PROJECT SUMMARY One of the fundamental problems in evolutionary biology is to understand the molecular genetic basis of speciation. Recent advances in speciation research have improved our understanding of interspecific divergence, but we still lack a comprehensive understanding of the molecular processes that diverge among incipient species. In the handful of cases where the genetic mechanisms of reproductive isolation have been elucidated, these invariably tackle late-evolving and/or hybrid dysfunction. This means that we still lack a general understanding of the molecular processes that govern pre-zygotic reproductive barriers, even though these are often important early in the speciation process. My lab will tackle this problem by identifying the molecular genetic basis of pre-zygotic and post-zygotic re- productive isolation between members of the Drosophila virilis species sub-group. This species group provides an especially unique opportunity to dissect the genetic and molecular mechanisms of pre-zygotic barriers, as members of this group are prone to evolve these types of barriers quickly between species and even among populations of the same species. Our overall approach integrates several strategies to answer the following questions: What are the genetic mechanisms that cause reproductive isolation between species? Which molec- ular and cellular processes are affected by divergence of these genetic mechanisms? What are the evolutionary forces that drive divergence of the relevant genes between species? What is the landscape of natural genetic variation within and between species that facilitates evolutionary divergence of these genes? The first project within this proposal will focus on post-mating pre-zygotic barriers (i.e., gametic incompatibil- ities). I have previously shown that gametic incompatibilities are rampant in the D. virilis sub-group, and that the genetic basis is moderately complex but highly tractable using a combination of molecular genetics techniques coupled with transcriptomic and proteomic analyses of reproductive traits. The second project will tackle the mechanisms of hybrid male sterility that are caused by incompatibilities between the Y and X chromosomes. The Drosophila Y chromosome carries several male fertility factors, but it has seldom been directly implicated in interspecific hybrid sterility between closely related species. Our preliminary data show that the Y chromosome is necessary and sufficient to cause sterility in hybrids. The research in this proposal will be innovative because we will deploy cutting edge tools in creative ways that will allow us to dissect complex genetic mechanisms in a newly established model system.