The damselflies Hetaerininae, a subfamily of Calopterygidae, comprise four genera distributed from North to South America: Hetaerina, Mnesarete, Ormenophlebia and Bryoplathanon. While several studies have focused on the intriguing behavioral and morphological modifications within Hetaerina, little of the evolutionary history of the group is well understood. Understanding the biogeographical history of Hetaerininae is further complicated by uncertainty in important geological events, such as the closure of the Central American Seaway (CAS). We generated a phylogenetic hypothesis to test the relationships and divergence times within Hetaerininae using IQtree and BEAST2 and found that Mnesarete and Ormenophlebia render Hetaerina paraphyletic. Reclassification of the genera within Hetaerininae is necessary based on our results. We also tested the fit to our dataset of two different hypotheses for the closure of CAS. Our results supported a gradual closure, starting in the Oligocene and ending in the Pliocene. Using Ancestral Character State Reconstruction, we found that the rubyspot, which is associated with higher fecundity in several species, was ancestral for Hetaerininae and subsequently lost four times. Estimates of diversification in association with the rubyspot are needed to understand the plasticity of this important character. Forest habitat was the ancestral state for Hetaerininae, with transitions to generalist species of Hetaerina found primarily in the Mesoamerican region. These results add to our understanding of the relationship between morphology, biogeography and habitat in a charismatic group of damselflies.
