Strain variation identifies a neural substrate for behavioral evolution in Drosophila.

Sexual selection acts on heritable differences within species, driving the parallel diversification of signal production in one sex and behavioral responses in the other. This coevolution implies that sensory preferences are themselves variable traits, yet the neural basis of such variation remains unclear. Here, we identify striking strain-specific differences in Drosophila melanogaster male mate preferences that arise from differential sensitivity to heterospecific female pheromones, revealing that the sensory tuning relevant to mate choice has not been fixed across extant populations of this species. We map this variation to alterations in the strength of an ascending inhibitory pathway targeting P1 neurons, a central circuit node known to dynamically pattern courtship. In selective males, heterospecific female pheromones robustly activate this inhibitory circuit, triggering transient suppression of courtship pursuit and promoting reorientations toward more suitable potential mates. Promiscuous males, by contrast, are insensitive to heterospecific pheromones and continue to chase inappropriate female targets due to attenuated inhibitory input. Ascending inhibition thus emerges as a key factor in shaping courtship dynamics and generating behavioral variation across members of a species. Notably, evolutionary divergence in the mate preferences of closely related Drosophila species maps to modifications at distinct nodes within the same chemosensory circuit. Our work thus reveals how the modular circuit logic controlling mate selection appears to facilitate behavioral diversification across both strains and species, pointing to inhibitory sensory pathways as a repeated substrate for evolutionary selection.