Effect of sympatry on discrimination of advertisement and aggressive signals by female chorus frogs

Behavioral isolation is a central driver of speciation, yet the mechanisms linking neural tuning to isolating behaviors remain poorly understood. We used intraspecific variation in the Upland chorus frog (Pseudacris feriarum) to test how reinforcement shapes female responses to divergent male advertisement (lower pulse rate; PR) and aggressive calls (higher PR). In populations sympatric with P. nigrita, male calls have evolved faster PRs and higher pulse numbers relative to allopatric populations, and auditory neurons show altered temporal selectivity. We predicted that (1) sympatric females would show stronger and narrower preferences for local advertisement calls. Due to shared tuning of auditory neurons to fast PRs, we also predicted that (2) allopatric females would be more likely to choose aggressive calls than sympatric females, and (3) neither population would discriminate between allopatric aggressive calls and sympatric advertisement calls. We conducted binary-choice phonotaxis experiments with females from one allopatric and one sympatric population. Sympatric females strongly preferred their local advertisement call over the allopatric advertisement call, confirming reproductive character displacement and narrowed preference functions in this population. Both populations found aggressive calls moderately attractive, but allopatric females chose them more frequently than sympatric females. Finally, females from both populations failed to discriminate between allopatric aggressive calls and sympatric advertisement calls, which share similar temporal features. These results demonstrate that reinforcement can alter female responses to both advertisement and aggressive signals. By narrowing preference functions and strengthening discrimination against heterospecific and conspecific signals, such behavioral shifts promote reproductive isolation during speciation. Behavioral premating isolation is a strong driver of speciation, yet the neural mechanisms of these behaviors remain understudied. We address this gap using Upland chorus frogs. Frog populations sympatric with a heterospecific have diverged in male calls and female preferences for mating calls relative to allopatric populations. We conducted behavioral experiments allowing females from a sympatric and allopatric population to choose among sympatric and allopatric mating and aggressive calls. We found that female chorus frogs sometimes chose aggressive calls over mating calls, and that allopatric females are more likely to do so than sympatric females. Both types of females did not discriminate between call types that have similar temporal features, supporting a novel model of neural evolution. Our results show that in sympatry, female discrimination against both heterospecific and divergent conspecific signals is sharpened, strengthening behavioral isolation during speciation.