Neottia (Orchidaceae), is an interesting genus where achlorophyllous leafless plants, next to leafless plants with a green stem and also potentially autotrophic leafy species are found. The leafless species with a green stem could thereby display a transition state in the evolution to achlorophyllous full mycoheterotrophic plants. Hence, we here investigated the C, N and H stable isotope patterns of two leafy (Neottia makinoana, Neottia puberula var. maculata), two leafless species with a green stem (Neottia furusei, Neottia listeroides) and two leafless achlorophyllous Neottia species (Neottia inagakii, Neottia acuminata) from Japan and Taiwan.
Relative C and N isotope natural abundances were measured in dual element analysis mode with an elemental analyser coupled to a continuous flow isotope ratio mass spectrometer via an open-split interface. The relative isotope abundances of H and O were measured using thermal conversion through pyrolysis coupled to a continuous flow isotope ratio mass spectrometer via an open-split interface.
Isotope abundances showed statistically significant enrichment in all leafless Neottiaspecies in 13C and 15N, whereas the Neottia species with green stems, were even more enriched than the achlorophyllous species. No enrichment in 13C and 15N was detectable in the leafy Neottiaspecies. The 2H isotope abundances confirmed the findings of the C and N measurements regarding the leafless Neottiaspecies but also revealed an enrichment in 2H in the two leafy Neottia species.
Despite the green stem, the two investigated species, at least in the sampled habitat, seem to be full mycoheterotrophic plants, displaying an obviously far advanced transition state to the full mycoheterotrophic achlorophyllous Neottia species. Furthermore, we were able to verify a partial C gain from fungi of the two leafy Neottia speciesN. makinoana and N. puberula var. maculata, due to an enrichment in 2H.