Cryptic diversity concealed in the Andean cloud forests: two new species of rainfrogs (Pristimantis) uncovered by molecular and bioacoustic data

Two new species of frogs previously confused with Pristimantis calcarulatus are identified using molecular phylogenetics and described using an integrative taxonomic approach. The species are distributed in montane cloud forests of the northwestern Ecuadorian Andes. Pristimantis pahuma n. sp. and Pristimantis cedros n. sp. differ from their closest relatives by strongly supported reciprocal monophyly in mitochondrial genetic data (16S). Additionally, the genetic distance among P. cedros n. sp., P. pahuma n. sp. and P. calcarulatus (sensu stricto) is 7.1–9.5%. The advertisement call of P. pahuma n. sp. also differs from that of P. calcarulatus; the former emits single-note calls irregularly, whereas P. calcarulatus always calls in a series of 8–24 notes (calls for P. cedros n. sp. not recorded). Morphologically, the three species are almost undistinguishable; however, P. cedros n. sp. differs in life by having an iris with more numerous and smaller black reticulations, whereas the other two species have larger and fewer reticulations. Biogeographically, P. cedros n. sp. is separated from P. pahuma n. sp. and P. calcarulatus by the climatically dry and low elevation Río Guayllabamba Valley, which acts as a strong barrier to dispersal in these frogs. The results of this and other studies suggest that the true species richness of Pristimantis in the Andes is vastly underestimated. This underestimation may lead to declining protection for such cryptic species, many of which have smaller ranges than previously assumed. Species distributed across potential geographic barriers should be studied to detect the existence of cryptic species.