Innovative evolutionary developments are often related to gene or genome duplications. The crop fungi of attine fungus‐growing ants are suspected to have enhanced genetic variation reminiscent of polyploidy, but this has never been quantified with cytological data and genetic markers. We estimated the number of nuclei per fungal cell for 42 symbionts reared by 14 species of Panamanian fungus‐growing ants. This showed that domesticated symbionts of higher attine ants are polykaryotic with 7–17 nuclei per cell, whereas nonspecialized crops of lower attines are dikaryotic similar to most free‐living basidiomycete fungi. We then investigated how putative higher genetic diversity is distributed across polykaryotic mycelia, using microsatellite loci and evaluating models assuming that all nuclei are either heterogeneously haploid or homogeneously polyploid. Genetic variation in the polykaryotic symbionts of the basal higher attine genera Trachymyrmex and Sericomyrmex was only slightly enhanced, but the evolutionarily derived crop fungi of Atta and Acromyrmex leaf‐cutting ants had much higher genetic variation. Our opposite ploidy models indicated that the symbionts of Trachymyrmex and Sericomyrmex are likely to be lowly and facultatively polyploid (just over two haplotypes on average), whereas Atta and Acromyrmex symbionts are highly and obligatorily polyploid (ca. 5–7 haplotypes on average). This stepwise transition appears analogous to ploidy variation in plants and fungi domesticated by humans and in fungi domesticated by termites and plants, where gene or genome duplications were typically associated with selection for higher productivity, but allopolyploid chimerism was incompatible with sexual reproduction.

Attine ants evolved farming 55–60 My before humans. Although evolutionarily derived leafcutter ants achieved industrial-scale farming, extant species from basal attine genera continue to farm loosely domesticated fungal cultivars capable of pursuing independent reproductive interests. We used feeding experiments with the basal attine Mycocepurus smithii to test whether reproductive allocation conflicts between farmers and cultivars constrain crop yield, possibly explaining why their mutualism has remained limited in scale and productivity. Stoichiometric and geometric framework approaches showed that carbohydrate-rich substrates maximize growth of both edible hyphae and inedible mushrooms, but that modest protein provisioning can suppress mushroom formation. Worker foraging was consistent with maximizing long-term cultivar performance: ant farmers could neither increase carbohydrate provisioning without cultivars allocating the excess toward mushroom production, nor increase protein provisioning without compromising somatic cultivar growth. Our results confirm that phylogenetically basal attine farming has been very successful over evolutionary time, but that unresolved host–symbiont conflict may have precluded these wild-type symbioses from rising to ecological dominance. That status was achieved by the evolutionarily derived leafcutter ants following full domestication of a coevolving cultivar 30–35 Mya after the first attine ants committed to farming. Significance Early subsistence farming implied significant physiological challenges for Neolithic farmers until they genetically isolated their crops through artificial selection and polyploidization. The attine ants faced analogous challenges when they adopted fungus farming 55–60 Mya. Whereas evolutionarily derived attine lineages irreversibly domesticated cultivars approximately 25 Mya and ultimately realized industrial-scale farming, basal lineages retained small-scale farming, diversified, and now coexist with advanced fungus-farmers in most New World tropical ecosystems. We show that management of independent sexual reproduction in cultivars constrained farming productivity, echoing early human farming of unspecialized low-productivity crops. Loss of cultivar gene exchange with nondomesticated relatives likely reduced host–symbiont conflict over reproduction, fostering the rise of ecologically dominant ant-agriculture.

Leaf-cutting ants employ diverse behavioral strategies for promoting the growth of fungal cultivars in a structure known as fungus-garden. As a nutritionally-rich resource for the ants, the fungal crop is threatened by microbial antagonists and pathogens. Strategies for protecting the garden against harmful microbes have been described in detail, though the process of microbial threat recognition is not fully understood. Here, we review the literature on leaf-cutting ants’ social immunity traits, in search of possibilities by which workers recognize harmful microbes in their system. Based on current data, we suggest mechanisms regarding (1) chemical recognition, where discrimination could be related to chemical cues from the antagonistic microbe or semiochemicals released by the fungus-garden during harmful interactions, or (2) through associative learning when workers would connect the microbe cues with a damage in the fungus-garden, developing a “colony-level memory” towards this threat. We also discuss evidence supporting ant-fungus communication as key for maintaining the health of the fungus-garden, as well as experimental setups for future evaluation of threat detection and recognition by leaf-cutter ants.