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