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In recent decades, multipartite mutualisms involving microorganisms such as fungi have been discovered in associations traditionally thought of as bipartite. Ant-plant mutualisms were long thought to be bipartite despite fungi being noticed in an epiphytic ant-plant over 100 years ago. We sequenced fungal DNA from the three distinct domatium chambers of the epiphytic ant-plant  Myrmecodia beccarii  to establish if fungal communities differ by chamber type across five geographic locations spanning 675 km. The three chamber types serve different ant-associated functions including ‘waste’ chambers, where ant workers deposit waste; ‘nursery’ chambers, where the brood is kept; and ‘ventilation’ chambers, that allow air into the domatium. Overall, fungi from the order Chaetothyriales dominated the chambers in terms of the proportion of operational taxonomic units (OTUs; 13.4%) and sequence abundances of OTUs (28% of the total); however a large portion of OTUs (28%) were unidentified at the order level. Notably, the fungal community in the waste chambers differed consistently from the nursery and ventilation chambers across all five locations. We identified 13 fungal OTUs as ‘common’ in the waste chambers that were rare or in very low sequence abundance in the other two chambers. Fungal communities in the nursery and ventilation chambers overlapped more than either did with the waste chambers but were also distinct from each other. Differences in dominance of the common OTUs drove the observed patterns in the fungal communities for each of the chamber types. This suggests a multipartite mutualism involving fungi exists in this ant-plant and that the role of fungi differs among chamber types.

Consistent patterns of fungal communities within ant-plants across a large geographic range strongly suggest a multipartite mutualism