The evolution of ant agriculture, as practised by the fungus-farming ‘attine’ ants, is thought to have arisen in the wet rainforests of South America about 55–65 Ma. Most subsequent attine agricultural evolution, including the domestication event that produced the ancestor of higher attine cultivars, is likewise hypothesized to have occurred in South American rainforests. The ‘out-of-the-rainforest’ hypothesis, while generally accepted, has never been tested in a phylogenetic context. It also presents a problem for explaining how fungal domestication might have occurred, given that isolation from free-living populations is required. Here, we use phylogenomic data from ultra-conserved element (UCE) loci to reconstruct the evolutionary history of fungus-farming ants, reduce topological uncertainty, and identify the closest non-fungus-growing ant relative. Using the phylogeny we infer the history of attine agricultural systems, habitat preference and biogeography. Our results show that the out-of-the-rainforest hypothesis is correct with regard to the origin of attine ant agriculture; however, contrary to expectation, we find that the transition from lower to higher agriculture is very likely to have occurred in a seasonally dry habitat, inhospitable to the growth of free-living populations of attine fungal cultivars. We suggest that dry habitats favoured the isolation of attine cultivars over the evolutionary time spans necessary for domestication to occur.

Evolutionary adaptations for maintaining beneficial microbes are hallmarks of mutualistic evolution. Fungus-farming “attine” ant species have complex cuticular modifications and specialized glands that house and nourish antibiotic-producing Actinobacteria symbionts, which in turn protect their hosts’ fungus gardens from pathogens. Here we reconstruct ant–Actinobacteria evolutionary history across the full range of variation within subtribe Attina by combining dated phylogenomic and ultramorphological analyses. Ancestral-state analyses indicate the ant–Actinobacteria symbiosis arose early in attine-ant evolution, a conclusion consistent with direct observations of Actinobacteria on fossil ants in Oligo-Miocene amber. qPCR indicates that the dominant ant-associated Actinobacteria belong to the genus Pseudonocardia. Tracing the evolutionary trajectories of Pseudonocardia-maintaining mechanisms across attine ants reveals a continuum of adaptations. In Myrmicocrypta species, which retain many ancestral morphological and behavioral traits, Pseudonocardia occur in specific locations on the legs and antennae, unassociated with any specialized structures. In contrast, specialized cuticular structures, including crypts and tubercles, evolved at least three times in derived attine-ant lineages. Conspicuous caste differences in Pseudonocardia-maintaining structures, in which specialized structures are present in worker ants and queens but reduced or lost in males, are consistent with vertical Pseudonocardia transmission. Although the majority of attine ants are associated with Pseudonocardia, there have been multiple losses of bacterial symbionts and bacteria-maintaining structures in different lineages over evolutionary time. The early origin of ant–Pseudonocardia mutualism and the multiple evolutionary convergences on strikingly similar anatomical adaptations for maintaining bacterial symbionts indicate that Pseudonocardia have played a critical role in the evolution of ant fungiculture.

The fungus‐growing ants and their fungal cultivars constitute a classic example of a mutualism that has led to complex coevolutionary dynamics spanning c. 55–65 Ma. Of the five agricultural systems practised by fungus‐growing ants, higher‐attine agriculture, of which leaf‐cutter agriculture is a derived subset, remains poorly understood despite its relevance to ecosystem function and human agriculture across the Neotropics and parts of North America. Among the ants practising higher‐attine agriculture, the genus Trachymyrmex Forel, as currently defined, shares most‐recent common ancestors with both the leaf‐cutter ants and the higher‐attine genera Sericomyrmex Mayr and Xerolitor Sosa‐Calvo et al. Although previous molecular‐phylogenetic studies have suggested that Trachymyrmex is a paraphyletic grade, until now insufficient taxon sampling has prevented a full investigation of the evolutionary history of this group and limited the possibility of resolving its taxonomy. Here we describe the results of phylogenetic analyses of 38 Trachymyrmex species, including 27 of the 49 described species and at least 11 new species, using four nuclear markers, as well as phylogenetic analyses of the fungi cultivated by 23 species of Trachymyrmex using two markers. We generated new genetic data for 112 ants (402 new gene sequences) and 95 fungi (153 new gene sequences). Our results corroborate previous findings that Trachymyrmex, as currently defined, is paraphyletic. We propose recognizing two new genera, Mycetomoellerius gen.n. and Paratrachymyrmex gen.n., and restricting the continued use of Trachymyrmex to the clade of nine largely North American species that contains the type species [Trachymyrmex septentrionalis (McCook)] and that is the sister group of the leaf‐cutting ants. Our fungal cultivar phylogeny generally corroborates previously observed broad patterns of ant–fungus association, but it also reveals further violations of those patterns. Higher‐attine fungi are divided into two groups: (i) the single species Leucoagaricus gongylophorus (Möller); and (ii) its sister clade, consisting of multiple species, recently referred to as Leucoagaricus Singer ‘clade B’. Our phylogeny indicates that, although most non‐leaf‐cutting higher‐attine ants typically cultivate species in clade B, some species cultivate L. gongylophorus, whereas still others cultivate fungi typically associated with lower‐attine agriculture. This indicates that the attine agricultural systems, which are currently defined by associations between ants and fungi, are not entirely congruent with ant and fungal phylogenies. They may, however, be correlated with as yet poorly understood biological traits of the ants and/or of their microbiomes.