The evolutionary history of fungus-farming ants has been the subject of multiple morphological, molecular phylogenetic, and phylogenomic studies. Due to its rarity, however, the phylogenetic position, natural history, and fungal associations of the monotypic genus Paramycetophylax Kusnezov have remained enigmatic. Here we report the first excavations of colonies of Paramycetophylax bruchi (Santschi) and describe its nest architecture and natural history. Utilizing specimens from these collections, we generated ultraconservedelement (UCE) data to determine the evolutionary position of Paramycetophylax within the fungus-farming ants and ribosomal ‘fungal barcoding’ ITS sequence data to identify the fungal cultivar. A maximum-likelihood phylogenomic analysis indicates that the genus Paramycetophylax is the sister group of the yeast-cultivating Cyphomyrmex rimosus group, an unexpected result that renders the genus Cyphomyrmex Mayr paraphyletic. A Bayesian divergence-dating analysis indicates that Paramycetophylax diverged from its sister group around 36 mya (30–42 mya, HPD) in the late Eocene-early Oligocene, a period of global cooling, expansion of grasslands, and large-scale extinction of tropical organisms. Bayesian analysis of the fungal cultivar ITS gene fragment indicates that P. bruchi practices lower agriculture and that the cultivar grown by P. bruchi belongs to the Clade 1 group of lower-attine fungi, a clade that, interestingly, also includes the C. rimosus-group yeast cultivars. Based on these results, we conclude that a better understanding of P. bruchi and its fungal cultivar, including whole-genome data, is critical for reconstructing the origin of yeast agriculture, a major transition in the evolution of fungus-farming ants.

Uncovering the evolutionary history of the subfamilies Ectatomminae and Heteroponerinae, or ectaheteromorphs, is key to understanding a major branch of the ant tree of life. Despite their diversity and ecological importance, phylogenetic relationships in the group have not been well explored. One particularly suitable tool for resolving phylogeny is the use of ultraconserved elements (UCEs), which have been shown to be ideal markers at a variety of evolutionary time scales. In the present study, we enriched and sequenced 2,127 UCEs from 135 specimens of ectaheteromorph ants and investigated phylogeny using a variety of model-based phylogenomic methods.Trees recovered from partitioned maximum-likelihood and species-tree analyses were well resolved and largely congruent.The results are consistent with an expanded concept of Ectatomminae that now includes the subfamily Heteroponerinae new synonym and its single tribe Heteroponerini new combination. Eleven monophyletic groups are recognized as genera: Acanthoponera, Alfaria status revived, Boltonia Camacho and Feitosa new genus, Ectatomma, Gnamptogenys, Heteroponera, Holcoponera status revived, Poneracantha status revived, Rhytidoponera, Stictoponera status revived, and Typhlomyrmex. The new phylogenetic framework and classification proposed here will shed light on the study of Ectatomminae taxonomy and systematics, as well as on the morphological evolution of the groups that it comprises.