Leafcutter Ant Farmers Prevent Loss of Edible Symbiotic Structures by Maintaining Allelic Diversity in Their Multinucleate Fungal Crop.

Leafcutter ants farm the domesticated fungal cultivar Leucoagaricus gongylophorus in subterranean nests containing up to hundreds of discrete garden chambers. The fungal cultivar produces edible symbiotic structures called gongylidia (nutritious swollen hyphal cells consumed by the ants) and expresses a form of polyploidy (multiple, genetically distinct nuclei per fungal cellular compartment). Yet, the fungus is also thought to lack the typical innate mechanisms for distributing these nuclei across its mycelial network of connected hyphal cells. Because new garden chambers are seeded by clonal fungal fragments from existing chambers, this raises questions about whether and how nuclear diversity is maintained across chambers within a colony. We hypothesized: (1) that mycelial fragmentation causes genetic and phenotypic instability in isolated fungal cultivar inoculates, but (2) that these variables remain stable when actively farmed by ants. We found that experimentally fragmented fungal isolates in Petri dishes lost gongylidium production and had higher growth rates. Microsatellite analyses confirmed that these phenotypic changes coincided with loss of alleles and fluorescence microscopy showed that gongylidium incompetency coincided with reduced nucleus numbers per cellular compartment. In contrast, natural fragmentation by ants in free-ranging rainforest colonies coincided with minimal genetic divergence across garden chambers, even as divergence increased slightly with chamber distance. By integrating field and laboratory data, these results support: (1) that gongylidium production depends on genetic heterozygosity and multinuclearity, and (2) that ant farmers can maintain this genetic diversity. Resolving these stabilizing mechanisms will be crucial for understanding the genome evolution underlying a naturally selected crop domestication system.