Functional unit of supergene in female-limited Batesian mimicry of Papilio polytes.

Supergenes are sets of genes and genetic elements that are inherited like a single gene and control complex adaptive traits, but their functional roles and units are poorly understood. In Papilio polytes, female-limited Batesian mimicry is thought to be regulated by a ∼130 kb inversion region (highly diversified region: HDR) containing 3 genes, UXT, U3X, and doublesex (dsx) which switches non-mimetic and mimetic types. To determine the functional unit, we here performed electroporation-mediated RNAi analyses (and further Crispr/Cas9 for UXT) of genes within and flanking the HDR in pupal hindwings. We first clarified that non-mimetic dsx-h had a function to form the non-mimetic trait in female and only dsx-H isoform 3 had an important function in the formation of mimetic traits. Next, we found that UXT was involved in making mimetic-type pale-yellow spots and adjacent gene sir2 in making red spots in hindwings, both of which refine more elaborate mimicry. Furthermore, downstream gene networks of dsx, U3X, and UXT screened by RNA sequencing showed that U3X upregulated dsx-H expression and repressed UXT expression. These findings demonstrate that a set of multiple genes, not only inside but also flanking HDR, can function as supergene members, which extends the definition of supergene unit than we considered before. Also, our results indicate that dsx functions as the switching gene and some other genes such as UXT and sir2 within the supergene unit work as the modifier gene.