1. Plants often rely on external, mutualistic partners to survive and reproduce in resource-limited environments or for protection from enemies. Such interactions, including mycorrhizal symbioses and ant–plant associations, are widespread and play an important role at the ecosystem and community levels. In ant–plant mutualisms, the plants may benefit from both the protection provided by the presence of ants and from the nutrients absorbed from insect debris. However, the role of third partners in plant nutrition, particularly ant-associated fungi, has never before been demonstrated.
2. We investigate this issue in the ant–plant Hirtella physophora. In this model system, Allomerus decemarticulatus ants are involved in two, highly specific interactions: first, with their host plant, and, secondly, with a fungus that they actively manipulate. Moreover, the ants combine both plant trichomes and fungal hyphae to make a trap to capture prey.
3. We empirically demonstrate the existence of a third type of interaction between the fungus and the plant through the use of both experimental enrichments with stable isotopes (15N) and histological approaches. The fungus growing in the galleries plays a role in providing nutrients to the host plant, in addition to the structural role it plays for the ants. Fungus-facilitated nitrogen uptake occurs mainly in old domatia, where abundant hyphae are in close contact with the plant cells. Whether the fungi inside the domatia and those in the galleries are the same is still uncertain.
4. Synthesis.Together, our results show that a fungal partner in an ant–plant mutualism can benefit the plant by improving its nutrient uptake, and they demonstrate the existence of a true tripartite mutualism in this system. Our results add further evidence to the notion that interpretations of some ant–plant symbioses as purely protective mutualisms have overlooked these nutritional aspects.