Vocal divergence is concordant with genomic evidence for strong reproductive isolation in grasshopper mice ().

Behavioral barriers to gene flow often evolve faster than intrinsic incompatibilities and can eliminate the opportunity for hybridization between interfertile species. While acoustic signal divergence is a common driver of premating isolation in birds and insects, its contribution to speciation in mammals is less studied. Here we characterize the incidence of, and potential barriers to, hybridization among three closely related species of grasshopper mice (genus ). All three species use long-distance acoustic signals to attract and localize mates; and are acoustically similar and morphologically cryptic whereas is larger and acoustically distinct. We used genotyping-by-sequencing (GBS) to test for evidence of introgression in 227 mice from allopatric and sympatric localities in the western United States and northern Mexico. We conducted laboratory mating trials for all species pairs to assess reproductive compatibility, and recorded vocalizations from and in sympatry and allopatry to test for evidence of acoustic character displacement. Hybridization was rare in nature and, contrary to prior evidence for / hybrids, only involved and . In contrast, laboratory crosses between and produced litters whereas and crosses did not. Call fundamental frequency in and was indistinguishable in allopatry but significantly differentiated in sympatry, a pattern consistent with reproductive character displacement. These results suggest that assortative mating based on a long-distance signal is an important isolating mechanism between and and highlight the importance of behavioral barriers in determining the permeability of species boundaries.