Divergence in advertisement calls, morphology and ecology of <i>Charadrahyla nephila</i> and <i>C. viruela</i> (Anura: Hylidae): implications for species boundaries

Understanding acoustic divergence is essential for delimiting species, as advertisement calls often function as primary signals for mate recognition and reproductive isolation in anurans. However, for many Neotropical frogs such data remain scarce. Here, we investigate acoustic, morphological and ecological divergence between Charadrahyla nephila and the recently described C. viruela occurring along an elevational gradient in the cloud forests of Oaxaca, Mexico. We recorded 106 advertisement calls from 21 males at two localities corresponding to C. nephila and C. viruela. Most acoustic variables showed significant differences between species. Individuals from C. nephila were smaller and lighter than individuals from C. viruela. C. nephila individuals also occupied thinner perches and occurred in cooler, drier environments consistent with elevational variation in microhabitats. The congruence across acoustic, morphological and ecological differences supports the hypothesis of two independent evolutionary lineages corroborating the existence of C. nephila and C. viruela species. These results highlight the taxonomic and conservation significance of acoustic divergence, as recognising distinct lineages may necessitate reassessment of the species’ IUCN Red List status due to narrower distribution ranges.