Project summary Drosophila (fruit flies) are an exceptional model for evolutionary genetics, with many species that can be subjected to the full suite of modern genetic and genomic methods that have been developed in the model species D. melanogaster. Hybrids between D. melanogaster and its sibling species D. sim- ulans are a long-standing model of speciation and our group has made several key contributions to understanding the genetic basis of hybrid incompatibilities between them. One major effort here will be to identify the maternal factors in D. simulans that interact with a large satellite block specific to D. melanogaster to cause F1 hybrid lethality. This will allow us to obtain a mechanistic understanding of how rapidly evolving non-coding DNA can lead to chromosome instability and lethality between spe- cies. Drosophila have also played a major role in the discovery of the phenomenon of meiotic drive, where different alleles segregate non-randomly from heterozygous parents in violation of Mendel’s first law of equal segregation. Meiotic drive can cause reductions in organismal fertility, drive rapid changes in karyotypes including of sex chromosomes, and contribute to species isolation. Yet little is known about fundamental questions such as the prevalence of meiotic drive in natural populartions or the differences in frequency between male and female meiotic drive. In a second project we will con- duct a high-throughput and sensitive screen for meiotic drive across all chromosomes in D. melano- gaster and D. simulans populations. In a third project we will investigate evolution of the primary sex determination signal. This signal comprises a group of positive and negative regulators of a central switch gene, that allow the developing Drosophila embryo to distinguish whether it has one or two X chromosomes and thus develop as a male or female. Surprisingly, there is substantial variation within D. melanogaster populations affecting the fidelity of the sex determination signal, which we will genet- ically map and characterize. We will also identify gains and losses of the signaling elements in ~100 fully sequenced species from across the Drosophila phylogeny. These approaches will provide data essential for understanding the evolutionary forces that drive changes in sex determination signals across animals. Our experience along with many reagents and methods we have previously devel- oped position us to accomplish these projects in Drosophila evolutionary genetics with the flexibility provided by the MIRA funding mechanism.