Eyespots originated multiple times independently across the Lepidoptera

Eyespot color patterns often function as a defense against predators and in mate choice. 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 28 separate origins of Discal eyespots (in the discal wing region) and 19 separate origins of Marginal eyespots (in the marginal wing region), including four separate instances where eyespots were preserved in most extant representatives of a species radiation. The first two eyespot radiations we observed are in the Nymphalidae, with a Marginal eyespot radiation occurring before a Discal one. While the remaining two eyespot radiations were observed in the Saturniidae, occurring in a reverse fashion, where a Discal eyespot radiation preceded a Marginal eyespot radiation. Even though eyespots do not appear to be homologous across Lepidoptera they may share a homologous gene-regulatory network. Our phylogenetic inference provides a roadmap for future developmental and functional studies addressing this hypothesis. This study therefore has implications for our understanding of the evolution of serial homologues and of convergent evolution of visual signals in insects. ### Competing Interest Statement The authors have declared no competing interest.