Network analysis is an effective tool to describe and quantify the ecological interactions between plants and root-associated fungi. Mycoheterotrophic plants, such as orchids, critically rely on mycorrhizal fungi for nutrients to survive, therefore, investigating the structure of those intimate interactions brings new insights into the plant community assembly and coexistence. So far, there is little consensus on the structure of those interactions, described either as nested (generalist interactions), modular (highly specific interactions) or of both topologies. Biotic factors (e.g., mycorrhizal specificity) were shown to influence the network structure, while there is less evidence of abiotic factor effects. By using next-generation sequencing of the orchid mycorrhizal fungal (OMF) community associated with 238 plant individuals belonging to 17 orchid species, we assessed the structure of four orchid-OMF networks in two European regions under contrasting climatic conditions (Mediterranean vs Continental). Each network contained four to 12 co-occurring orchid species, including up to eight species shared among the sites. All four networks were both nested and modular, and fungal communities were different between co-occurring orchid species, despite multiple sharing of fungi across some orchids. Co-occurring orchid species growing in Mediterranean climates were associated with more dissimilar fungal communities, consistent with a greater modular structure compared to the Continental ones. The OMF diversity was comparable among orchid species since most orchids were associated with multiple rarer fungi and with only a few highly dominant ones in the roots. Our results provide useful highlights on potential factors involved in structuring plant-mycorrhizal fungi interactions in different climatic conditions.

To assess the fungal communities associated with several orchid species, root samples were collected from 238 plant individuals belonging to 17 orchid species in May-June 2018 in four species-rich grasslands located in two Europeans regions (Czech Republic and Southern France). Fungal DNA was extracted by the CTAB method, then the ITS2 nuclear rDNA region was amplified using two barcode-tagged primer pairs to cover the whole fungal diversity: the forward primer 5.8S-OF (5’-CATYGARTYKTTGAACGCA-3’) (Vogt-Schilb et al., 2020) complemented with the reverse primers (i) ITS4OF (5’-GTTACTAGGGGAATCCTTGTT-3’) and (ii) ITS4Tul (5’-CCGCCAGATTCACACATTGA-3’) (Taylor & McCormick, 2008). After bioinformatic analysis, the obtained reads were assigned to OTUs taxa when belonging to Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae families with at least 85% of the similarity threshold (other fungal taxa known to form orchid mycorrhiza like ectomycorrhizal and saprobic fungi were kept for further analysis). Sequencing was performed on a MiSeq Illumina by the SEQme Company (Dobříš, Czech Republic).