Identity and specificity of the fungi forming mycorrhizas with the rare mycoheterotrophic orchid Rhizanthella gardneri

Fully subterranean Rhizanthella gardneri (Orchidaceae) is obligately mycoheterotrophic meaning it is nutritionally dependent on the fungus it forms mycorrhizas with. Furthermore, R. gardneri purportedly participates in a nutrient sharing tripartite relationship where its mycorrhizal fungus simultaneously forms ectomycorrhizas with species of Melaleuca uncinata s.l. Although the mycorrhizal fungus of R. gardneri has been morphologically identified as Thanatephorus gardneri (from a single isolate), this identification has been recently questioned. We sought to clarify the identification of the mycorrhizal fungus of R. gardneri, using molecular methods, and to identify how specific its mycorrhizal relationship is. Fungal isolates taken from all sites where R. gardneri is known to occur shared almost identical ribosomal DNA (rDNA) sequences. The fungal isolate rDNA most closely matched that of other Ceratobasidiales species, particularly within the Ceratobasidium genus. However, interpretation of results was difficult as we found two distinct ITS sequences within all mycorrhizal fungal isolates of R. gardneri that we assessed. All mycorrhizal fungal isolates of R. gardneri readily formed ectomycorrhizas with a range of M. uncinata s.l. species. Consequently, it is likely that R. gardneri can form a nutrient sharing tripartite relationship where R. gardneri is connected to autotrophic M. uncinata s.l. by a common mycorrhizal fungus. These findings have implications for better understanding R. gardneri distribution, evolution and the ecological significance of its mycorrhizal fungus, particularly in relation to nutrient acquisition.