ABSTRACT Background: The importance of genetic exchange between species has been a constant subject of debate among evolutionary biologists. Genomic data has demonstrated that gene exchange is much more common than previously thought and might even serve as a source of adaptation. Broad, long-term objective: The proposed study of naturally occurring hybrid zones will leverage the natural experiment that occurs in hybrid zones to find admixed genomes with which then can be used to dissect the genetics of behavioral traits that differ between two recently diverged Drosophila species. The goal of this supplement is to facilitate the professional development of Ms. Heidi Mavengere. Specific aims: Aim 1 of the study uses admixed flies from a hybrid zone to map genes underlying the ecologically important trait of temperature preference. This trait is an important source of reproductive isolation among Drosophila species. Admixture mapping provides a unique opportunity to identify the genes and processes that ultimately have led to divergence in ecological niche. Aim 2 will test genomic predictions using functional genetics tests to confirm whether the alleles associated with the phenotypic difference have caused the change. Method: Fruit flies from the genus Drosophila can be collected in their natural habitat and also maintained under laboratory conditions. In the last few years, we have improved our sequencing approaches and have also developed the ability to generate precise gene knock-outs in multiple species of Drosophila. This proposal leverages the identification of introgressed alleles in the genome to reveal the genetic basis of interspecific differences. The genes identified in these admixture analyses will be functionally validated with CRISPR- mediated gene replacements. Our preliminary results for admixture mapping and genome editing indicate we have the ability to execute the research proposal. Health-relatedness: Admixture mapping in humans is a primary approach used for the identification of disease-causing gene variants. However, follow-up experiments to test the function of genes correlated with disease are difficult to perform in humans. Admixture mapping protocols have therefore never been validated end-to-end from the population level at which variants correlated with disease are identified, to the functional molecular genetic level where the phenotypic state is manifested. This proposal will study traits involved in ecological reproductive isolation using admixture mapping and will connect inferences from statistical genomics to measurable phenotypic differences via genome editing. Impact: The results from this study will provide the first broad view of patterns of introgression in cases where species interbreed in nature, and will leverage those patterns of admixture to dissect the genetic basis of behavioral differences between species that cause reproductive isolation.