Exploring fungus-plant N transfer in a tripartite ant-plant-fungus mutualism.

Background and Aims The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a N-15-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added N-15 from the fungus to plant tissues. Key Results The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The N-15 provisioning of the fungus in the galleries resulted in significant enrichment of the N-15 signature of the plant's leaves 1 d after the N-15-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from N-15-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.