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Abstract Rivers are prominent landscape features, acting as key promoters of diversification among freshwater organisms. Albeit generally considered potential barriers to species movement, they may also facilitate gene flow and structure populations of semiaquatic species (Riverine Thruway Hypothesis, RTH). We evaluated the role of rivers on the processes responsible for current genetic variation in the semiaquatic frog Pseudis bolbodactyla, testing whether each hydrographic basin harbours distinct genetic lineages. We sequenced three markers on 166 samples from 13 localities along the Paraná (PR), Araguaia–Tocantins (AT), and São Francisco (SF) River basins in Brazil. We recovered three populations geographically matching each hydrographic basin. Our results indicate migration among basins, with the best model selected using approximate Bayesian computation, including migration between AT and SF and ancient gene flow from PR to the AT–SF ancestor. Our findings are likely related to the orogenic events in Central Brazil dating to the Late Miocene (5 Mya), when hydrographic basins and the geomorphological features of the Brazilian Shield were formed. This suggests that P. bolbodactyla probably represents a species complex, with each lineage occurring in a distinct hydrographic basin, matching the predictions of the RTH. 

ABSTRACT Understanding Neotropical megadiversity remains challenging due to fundamental taxonomic issues, including identifying and describing cryptic species and their distribution, and the limited knowledge of key factors driving biological diversification. Such challenges are especially prominent in diverse clades with high levels of cryptic species, such as many Neotropical frogs. Herein, we conduct a mitochondrial phylogenetic study on monkey treefrogs in the genusPithecopusand a multilocus analysis of theP. hypochondrialisspecies group throughout its distribution in the South American dry diagonal (DD). Our goals were to infer the main lineages and identify historical factors that led to their divergence. Among nine currently recognised species, we found 14 well‐structured mitochondrial lineages in two main clades, which diversified during the Paleogene/Neogene transition. The lowland clade is widely distributed in the DD, and our multilocus analysis suggests it comprises seven geographically structured lineages:P. azureus, two lineages ofP. hypochondrialis, two species and one lineage for theP. nordestinuscomplex, andPithecopussp. Considering the geomorphological history of the region, diversification may have been promoted by allopatric speciation during Miocene paleogeographic events, especially the Brazilian Plateau compartmentalization and fluctuations in the São Francisco River water volume. Furthermore, gene flow between some lineages may be explained by niche conservatism and demographic expansions during the late Pleistocene, a period marked by climatic fluctuations and biome shifts. Our results reinforce the importance of both Neogene tectonic activity and Quaternary climatic fluctuations in shaping the Neotropical biota.