The Developmental Basis And Evolution Of Wing Color Pattern In Butterflies

Evolutionary biologists look to understand the processes underlying biodiversity. One aim of the field is to link the phenotype to its instructive genetic code, and to take advantage of homologies between species to ask what genetic changes generate the diversity of forms and shapes we see in nature. In this work, I study the evolution of wing color patterns in butterflies-a system famous for its striking variation in form and complexity. The specific goals of my work were to test Wnt gene functions in patterning wing colors, and to characterize the regulatory architecture modulating color pattern-related function of the WntA gene. Our results revealed that WntA is essential to establish multiple pattern elements, even in highly divergent species of nymphalids and papilionids-two distant and very diverse families of butterflies. In nymphalids, I observed lineage-specific effects in various wing regions, including spatial shifts in gene expression and novel expression domains, that are associated with color pattern evolution. In papilionids, a basal butterfly family, WntA function is restricted to an expanded wing margin system that largely dominates Papilio wing patterning. Interestingly, I confirmed that the main pattern systems driving nymphalid and Papilio wing diversity, i.e., the central symmetry system and the glauca, respectively, are not homologous. In Papilio, I identified a new set of Wnt6$-related wing pattern elements, the submarginal spots, that show substantial variation between papilionid species and deserve further characterization in future studies. WntA's versatility and flexibility in patterning a diverse repertoire of elements prompted me to explore this gene's cis-regulatory landscape. I implemented a novel 'shotgun' CRISPR/Cas9 strategy to produce mosaic knock-outs of WntA cis-regulatory elements across multiple species. The results contrasted with traditional postulates about gene regulatory modularity, and instead pointed to a highly interdependent network of both ancestral and recently evolved cis-regulatory elements that interact to generate WntA patterns. Monarch butterflies stood out from other nymphalids as having a highly divergent cis-regulatory architecture, likely associated with its distinctive WntA wing pattern. Finally, the implementation of a 'shotgun' deletion strategy revealed that most CREs have the potential to act as both enhancers or silencers, which is new evidence for our emerging understanding of regulatory element functionality.