1. Orchid mycorrhiza forms unique symbiotic associations between members of the Orchidaceae and multiple ecological guilds of fungi. Because orchids associate with a wide variety of fungi with different ecological functions, they represent an ideal study system to address fundamental questions about the evolution and ecophysiology of mycorrhizal symbiosis. Although it is well established that shifts in mycorrhizal associations are linked to transitions in plant trophic mode, it remains unclear what ecological drivers promote these evolutionary changes.2. Here, we investigated mycorrhizal communities and isotope signatures across six populations of the terrestrial orchid Neottia ovata growing under contrasting light conditions in temperate Europe. We hypothesized that plants growing in forests would associate with different mycorrhizal fungi than plants occurring in grasslands and that the limited light availability in forests leads to a higher contribution of fungi to the carbon budget of orchids.3. Our results showed that N. ovata predominantly associated with rhizoctonia fungi of the family Serendipitaceae in both habitats, but plants in forests also recruited ectomycorrhizal fungi. Root communities highly resembled soil communities and variation in root communities was significantly related to habitat type and edaphic factors. In contrast, isotope signatures ( 13 C, 15 N, 2 H and 18 O) and N concentration showed no significant relationship with habitat type. In addition, both 13 C and 2 H were not significantly correlated to habitat's light availability. 4.Although it has been suggested that the presence of a wide variety of ectomycorrhizal fungi in root communities of orchids can serve as a precursor for evolutionary shifts to partial mycoheterotrophy (mixotrophy), the presence or absence of | 1949Functional Ecology WANG et al.

Background and Aims The chlorophyllous, terrestrial orchid Cremastra appendiculata from East Asia is unique concerning its fungal mycorrhiza partners. The initially mycoheterotrophic protocorms exploit rather specialised non-rhizoctonia saprotrophic Psathyrellaceae. Adult individuals of this orchid species are either linked to Psathyrellaceae being partially mycoheterotrophic or form mycorrhiza with fungi of the ubiquitous saprotrophic rhizoctonia group. This study provides new insights on nutrition mode, subterranean morphology, and fungal partners across different life-stages of C. appendiculata. Methods We compared different development stages of C. appendiculata to surrounding autotrophic reference plants based on multi-element natural abundance stable isotope analyses (δ 13C, δ 15N, δ 2H, δ 18O) and total N concentrations. Site- and sampling-time-independent enrichment factors of stable isotopes were used to reveal trophic strategies. We determined mycorrhizal fungi of C. appendiculata protocorm, seedling, and adult samples using high-throughput DNA sequencing. Key Results We identified saprotrophic non-rhizoctonia Psathyrellaceae as dominant mycorrhizal fungi in protocorm and seedling rhizomes. In contrast, the roots of seedlings and mature C. appendiculata were mainly colonised with fungi belonging to the polyphyletic assembly of rhizoctonia (Ceratobasidium, Thanatephorus and Serendipitaceae). Mature C. appendiculata did not differ in isotopic signature from autotrophic reference plants suggesting a fully autotrophic nutrition mode. Characteristic of orchid specimens entirely relying on fungal nutrition, C. appendiculata protocorms were enriched in 15N, 13C and 2H compared to reference plants. Seedlings showed an intermediate isotopic signature, underpinning the differences in the fungal community depending on their subterranean morphology. Conclusions In contrast to the suggestion that C. appendiculata is a partially mycoheterotrophic orchid species, we provide novel evidence that mature C. appendiculata with rhizoctonia mycobionts can be entirely autotrophic. Besides an environmentally driven variability among populations, we suggest high within-individual flexibility in nutrition and mycobionts of C. appendiculata, which is subject to the ontogenetic development stage.