and meanwhile the deep conservation of its crucial function in sex determination across most investigated taxa, the tra gene has attracted attention as the key gene for evolutionary change in sex-determining mechanisms. In contrast to this conventional insight, unexpectedly, we discovered that the number of tra genes has expanded through gene duplication in stag beetles (Lucanidae, Coleoptera). In addition, we discovered that the tra gene in several species of stag beetles has the same splicing variant in both sexes. Our findings suggest that the tra gene in the Lucanidae family deviates from the basic pattern observed in other insects regarding copy number and expression profiles. The discovery of tra expansion and expression pattern divergence in the family Lucanidae may provide new insights into the evolutionary diversity of the sex differentiation cascade.