Conserved roles of Distal-less and spalt in regulating butterfly melanic patterns

Simple spot patterns are proposed to be ancestral to more complex eyespot patterns found on the wings of butterflies. Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in two separate functions in establishing nymphalid butterfly eyespots: in the differentiation of their central signaling cells, or foci, necessary for eyespot ring formation, and in scale melanisation. However, little is known about the functions of these genes in the development of more primitive spot patterns. Here, we study the roles of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. We examined the expression pattern of both genes in developing larval and pupal wings and explored their functions using the CRISPR/Cas9 system. In P. canidia, both Dll and sal are expressed in the tips and along the margin of pupal wings, areas of future scale melanisation. Additionally, sal alone, is expressed in the future black spot patterns. CRISPR knockouts of Dll and sal showed that both genes are required for promoting melanic scales in the areas where they are expressed. Both Dll and sal also function as repressors of the pteridine pigment pathway. We conclude that both genes share a conserved role in promoting scale melanisation, across pierid and nymphalid butterflies, but are unlikely to be involved in differentiating spot centers. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct and independently evolved in each lineage.