Premise: Orchids are obligately dependent on orchid mycorrhizal fungi (OMF) for nutrition, growth, and establishment. Varying degrees of mycorrhizal specificity have been observed in orchids, with generalists associating with a range of fungi and specialists with a few species. The leafless, epiphytic orchid alliance specifically associates with Ceratobasidiaceae fungi, although such specificity remains understudied in relation to remote island colonization. 

Methods: We investigated the OMF of Taeniophyllum marianense, a leafless, epiphytic orchid native to Guam. Samples from 189 seedlings and mature T. marianense growing on 26 different host tree species were collected across 10 sites in Guam. OMF was identified using Basidiomycota, Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae-specific primers for coverage of major mycorrhizal groups. Identified OMF were assigned to operational taxonomic units (OTUs) based on 97% sequence similarity. Mycorrhizal compatibility with T. marianense was assessed in isolated OMF by in vitro symbiotic germination experiments.

Results: T. marianense in Guam associated with a diversity of OMF encompassing 5 Tulasnellaceae, 6 Ceratobasidiaceae, and 4 Serendipitaceae OTUs. Two Tulasnellaceae OTUs accounted for 64.6% of fungal sequences across seedling and mature orchids from different host tree species and habitats, suggesting their role as a major mycorrhizal partner. Ceratobasidiaceae accounted for only 13.1% of sequences, suggesting their minor, opportunistic role.

Conclusions: While the leafless, epiphytic orchid alliance specifically associates with Ceratobasidiaceae, T. marianense in Guam primarily associates with two globally distributed Tulasnellaceae partners. Our findings suggest that remote island colonization is not limited by mycorrhizal specificity and expand our understanding of symbioses in the context of island biogeography.

Nuclear ribosomal DNA sequences from the three data sets (Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae) were aligned using MAFFT. Phylogenetic analyses were performed using RAxML-NG. The phylogenetic tree was constructed using the maximum likelihood method based on the GTR+G+I substitution model. The relative robustness of each branch was evaluated using the bootstrap method with 1000 replicates.