Cis-regulatory evolution of Wnt family genes contributes to a morphological difference between silkworm species.

Closely related species often exhibit distinct morphologies that can contribute to species-specific adaptations and reproductive isolation. One example is Lepidopteran caterpillar appendages, such as the "caudal horn" of Bombycoidea moths, which have evolved substantial morphological diversity among species in this group. Using interspecific crosses, we identify the genetic basis of the caudal horn size difference between Bombyx mori and its closest relative Bombyx mandarina. The three largest of eight QTL account for one third the mean horn length difference between the species. The largest of these, on chromosome 4, encompasses a conserved Wnt family gene cluster, key upstream regulators that are well-known for their roles in morphological diversification in animals. Using allele-specific expression analysis and CRISPR/Cas9 knockouts, we show that tissue-specific cis-regulatory changes to Wnt1 and Wnt6 contribute to the species difference in caudal horn size. This kind of modularity enables highly pleiotropic genes, including key upstream growth regulators, to contribute to the evolution of morphological traits without causing widespread deleterious effects.