Stable isotope signatures of underground seedlings reveal the organic matter gained by adult orchids from mycorrhizal fungi

1. Orchids produce dust seeds dependent on the provision of organic carbon by mycorrhizal fungi for their early development stages. Hence, all chlorophyllous orchids experience a dramatic switch in trophic strategies from initial mycoheterotrophy to either autotrophy or partial mycoheterotrophy during ontogeny. Yet, the degree to which partially mycoheterotrophic orchids gain carbon from their mycorrhizal fungi is unclear based on existing approaches. 2. Here, we propose a novel approach to quantify the fungal-derived organic matter gain of chlorophyllous mature orchids mycorrhizal with rhizoctonia fungi, using the stable isotope signatures of their fully mycoheterotrophic (FMH) seedlings in a linear two-source mixing model. 3. We conducted a field germination experiment with seven orchid species and measured carbon, nitrogen and hydrogen stable isotope natural abundances and nitrogen concentrations of mature orchids, underground seedlings, and autotrophic references. 4. After in situ burial for 19-30 months, germination rates varied considerably among five orchid species and failed for two. On average, underground seedlings were enriched in C-13 and N-15 relative to mature orchids and had higher nitrogen concentrations. Using the mean enrichment factors epsilon C-13 and epsilon H-2 of seedlings as FMH endpoint, the organic matter gain derived by mature orchids from mycorrhizas was c. 20%. 5. Chlorophyllous orchids mycorrhizal with rhizoctonias are predisposed to partially mycoheterotrophic nutrition due to their initially mycoheterotrophic seedling stage. We show that the carbon and hydrogen isotope abundances of underground seedlings can be used in an improved mixing-model to identify a significant proportion of fungal-derived organic matter in mature orchids.