The evolutionary basis for high species diversity in tropical regions of the world remains unresolved. Much research has focused on the biogeography of speciation in the Amazon Basin, which harbors the greatest diversity of terrestrial life. The leading hypotheses on allopatric diversification of Amazonian taxa are the Pleistocene refugia, marine incursion, and riverine barrier hypotheses. Recent advances in the fields of phylogeography and species-distribution modeling permit a modern re-evaluation of these hypotheses. Our approach combines comparative, molecular phylogeographic analyses using mitochondrial DNA sequence data with paleodistribution modeling of species ranges at the last glacial maximum (LGM) to test these hypotheses for three co-distributed species of leafcutter ants (Atta spp.). The cumulative results of all tests reject every prediction of the riverine barrier hypothesis, but are unable to reject several predictions of the Pleistocene refugia and marine incursion hypotheses. Coalescent dating analyses suggest that population structure formed recently (Pleistocene-Pliocene), but are unable to reject the possibility that Miocene events may be responsible for structuring populations in two of the three species examined. The available data therefore suggest that either marine incursions in the Miocene or climate changes during the Pleistocene—or both—have shaped the population structure of the three species examined. Our results also reconceptualize the traditional Pleistocene refugia hypothesis, and offer a novel framework for future research into the area.

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus-growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher-attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower-attine lineages. Higher-attine fungi form two clades, Clade-A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade-B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade-A fungi because some leafcutter species ranging across South America cultivate Clade-B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade-B fungi because some Trachymyrmex species cultivate Clade-A fungi and other Trachymyrmex species cultivate fungi known so far only from lower-attine ants; (iii) in some locations, single higher-attine ant species or closely related cryptic species cultivate both Clade-A and Clade-B fungi; and (iv) ant-fungus co-evolution among higher-attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade-A or Clade-B fungi, sustaining with either cultivar-type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade-A or Clade-B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade-A), but must derive from ant-fungus synergisms and unique ant adaptations.