Butterfly wing pattern evolution is associated with changes in a Notch/Distal-less temporal pattern formation process

In butterflies there is a class of intervein wing patterns that have lines of symmetry halfway between wing veins. These patterns occur in a range of shapes, including eyespots, ellipses, and midlines, and were proposed to have evolved through developmental shifts along a midline-to-eyespot continuum. Here we show that Notch (N) upregulation, followed by activation of the transcription factor Distal-less (DII), is an early event in the development of eyespot and intervein midline patterns across multiple species of butterflies. A relationship between eyespot phenotype and N and DII expression is demonstrated in a loss-of-eyespot mutant in which N and DII expression is reduced at missing eyespot sites. A phylogenetic comparison of expression time series from eight moth and butterfly species suggests that intervein N and DII patterns are a derived characteristic of the butterfly lineage. Furthermore, prior to eyespot determination in eyespot-bearing butterflies, N and DII are transiently expressed in a pattern that resembles ancestral intervein midline patterns. In this study we establish N upregulation as the earliest known event in eyespot determination, demonstrate gene expression associated with intervein midline color patterns, and provide molecular evidence that wing patterns evolved through addition to and truncation of a conserved midline-to-eyespot pattern formation sequence.