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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., Competing Interests: The authors declare no conflict of interest.

Background: Xyloglucan is an important component in plant cell walls that herbivores cannot digest without microbial symbionts. Leaf-cutting ants are major insect herbivores in the Neo-Tropics that rely on fungus-garden enzymes for degrading plant cell walls. However, many of these ants discard much of their harvested plant material after partial degradation, which has led to the hypothesis that the fungal symbionts are primarily producing cell wall degrading enzymes to gain access to intracellular nutrients rather than for obtaining sugars from recalcitrant cell wall polymers, such as (hemi-)cellulose., Results: The fungal symbiont provides a single xyloglucanase (Xeg1) to its ant farmers by upregulating the expression of this protein in the inflated hyphal tips (gongylidia) that the ants ingest. Similar to other enzymes ingested this way, also Xeg1 is not digested but vectored to the fresh leaf-fragment pulp at the top of fungus gardens via ant fecal fluid. Xeg1 is 4-5 times more active in fecal fluid when ants ingest their normal fungal food, compared to a sucrose control diet, as expected when they cannot produce Xeg1 themselves. We confirm substrate specificity of fungal Xeg1 towards xyloglucan by heterologous expression in yeast and show that xyloglucanase activity is higher in the oldest, bottom layers of fungus gardens and in discarded debris material than in the upper and middle layers of fungus gardens., Conclusion: Our results are consistent with Xeg1 playing a role in the initial breakdown of plant cell wall hemicellulose to provide sugars for aggressive hyphal growth before intracellular proteins become available. Xeg1 does not play a major decomposition role in the middle layer of fungus gardens where it is produced by the gongylidia. Overall high xyloglucanase activity in old mycelium that is (about to be) discarded is striking and quite possibly serves defensive purposes by precluding that competing microorganisms can grow. Our results support the hypothesis that the ant-fungus symbiosis prioritizes access to the protein-rich contents of live plant cells and that carbohydrates are not a limiting resource.

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5-7, but the aspartic proteases were inactive across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.