Partial mycoheterotrophy is more widespread among orchids than previously assumed.

With > 22 000 species in 880 genera occurring on all continents in almost every habitat, the Orchidaceae is considered the most diverse andmost widely distributed plant family on Earth (Merckx et al., 2013). All orchids produce tiny, dust-like seeds (Arditti & Ghani, 2000) that require carbon (C) and nutrient supply by mycorrhizal fungi for germination and in the early seedling development phase (Smith&Read, 2008). This kind of nutrition is called initialmycoheterotrophy (Merckx, 2013). Later themajority of orchids develop green leaves and photosynthesize, yet remain mycorrhizal. There are, however, also > 200 achlorophyllous orchid species. These orchids require complete C and presumably mineral nutrient supply from mycorrhizal fungi and are known as full mycoheterotrophs (Leake, 1994; Merckx, 2013). Owing to their use of a C(Gleixner et al., 1993) and N-enriched (Gebauer & Dietrich, 1993) fungal source for nutrition, mycoheterotrophic orchids are significantly enriched in C and N, when compared with autotrophic plants from the same microhabitats (Gebauer &Meyer, 2003; Trudell et al., 2003; Hynson et al., 2013). Based on their isotopic positioning between autotrophic nonorchids and achlorophyllous orchids, during the last decade a couple of chlorophyllous orchids have been found to acquire C from both autotrophic photosynthates and associated fungi (Gebauer & Meyer, 2003; Hynson et al., 2013). This mode of mixotrophic C gain is called partialmycoheterotrophy (Gebauer& Meyer, 2003; Merckx, 2013). In the past, the use of C stable isotope natural abundance played an important role in elucidating C gain by higher plants from two origins. This approach was highly successful when the two C sources of interest were sufficiently distinguished in their isotope composition, for example, in the case of hemiparasites with C3 photosynthesis simultaneously utilizing host plants with C4 or CAM photosynthesis (Press et al., 1987; Schulze et al., 1991; Ziegler, 1996). Limitations of this approach emerged in cases with two potential C sources not sufficiently distinguished in their C isotope composition, for example in the case of C3 hemiparasites utilizing C3 host plants (Schulze et al., 1991; Ziegler, 1994). Since early investigations by Bernard (1909), the majority of chlorophyllous orchids are considered as formingmycorrhizas with fungi that belong to the phylogenetically heterogeneous assemblage of rhizoctonias (Dearnaley et al., 2012). Interestingly, all known partially mycoheterotrophic orchids are additionally or exclusively associated with fungi that simultaneously form ectomycorrhizas with neighbouring trees (Selosse et al., 2002; Bidartondo et al., 2004; Hynson et al., 2013). Regarding the C and nitrogen (N) isotope abundance pattern, rhizoctonia-associated orchids do not provide a uniform picture. Hynson et al. (2013) speculated that a partially mycoheterotrophic C gain may be hidden behind findings of almost uniform N enrichment and inconsistent C positioning (Bidartondo et al., 2004; Liebel et al., 2010; Hynson et al., 2013) and called these orchids ‘cryptic partial mycoheterotrophs’. In order to elucidate this phenomenon of cryptic C gain from fungal sources, St€ockel et al. (2014) investigated C and N isotope abundances in initially mycoheterotrophic orchid seedlings and in mycoheterotrophic, partially mycoheterotrophic and putatively autotrophic orchid adults. It emerged that mycoheterotrophic seedlings of ectomycorrhiza-associated orchids were enriched in C and N in the same manner as mycoheterotrophic adults. However, mycoheterotrophic seedlings of rhizoctonia-associated orchids were by far less enriched in C and N than were mycoheterotrophic seedlings of orchids with ectomycorrhizal fungi. St€ockel et al. (2014) concluded that C and N stable isotope abundance alone is not suited to unequivocally identify partially mycoheterotrophic species among adults of rhizoctonia-mycorrhizal chlorophyllous orchids. Here we provide a new approach to detect partial mycoheterotrophy among rhizoctonia-mycorrhizal orchids. In addition to C and N stable isotope abundance, we analysed hydrogen (H) stable isotope natural abundance in dry tissues of an achlorophyllous orchid, chlorophyllous orchids associated either with ectomycorrhizal fungi or with rhizoctonias, autotrophic reference plants and sporocarps of ectomycorrhizal and saprotrophic fungi growing together in the understorey of a beech forest. We hypothesized that H isotope abundance can be used as a substitute for C isotope abundance in cases of limited information available fromC isotope composition, because all organic compounds exchanged between fungi and plants are composed of C andH atoms.We furthermore hypothesized that the relative abundance of the heavy isotope H increases proportionally with the heterotrophic C gain of the investigated species. Thus, partially mycoheterotrophic orchids should be positioned in their H isotope abundance between autotrophic and fully mycoheterotrophic plants irrespective of their fungal partner. The second hypothesis is based on the conclusion of Yakir (1992) that secondary heterotrophic organic compounds, that is in our case organic compounds of fungal origin, are enriched in H compared with primary photosynthetic organic compounds. In order to determine whether transpiration (Ziegler, 1988) is responsible for differences in H isotope abundance of orchids and reference plants, we analysed oxygen (O) isotope abundances. Transpiration affects H and O isotope abundances in the same direction (Sternberg, 1988).