Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.
Butterfly eyespots play an essential role in natural and sexual selection, yet the evolutionary origins of eyespots and of their underlying gene regulatory network remain unknown. By scoring phenotypes and wing expression of five genes in 399 and 21 nymphalid species, respectively, we tested when eyespots and expression of their associated genes evolved. We found that the origin of eyespots was concurrent with the origin of the gene expression patterns, approximately 90 million years ago. Following this event, many genes expressed in eyespot development were lost in some lineages without a corresponding loss of eyespots, indicating substantial evolution in the cluster of genes associated with eyespots. This finding suggests that complex traits such as butterfly eyespots may initially evolve by re-deploying pre-existing gene regulatory networks, which are subsequently trimmed of genes that are unnecessary in the novel context.