Eyespots originated multiple times independently across the Lepidoptera

Eyespot colour patterns function as an effective defence against predators and have evolved numerous times independently across animals. In Nymphalid butterflies, eyespots have a single evolutionary origin close to the base of this clade, but eyespots are also present in many other lepidopteran lineages and may have multiple independent origins. Here we use phylogenetic comparative methods to investigate the evolution of eyespots across a multi-superfamily phylogeny of Lepidoptera, and to pinpoint lineages in which eyespots likely originated independently. We find a total of 30 separate origins of Discal eyespots (in the discal wing region) and 21 separate origins of Marginal eyespots (in the marginal wing region), a type of serial homologue. In four instances eyespots were preserved in most extant representatives of a subsequent species radiation. In one such instance, in the Nymphalidae, we found a Marginal eyespot common ancestor predating the origin of a Discal eyespot baring ancestor, while in the Saturniidae we observed the opposite. We conclude that eyespots do not appear to be homologous across the Lepidoptera. However, our phylogenetic inference provides a roadmap for future developmental and functional studies that can address whether discal and marginal eyespots share a homologous gene-regulatory network. This study, therefore, has implications for our understanding of the evolution of serial homologues and of convergent evolution of visual signals in insects.