Project summary “Evolutionary innovation” refers to the origin of entirely new traits, as opposed to the modification of existing traits. Although such novelties are relatively rare, all the complexity and diversity of life have ultimately been shaped by evolutionary innovations that occurred in a nested pattern, with every innovation dependent on many earlier novelties. Despite their critical importance for everything from geological nutrient cycles that support all life on Earth to human sentience that distinguishes us from our closest relatives, the molecular mechanisms of evolutionary innovations are understood far less than the evolution of existing traits. This is true at all levels of biological organization, from single molecules to the most complex features of animal form and function. We understand the evolution of existing organs and cell types better than the origin of new ones; quantitative variation in gene expression has been explored in greater detail than the origin of new regulatory pathways; much more is known about the evolution of existing genes than about the origin of new genes, and so on. It is this gap in knowledge that motivates our work. To achieve a comprehensive understanding of evolutionary innovations, it is necessary to connect novelties at all levels of biological organization: from new functional elements in the genome, to new genetic pathways, to new morphological structures. Research in our lab will advance in several directions, using the Drosophila model system. First, we will identify the key DNA sequence changes responsible for the origin of a new morphological structure that evolved recently in Drosophila. Second, we will characterize the cell differentiation pathway that translates these changes into novel morphologies and reconstruct how this pathway was assembled in the course of evolution. Third, we will look more broadly at how ancestral genes acquire new expression patterns. We will test whether cooption of old genes into new tissues involves modification of existing regulatory sequences or the origin of new regulatory elements. Finally, we will reconstruct the origin of new regulatory elements from non-functional ancestral sequences. By focusing on new genes that evolved within natural populations of a single species, we will identify the series of mutations that create new regulatory elements in the genome. Together, these approaches will promote a deep mechanistic understanding of evolutionary innovations.