Mycorrhizal specialization for Tulasnellaceae fungi in Taeniophyllum marianense, a leafless epiphytic orchid native to Guam

Orchids are obligately dependent on orchid mycorrhizal fungi (OMF) for nutrition, growth, and establishment. The degree of mycorrhizal specificity varies, from generalists associating with several fungi to specialists relying on a few species. Many leafless epiphytic orchids specifically associate with Ceratobasidiaceae fungi. However, the mycorrhizal associates and specificity of Taeniophyllum marianense, a leafless epiphytic orchid native to the remote island of Guam, remain unknown. To address this knowledge gap, we investigated 189 seedling and mature individuals of T. marianense growing on 26 host tree species across 10 sites in Guam. OMF were identified using fungal-specific primers targeting Ceratobasidiaceae, Serendipitaceae, Tulasnellaceae, and general Basidiomycota. Sequences were grouped into operational taxonomic units (OTUs) based on 97% similarity. T. marianense in Guam associated with a diversity of OMF, including five Tulasnellaceae, six Ceratobasidiaceae, and four Serendipitaceae OTUs. Two Tulasnellaceae OTUs (TU1 and TU2) were the most dominant, comprising 64.6% of fungal sequences and occurring consistently across seedling and mature orchids from different host tree species and habitats, suggesting their role as primary mycorrhizal associates. In contrast, Ceratobasidiaceae and Serendipitaceae were less common and may play minor or opportunistic roles. The mycorrhizal capacity of TU1 and TU2 isolates was confirmed in vitro, where both strains significantly promoted seed germination and protocorm development. While other leafless epiphytic orchids typically associate with Ceratobasidiaceae, T. marianense in Guam specifically associates with Tulasnellaceae fungi closely related to globally distributed species. These findings suggest that mycorrhizal specialization may persist in island ecosystems through flexible associations with widespread, locally available fungal associates.