Compositional heterogeneity and outgroup choice influence the internal phylogeny of the ants.

Knowledge of the internal phylogeny and evolutionary history of ants(Formicidae), the world's most species-rich clade of eusocialorganisms, has dramatically improved since the advent of molecular phylogenetics. A number of relationships at the subfamilylevel, however, remain uncertain. Key unresolved issues include placement of the root of the ant treeof lifeand the relationships among the so-called poneroid subfamilies. Here we assemble a new data set to attempt a resolution of these two problems and carry out divergence dating, focusing on the age of the root node of crown Formicidae. For the phylogenetic analyses we included data from 110 antspecies, including the key species Martialis heureka. We focused taxon sampling on non-formicoid lineages of antsto gain insight about deep nodes in the antphylogeny. For divergence dating we retained a subset of 62 extant taxa and 42 fossils in orderto approximatediversified sampling in the context of the fossilized birth-death process. We sequenced 11 nuclear genefragments for a total of ∼7.5 kb and investigated the DNA sequence data for the presence of among-taxon compositional heterogeneity, a property known to mislead phylogenetic inference, and for its potential to affect the rooting of the antphylogeny. We found sequences of the Leptanillinaeand several outgrouptaxa to be rich in adenine and thymine (51% average AT content) compared to the remaining ants(45% average). To investigate whether this heterogeneity could bias phylogenetic inference we performed outgroupremoval experiments, analysis of compositionally homogeneous sites, and a simulation study. We found that compositional heterogeneity indeed appears to affect the placement of the root of the anttree but has limited impact on more recent nodes. Our findings have implications for outgroupchoice in phylogenetics, which should be made not only on the basis of close relationship to the ingroup, but should also take into account sequence divergence and other properties relative to the ingroup. We put forward a hypothesis regarding the rooting of the antphylogeny, in which Martialis and the Leptanillinaetogether constitute a clade that is sister to all other ants. After correcting for compositional heterogeneity this emerges as the best-supported hypothesis of relationships at deep nodes in the anttree. The results of our divergence dating under the fossilized birth-death processand diversified sampling suggest that the crown Formicidae originated during the Albian or Aptianages of the Lower Cretaceous (103-124 Ma). In addition, we found support for monophyletic poneroids comprising the subfamilies Agroecomyrmecinae, Amblyoponinae, Apomyrminae, Paraponerinae, Ponerinae, and Proceratiinae, and well-supported relationships among these subfamiliesexcept for the placement of Proceratiinaeand ( Amblyoponinae+ Apomyrminae). Our phylogeny also highlights the non- monophylyof several antgenera, including Protanillaand Leptanillain the Leptanillinae, Proceratiumin the Proceratiinae, and Cryptopone, Euponera, and Mesoponerawithin the Ponerinae.