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Abstract Species interactions drive diverse evolutionary outcomes. Speciation by cascade reinforcement represents one example of how species interactions can contribute to the proliferation of species. This process occurs when the divergence of mating traits in response to selection against interspecific hybridization incidentally leads to reproductive isolation among populations of the same species. Here, we investigated the population genetic outcomes of cascade reinforcement in North American chorus frogs (Hylidae:Pseudacris). Specifically, we estimated the frequency of hybridization among three taxa, assessed genetic structure within the focal species,P.feriarum, and ascertained the directionality of gene flow withinP.feriarumacross replicated contact zones via coalescent modeling. Through field observations and preliminary experimental crosses, we assessed whether hybridization is possible under natural and laboratory conditions. We found that hybridization occurs amongP.feriarumand two conspecifics at a low rate in multiple contact zones, and that gene flow within the former species is unidirectional from allopatry into sympatry with these other species in three of four contact zones studied. We found evidence of substantial genetic structuring withinP.feriarumincluding a divergent western allopatric cluster, a behaviorally‐distinct sympatric South Carolina cluster, and several genetically‐overlapping clusters from the remainder of the distribution. Furthermore, we found sub‐structuring between reinforced and nonreinforced populations in the two most intensely‐sampled contact zones. Our literature review indicated thatP.feriarumhybridizes with at least five heterospecifics at the periphery of its range providing a mechanism for further intraspecific diversification. This work strengthens the evidence for cascade reinforcement in this clade, revealing the geographic and genetic landscape upon which this process can contribute to the proliferation of species. 

Background: The origin and maintenance of species is a unifying theme in evolutionary biology. Mate choice and selection on sexual signals have emerged as powerful drivers of reproductive isolation – the key pillar of the biological species concept. The mechanistic underpinnings of isolating behaviors lie in the circuit- and cellular-level properties of the brain and remain relatively understudied. Summary: Here, I argue that temporal auditory selectivity in anuran amphibians offers a window into the proximate mechanisms of reproductive isolation. First, I discuss anuran behaviors as a longstanding neuroethological model with which to examine behavioral reproductive isolation and its neural correlates. Next, I review how modern neurobiological techniques are revealing the proximate mechanisms of the evolution of divergent mate preferences in anurans, highlighting cellular-level neural shifts in temporal coding. Finally, I discuss future research directions to reveal the neural mechanisms through which behavioral isolation is generated and maintained in anuran model systems. Key Messages: Anurans offer a powerful model for addressing questions about how neural barriers to gene flow arise across biological scales and how changes in the brain contribute to speciation. Modern evolutionary neurobiology will benefit from applying new tools to this longstanding neuroethological model clade. 

Many traits important for reproductive isolation are environmentally responsive. However, most studies examining reproductive isolation do not explicitly take into consideration environmental variation. Temperature can have a particularly large effect on reproductive behaviours, especially in ectotherms. Here, we tested whether temperature affects the degree of reproductive isolation between the upland chorus frog, Pseudacris feriarum, and its congener Pseudacris nigrita. These two species engage in costly hybridization in nature, leading to reinforcement of male signals and female preferences in sympatry. However, male advertisement calls vary with temperature such that the difference between the advertisement calls of the two species is narrower at cold temperatures than at warm temperatures. To examine the effect of temperature on reproductive isolation in this system, we performed six binary choice preference trials using sympatric female P. feriarum. In these experiments, we acclimated females to either warm (20 °C) or cool (10 °C) temperatures and gave them a choice between heterospecific and conspecific advertisement calls appropriate for the temperature. We also conducted experiments where we gave females a choice between more similar stimuli at warm temperatures to test whether females have narrower preferences at cold temperatures. We found that females always had a significant preference for the conspecific advertisement call, regardless of temperature or signal similarity. However, females took twice as long to make a choice in suboptimal conditions. This time delay may lead to increased risk of hybridization, despite females’ ability to discriminate between calls. Our results highlight the importance of considering environmental context when examining traits involved in reproductive isolation.