• Amazonia;
  • biodiversity;
  • crown ages;
  • diversification;
  • Lepidoptera;
  • mitochondrial DNA;
  • Neogene;
  • Neotropics;
  • Pleistocene refugium hypothesis


Rejection of the Pleistocene refugium hypothesis (PRH) as an explanation for the high biodiversity of Neotropical forest is based in part on the assertion that biotic elements of these forests evolved during the Neogene. That argument is justified, in turn, by the ages of crown groups (the age of the most recent common ancestor of extant species of a clade). We consider the use of crown ages as a metric to reject the PRH to be an unfair test, because the circumscription of crown groups of interest is arbitrary, and their ages represent overestimates of the time of species formation. We present divergence times between pairs of sister species (131 pairs), and among pairs of sister species and their closest relative (56 triplets), from 35 genera of Neotropical butterflies. Our aim is to refocus the discussion about the timing of diversification of the Neotropical biota on the time of the formation of extant species, a metric that is consistent and comparable across taxa. Our results show that 72% of speciation events leading to the formation of butterfly sister species occurred within the last 2.6 Myr, a result consistent with the temporal predictions of the PRH, suggesting that the PRH cannot be completely discarded as a driver of Neotropical diversification.