Deep divergence and hybridization among sympatric Neotropical toads

Introgression following hybridization is increasingly accepted as a widespread evolutionary phenomenon in animal species. Previous studies have suggested an extensive unidirectional introgression between two common Neotropical toads (Rhinella marina and R. schneideri). Here, we sought to investigate the extent of hybridization between these species and the evolutionary consequences of this process. For this, we used a comprehensive suite of molecular markers (one mtDNA, three scnDNA, and seven microsatellites) and an expanded sample. Our multilocus approach revealed a complex pattern of hybridization, involving an undocumented cryptic evolutionary lineage (RAB2). This new lineage is likely to have had a long, independent, and evolutionary history, as revealed by the split time from both R. marina and R. schneideri that was inferred to have occurred in the Late Miocene/Early Pliocene (similar to 6 Myr, 95% CI; 3.79-8.32). Interestingly, RAB2 occurs in sympatry/syntopy with some populations of R. marina from RAB, where cases of introgression were apparent at both the mtDNA and nuclear levels. Unexpectedly, however, no evidence of ongoing gene flow between R. marina and R. schneideri was detected, suggesting that the extensive cytonuclear discordance found in R. schneideri probably reflects an ancient introgression following hybridization with R. marina. Overall, this study showed that potentially overlooked cryptic lineages might contribute to biased inferences in the patterns of introgressive hybridization. These findings highlight the pitfalls of inferring evolutionary processes in groups of morphologically cryptic species.