Accessory gland proteins (Acps) are important components of seminal fluid, influencing female physiology and affecting male fitness. Many Acps appear to be under positive selection, potentially driven by sexual conflict and are thus ideal candidates to study coevolutionary process. Rapid amino acid substitutions, however, can make the identification of divergent orthologs difficult using only nucleotide sequence data. Thus, more targeted approaches are required to properly identify Acps, even in species with completed genomes. In this study I propose to create four accessory gland specific cDNA libraries to identify Acps in Drosophila sechellia, D. mauritiana, D. yakuba and D. miranda, I will then annotate these genes and use the D. miranda Acps to determine those present in the full genome of its close relative D. pseudoobscura. Combining this data with the Acps already identified in D. melanogaster and D. simulans will produce a total of seven species, I will then use maximum likelihood fit evolutionary models to the divergence data to characterize the selective pressures influencing the evolution of seminal fluid proteins across these seven species. Sites identified with excess amino acid substitutions will be strong candidates for evidence of positive selection. Parameterization of these models using seven species will provide excellent power to detect evolutionary forces acting on specific lineages or on specific functional classes of genes. Finally, I will then determine the functional conservation of Acps using transgenic D. melanogaster expressing heterospecific accessory gland proteins.
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