The swallowtail butterfly Papilio polytes is sexually dimorphic and exhibits female-limited Batesian mimicry. This species also has two female forms, a non-mimetic form with male-like wing patterns, and a mimetic form resembling an unpalatable model, Pachliopta aristolochiae . The mimicry locus H constitutes a dimorphic Mendelian ‘supergene’, including a transcription factor gene doublesex ( dsx ). However, how the mimetic-type dsx ( dsx-H ) orchestrates the downstream gene network and causes the mimetic traits remains unclear. Here we performed RNA-seq-based gene screening and found that Wnt1 and Wnt6 are up-regulated by dsx-H during the early pupal stage and are involved in the red/white pigmentation and patterning of mimetic female wings. In contrast, a homeobox gene abdominal-A is repressed by dsx-H and involved in the non-mimetic colouration pattern. These findings suggest that dual regulation by dsx-H , induction of mimetic gene networks and repression of non-mimetic gene networks, is essential for the switch from non-mimetic to mimetic pattern in mimetic female wings.

Genetic polymorphisms underlie the convergent and divergent evolution of various phenotypes. Diverse colour patterns on caterpillars, which are ecologically important, are good models for understanding the molecular backgrounds of phenotypic diversity. Here we show that a single evolutionarily conserved gene apontic-like (apt-like) encoding for a putative transcription factor accounts for the silkworm p locus, which causes at least 15 different larval markings involved in branch-like markings and eye-spot formation. The expression of apt-like and melanin synthesis genes are upregulated in association with pigmented areas of marking mutants Striped (p S ) and normal ( þ p ) but not in the non-marking allele plain (p). Functional analyses, ectopic expression, RNAi and TALEN, demonstrate that apt-like causes melanin pigmentation in a cell-autonomous manner. These results suggest that variation in p alleles is caused by the differential expression of the gene apt-like which induces targeted elevation of gene expressions in the melanin synthesis pathway.

The authors note that two references were omitted from the article. The complete references appear below.Citations to refs. 42 and 43 should be included on page 8340, left column, second paragraph, line 23, where the reference callout "(33, 34)" should instead appear as " (33,34, 42, 43)." Also, on the same page, right column, first full paragraph, line 4, the reference callout "(36)" should instead appear as "(36, 42)." Lastly, in the same paragraph, line 9, the reference callout "(33, 34)" should instead appear as " (33,34, 42, 43)."

In a Batesian mimic butterfly Papilio polytes, mimetic females resemble an unpalatable model, Pachliopta aristolochiae, but exhibit a different color pattern from nonmimetic females and males. In particular, the pale-yellow region on hind wings, which correspondingly sends important putative signals for mimicry and mate preference, is different in shape and chemical features between nonmimetic and mimetic morphs. Recently, we found that mimetic-type doublesex [dsx (H)] causes mimetic traits; however, the control of dimorphic pale-yellow colors remains unclear. Here, we revealed that dsx (H) switched the pale-yellow colors from UV-excited fluorescent type (nonmimetic) to UV-reflecting type (mimetic), by repressing the papiliochrome II synthesis genes and nanostructural changes in wing scales. Photoreceptor reactivities showed that some birds and butterflies could effectively recognize mimetic and nonmimetic pale-yellow colors, suggesting that a genetic switch in the UV response of pale-yellow colors may play essential roles in establishing the dimorphic female-limited Batesian mimicry.

In recent years, many examples have been reported in which complex adaptive traits are regulated by supergene units consisting of multiple genes adjacent to each other on the chromosome. However, few supergene units have been functionally characterized, and it has not been demonstrated that multiple genes in a supergene unit are involved in the adaptive trait. The H locus on chromosome 25 has been shown to be responsible for the female-limited Batesian mimicry in Papilio polytes. The mimetic H locus, about 130 kb, is inverted at both ends from the non-mimetic h locus, and the sequences between H and h locus are diversified (Highly Diversified Region: HDR). The HDR of the H locus contains three genes, doublesex (dsx), U3X, and UXT, and RNAi analyses have shown that dsx is involved in the mimetic pattern formation. Here, we showed that knockdown by RNAi and knockout by Crspr/Cas9 of the transcriptional regulator UXT altered the mimetic pattern to the non-mimetic like pattern in hindwings. In addition, it was also shown by RNAi that long non-coding RNA U3X was functionally involved in the mimetic pattern formation, and by RNA-seq analysis that U3X induced the dsx expression and repressed the UXT expression. In addition, the knockdown of prospero and sir2 genes adjacent to the HDR also affected the hindwing pattern formation. These results indicate that not only genes within the HDR but also adjacent genes outside the HDR may be involved in the mimicry supergene function. This suggests that cis-elements regulating multiple gene expression exists within the HDR of the H-allele, and the need to consider a more extended functional unit of the supergene. This paper shows that multiple genes inside and outside the HDR are functionally involved in the formation of complex adaptive traits, and provides a new perspective on the evolution of the structure and functional units of the supergene.