Project Summary Animals, including humans, contain a small number of cells in their gonads called germline stem cells (GSCs). These cells are essential for making sperm or eggs throughout the lifetime of the animal. The decision for a dividing stem cell to renew as a stem cell or to differentiate, and the regulation of early divisions during GSC are some of the most critical decisions in development. Studies in the fruit fly, Drosophila melanogaster, have identified many of the key genes involved in the cellular signaling for GSC maintenance and differentiation. Several of the key GSC regulatory genes are rapidly evolving under adaptive evolution in two closely related species of Drosophila (D. melanogaster and its sibling species D. simulans) but not in several additional closely related species. The key “switch” gene for GSC differentiation, bag of marbles (bam) has accumulated a highly significant excess of amino-acid changes between these two closely related species (13%). This proposal focuses on identifying the functional consequences of these changes and gaining insight into possible evolutionary forces driving these changes, including interactions with the endosymbiotic bacteria Wolbachia. This knowledge is essential for understanding how GSCs are regulated during normal development and how their misregulation due to mutation and interaction with germline parasites can lead to infertility, germline cancers, and reproductive isolation. Our proposed studies provide a framework with which to test the functional consequences and evolutionary forces driving these evolutionary changes in genes controlling stem cell fate decisions in Drosophila as well as in other organisms, including humans.