Reduced fungal dependency during in situ germination of the terrestrial orchid Bletilla formosana

Orchid seed germination is generally considered to be obligatorily dependent on mycorrhizal fungi. However, some terrestrial orchids are capable of asymbiotic germination in water agar under laboratory conditions, raising questions about the degree of fungal dependency during germination in nature. Here, we investigated in situ germination and fungal associations of the terrestrial orchid Bletilla formosana in Taiwan using seed packet experiments, high-throughput sequencing, ink staining, and stable isotope analyses. B. formosana exhibited rapid germination and a relatively high germination percentage under natural conditions, with seeds germinating within one month and median germination reaching 43.4%. Germination varied among sites without a significant overall site effect and was not affected by proximity to conspecific plants. High-throughput ITS amplicon sequencing revealed that germinated protocorms associated with diverse fungi across Basidiomycota, including Oxyporus, Serendipita, and Ceratobasidium. Although fungal pelotons were detected in approximately 61.7% of protocorms, colonization levels were low, with only 1.96% of protocorm cells colonized. Stable isotope analyses revealed significant ¹³C enrichment in protocorms relative to surrounding autotrophic plants, indicating fungal-derived carbon acquisition during germination. These results demonstrate that B. formosana maintains fungal associations during germination in nature but exhibits substantially reduced fungal dependency compared with most terrestrial orchids.