The origin of a novel complex trait: butterfly eyespots

An important and still largely unanswered question in the field of evo-devo concerns the molecular changes that lead to the origin of novel complex traits. One such trait is the eyespot color patterns on the wings of nymphalid butterflies. My lab is combining a series of evo-devo approaches to identify the molecular and developmental changes underlying eyespot origins.  A multi-species comparative phylogenetic approach led to the identification of the ancestral lineage where eyespots likely evolved, and also where four genes started becoming expressed in eyespot centers. A CRISPR/Cas9 gene editing approach identified three genes required for eyespot development that when mutated lead to wings without eyespots. Two of the genes evolved their new expression pattern concurrently with eyespot origins whereas the third gene evolved its expression at a later date in a subset of butterfly lineages. This means that genes that become associated with the development of a novel trait at a late stage in their evolution can become essential developmental genes. In addition, a combination of in situ gene expression with modeling work, led to reconstructions of mosaic mutant wing patterns and to the identity of two potential ligands involved in eyespot center differentiation via simple reaction-diffusion mechanisms. FAIRE-Seq further identified regulatory sequences flanking one of the genes, Distal-less, which is potentially involved in this reaction-diffusion network. When these regulatory sequences were deleted they led to eyespot deletions as well as disruptions of multiple other traits in the butterfly, indicating regulatory sequences with pleiotropic functions. This suggests that eyespots may have originated from co-opted limbs.