The research proposed by Daniel Howard and John Doherty addresses some of the most difficult problems in biology; namely, how does reproductive isolation (the absence of interbreeding between individuals of different species) develop as one species splits into two and, in particular, what is the role of selection against hybridization in the evolution of reproductive barriers? Answers to these questions are central to an understanding to the development and conservation of biological diversity on earth. The questions will be approached by studying ground crickets that are in the process of splitting to form new species. Answers obtained from the study will be relevant to all sexually reproducing organisms. Research over the past three years has identified male calling song differences as a potential reproductive barrier between the newly forming species, Allonemobius fasciatus and A. socius. The identification of this potential barrier during a critical stage of species formation provides a rare opportunity to study the evolution of reproductive isolation during a speciation event. During the next three years, the hypothesis of reinforcement will be tested. This hypothesis states that the calling song displacement exhibited by A. fasciatus in areas where it occurs with A. socius represents a reproductive barrier that has evolved as a consequence of selection against hybridization. The work will include investigations of : (1) geographic variation in male calling song, (2) female response to song, (3) the genetics of song differences, (4) the cost of a hybrid mating, and (5) sperm competition. The proposed studies will not only provide a rigorous test of the hypothesis of reinforcement, but they will provide fundamental information on a trait potentially responsible for reproductive isolation. Only by combining studies of the genetics, behavioral ecology, and developmental biology of specific traits serving as reproductive barriers with analyses of geographic distribution patterns, will biologists arrive at an understanding of the process of speciation. The proposed research should lead into even more rigorous studies of the acquisition of reproductive isolation between A. fasciatus and socius. These species are rapidly becoming a model system for the study of biological processes. From an applied perspective, the family Gryllidae (crickets) includes some very serious agricultural pests. The proposed research will provide badly needed information on the reproductive biology of crickets - information that can be used to help develop biological control programs.