Eyespots originated multiple times independently across the Lepidoptera

Eyespot colour patterns demonstrate an effective defence against predators and are found in many animal lineages. 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 32 separate origins of Discal eyespots (in the discal wing region) and 21 separate origins of Marginal eyespots (in the marginal wing region). In two instances, eyespots were preserved in most extant representatives of a subsequent species radiation. In one such instance, in the Nymphalidae, ancestral state reconstructions suggest that an ancestor with Marginal eyespots predated the origin of one or more lineages with Discal eyespots, while in the Saturniidae we observed the opposite. We conclude that eyespots do not appear to be homologous across the Lepidoptera. Our phylogenetic inference provides a roadmap for future developmental and functional studies that can address whether Discal and Marginal eyespots share the same 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.