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Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria ( Pseudonocardia ). Specifically, we examine conflict between the ants and their  Pseudonocardia  symbionts maintained to derive antibiotics against parasitic microfungi ( Escovopsis ) infecting the ants' fungus garden. Symbiont assays pairing isolates of  Pseudonocardia  spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with  Acromyrmex  leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of  Pseudonocardia  symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of  Pseudonocardia  within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.

Antagonistic Bacterial Interactions Help Shape Host-Symbiont Dynamics within the Fungus-Growing Ant-Microbe Mutualism