Carbon and nitrogen gain during the growth of orchid seedlings in nature.

For germination and establishment, orchids depend on carbon (C) and nutrients supplied by mycorrhizal fungi. As adults, the majority of orchids then appear to become autotrophic. To compare the proportional C and nitrogen (N) gain from fungi in mycoheterotrophic seedlings and in adults, here we examined in the field C and N stable isotope compositions in seedlings and adults of orchids associated with ectomycorrhizal and saprotrophic fungi. Using a new highly sensitive approach, we measured the isotope compositions of seedlings and adults of four orchid species belonging to different functional groups: fully and partially mycoheterotrophic orchids associated with narrow or broad sets of ectomycorrhizal fungi, and two adult putatively autotrophic orchids associated exclusively with saprotrophic fungi. Seedlings of orchids associated with ectomycorrhizal fungi were enriched in (13) C and (15) N similarly to fully mycoheterotrophic adults. Seedlings of saprotroph-associated orchids were also enriched in (13) C and (15) N, but unexpectedly their enrichment was significantly lower, making them hardly distinguishable from their respective adult stages and neighbouring autotrophic plants. We conclude that partial mycoheterotrophy among saprotroph-associated orchids cannot be identified unequivocally based on C and N isotope compositions alone. Thus, partial mycoheterotrophy may be much more widely distributed among orchids than hitherto assumed.

The degree of mycoheterotrophic carbon gain in green, variegated and vegetative albino individuals of Cephalanthera damasonium is related to leaf chlorophyll concentrations.

• Achlorophyllous variants of some forest orchids are known to reach almost the same size as their green forms. These vegetative albino forms cover their entire carbon (C) demand through fungi that simultaneously form ectomycorrhizae with trees, while green variants partially draw on C from photosynthesis and C from fungal hosts. Here, we investigate whether the amount of C derived from either source is proportional to leaf chlorophyll concentration. The discovery of two Cephalanthera damasonium populations with variegated leaves enabled a continuous bridging of leaf chlorophyll concentrations between green and albino forms. • Leaves of 27 green, variegated and albino individuals of C. damasonium were compared for chlorophyll concentrations, C sources (as characterized by (13)C abundances) and total C and nitrogen (N) concentrations. • We found a linear relationship between leaf chlorophyll concentrations and the proportional reliance on fungi as a C source. Furthermore, we show that the shift in C gain through mycoheterotrophic means significantly changes leaf total C and N concentrations. • Our results document that partial mycoheterotrophy in C. damasonium is not a static nutritional mode but a flexible mechanism related inter alia to leaf chlorophyll concentrations. The change in proportional reliance on fungi as a C source affects leaf chemical composition.

15N and 13C natural abundance of two mycoheterotrophic and a putative partially mycoheterotrophic species associated with arbuscular mycorrhizal fungi.

• In contrast to mycoheterotrophs that associate with ectomycorrhizal and saprotrophic fungi, we know little about the ecophysiology of arbuscular mycorrhizal mycoheterotrophs. Here, we identify the mycorrhizal fungi of two unrelated mycoheterotrophs and one putative partial mycoheterotroph that form arbuscular mycorrhizas, and analyse their carbon (C) and nitrogen (N) isotope signatures. • We used molecular methods to identify the mycorrhizal fungi of Dictyostega orobanchoides, Burmannia capitata (Burmanniaceae) and Voyria aphylla (Gentianaceae). Their C and N sources were investigated by analysing their stable isotope natural abundances (δ(13)C and δ(15)N). In addition, four putative partially mycoheterotrophic Burmannia species were grown ex situ. • We found that both mycoheterotrophs and a green Burmannia species are associated with nonoverlapping Glomeromycota fungi. The investigated mycoheterotrophs are significantly more enriched in (13)C than co-occurring autotrophic plants but lack significant (15)N enrichment. The green Burmannia species is not significantly enriched in (13)C and (15)N compared with surrounding plants and can grow fully autotrophically under controlled conditions. • Our results suggest that mycoheterotrophic Burmanniaceae and Gentianaceae are able to exploit arbuscular mycorrhizal fungi. Green relatives of mycoheterotrophic Burmanniaceae from high-light grassland sites also associate with arbuscular mycorrhizal fungi but we found no evidence that they receive detectable amounts of C from fungi.

C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia.

We compared the nutritional modes and habitats of orchids (e.g., autotrophic, partially or fully mycoheterotrophic) of the Mediterranean region and adjacent islands of Macaronesia. We hypothesized that ecological factors (e.g., relative light availability, surrounding vegetation) determine the nutritional modes of orchids and thus impose restrictions upon orchid distribution. Covering habitats from dark forests to open sites, orchid samples of 35 species from 14 genera were collected from 20 locations in the Mediterranean and Macaronesia to test for mycoheterotrophy. Mycorrhizal fungi were identified via molecular analyses, and stable isotope analyses were applied to test whether organic nutrients are gained from the fungal associates. Our results show that orchids with partial or full mycoheterotrophy among the investigated species are found exclusively in Neottieae thriving in light-limited forests. Neottioid orchids are missing in Macaronesia, possibly because mycoheterotrophy is constrained by the lack of suitable ectomycorrhizal fungi. Furthermore, most adult orchids of open habitats in the Mediterranean and Macaronesia show weak or no N gains from fungi and no C gain through mycoheterotrophy. Instead isotope signatures of some of these species indicate net plant-to-fungus C transfer.