Molecular Evolution of Flightin: Patterns of constraint and divergence suggest dual functional roles. Fligthin is a hyperphosphorylated myosin binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscle (IFM) . In this species, Flightin is essential for flight and the stretch activation response responsible for producing fast wing beats. Analysis of genome sequences indicate that the central region (~ 52 amino acids) of flightin (refered to as WYR), is conserved among insects and crustaceans. Based on its high conservation, the WYR region is hypothesized to fulfill an essential function that may include myosin binding. In contrast, the N-terminal and C-terminal regions of flightin are not well conserved, with the N-terminal region (~ 80 amino acids) showing the most variability. Preliminary analyses of the flightin sequence from twelve Drosophila species indicate that the N-terminus of flightin is evolving at a rate faster than the rest of the protein and faster than that observed for other muscle proteins like the myosin regulatory light chain. One goal of this study is to characterize the rate of evolution of flightin by sampling a larger set of Drosophila species. In addition, it has been identified that there are seven phosphorylation sites that reside within the variable N-terminus of D. melanogaster, and several of these sites are not conserved among Drosophila species. We hypothesize that the variability in N-terminal sequence and phosphorylation potential may influence myofilament stiffness, which in turn dictates wing beat frequency. It is known that an important courtship behavior of Drosophila is the male mating song produced by wing beats. The song type and number of songs produced vary among drosophila lineages. As the flies utilize their musculature in the thorax for beating the wings during the production of their species-specific mating song, and as Flightin is a very important protein in the sarcomere for both structure and function, we think that Flightin may be sexually selected for species-specific role. Thus in this study, we will test the hypothesis that Flightin is under dual evolutionary constraints, purifying selection (WYR) to fulfill an essential muscle physiological function, and positive selection ((N-terminus) to fulfill a behavior-associated function.