Species-specific coordinated gene expression and trans-regulation of larval color pattern in three swallowtail butterflies.

The diversity of butterfly larval color pattern has been attracted to people since Darwin's time; however, its molecular mechanisms still remain largely unknown. Larval body markings often differ completely between closely related species under natural selection. The final instar larvae of the swallowtail butterflies Papilio xuthus and Papilio polytes show a green camouflage pattern, whereas those of Papilio machaon show a warning color pattern, although P. xuthus and P. machaon are closely related species. To identify the genes that contribute to species divergence, we compared the expression pattern of eight pigment-associated genes between three Papilio species. The spatial expression pattern of melanin-related genes coincided with the species-specific cuticular markings. We newly found that the combination of bilin-binding protein and yellow-related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration. To distinguish whether the interspecific differences in pigment-associated genes are caused by cis-regulatory changes or distribution differences in trans-regulatory proteins, we compared species-specific mRNA expression in an F1 hybrid specimen. Px-YRG and Pp-YRG showed a similar expression pattern, suggesting that the change in expression of YRG is caused mainly by changes in the distribution of trans-regulatory proteins. Our findings shed light on the gene regulatory networks for butterfly larval color pattern.