Population genetic structure and the evolution of specialization within a multi-species ant-microbe symbiosis

The fungus-growing ant, Apterostigma dentigerum, is engaged in an obligate mutualism with fungi the ants cultivate as their primary food source. These fungal cultivars are attacked by ascomycete fungi (Escovopsis) capable of rapidly consuming the fungal garden. To manage these garden pathogens, the ants employ Actinobacteria (Pseudonocardia), which produce a novel antibiotic compound (dentigerumycin) with the potential to inhibit Escovopsis growth. Here, we use A.dentigerum, and three of its microbial symbionts to understand how geographic population structure impacts the evolution of specialization, and how the evolutionary dynamics of specialization operate within a quadripartite symbiosis. Population genetic and bioassay analyses showed that fungal cultivars are not co-adapting to their most common Escovopsis species across populations in Central America. Pseudonocardia, however, may be the specialized against Escovopsis. In contrast to cultivar-Escovopsis interactions, Pseudonocardia-Escovopsis bioassay experiments showed that the symbiotic bacteria’s ability to inhibit pathogen growth is highly variable. Pseudonocardia’s ability to inhibit Escovopsis was significantly correlated with Escovopsis abundance - the bacteria were better at inhibiting the most abundant Escovopsis species, whereas less common species were inhibited less, suggesting specialization against Escovopsis. Multi-locus population genetic and phylogenetic analyses showed that ant-associated Pseudonocardia are structured clonally and follow a pattern of isolation by distance over geographic space. Moreover, the geographic structure of Pseudonocardia may facilitate localized adaptation to Escovopsis. Overall, it appears that Pseudonocardia may be functioning as the indirect ‘host’ responsible for defending cultivars against Escovopsis in the association with A. dentigerum.