Spalt expression and the development of melanic color patterns in pierid butterflies

We use immunohistochemistry targeting Sal proteins across several pierids and show that Sal is associated with dense patches of melanization across species but is not associated with vein-melanization or diffuse melanization on the wing. In addition, Sal is expressed along cross-veins and wing compartment midlines that do not develop melanization. Male and female P. rapae spots are sexually dimorphic in size and this dimorphism is also present in the domains of Sal expression. Finally, by disrupting cells positioned in the center of the anterior black spots of P. rapae, before and during the time of Sal expression, spot size was reduced.Our results suggest, but do not conclusively show, that pierid spots may develop in a manner similar to that of nymphalid eyespots, that is, containing a group of signaling cells at the center of the pattern responsible for the differentiation of the complete spot, and that spots and eyespots share at least one signal-response gene in common, the transcription factor Sal. We propose that focal differentiation and focal signaling mechanisms evolved prior to the split of the nymphalid and pierid lineages.