Biogeographic evidence supports the Old Amazon hypothesis for the formation of the Amazon fluvial system.

The Amazon has high biodiversity, which has been attributed to different geological events such as the formation of rivers. The Old and Young Amazon hypotheses have been proposed regarding the date of the formation of the Amazon basin. Different studies of historical biogeography support the Young Amazon model, however, most studies use secondary calibrations or are performed at the population level, preventing evaluation of a possible older formation of the Amazon basin. Here, we evaluated the fit of molecular phylogenetic and biogeographic data to previous models regarding the age of formation of the Amazon fluvial system. We reconstructed time-calibrated molecular phylogenies through Bayesian inference for six taxa belonging to Amphibia, Aves, Insecta and Mammalia, using both, nuclear and mitochondrial DNA sequence data and fossils as calibration points, and explored priors for both data sources. We detected the most plausible vicariant barriers for each phylogeny and performed an ancestral reconstruction analysis using areas bounded by major Amazonian rivers, and therefore, evaluated the effect of different dispersal rates over time based on geological and biogeographical information. The majority of the genes analyzed fit a relaxed clock model. The log normal distribution fits better and leads to more precise age estimations than the exponential distribution. The data suggested that the first dispersals to the Amazon basin occurred to Western Amazonia from 16.2-10.4 Ma, and the taxa covered most of the areas of the Amazon basin between 12.2-6.2 Ma. Additionally, regardless of the method, we obtained evidence for two rivers: Tocantins and Madeira, acting as vicariant barriers. Given the molecular and biogeographical analyses, we found that some taxa were fitted to the "Old Amazon" model.