Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural 13C and 15N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analyzed mycorrhizal associations and 13C and 15N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and non-green (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with non-ectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia-associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus-avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales, and that the association with non-ectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia-associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre-adaptation to mycoheterotrophy in the whole Neottieae.

植物依赖真菌碳源的真菌异养（mycoheterotrophy）策略已多次独立演化。在兰科植物中，该策略与菌根共生类型从丝核菌属（rhizoctonia）向外生菌根真菌（ectomycorrhizal fungi）的转变，以及自然状态下较高的碳13（13C）和氮15（15N）丰度密切相关。部分真菌异养物种的光合自养近缘类群也呈现出相似的同位素丰度特征，但此类类群大多尚未被研究，这模糊了我们对真菌异养演化路径的认知。本研究针对兼具光合自养与非光合（真菌异养）物种的鸟巢兰属（Neottia），选取其两个光合自养物种——凹唇鸟巢兰（Neottia ovata）与心叶鸟巢兰（Neottia cordata）（隶属于鸟巢兰族（Neottieae）），分析了二者的菌根共生关系以及生物量中的碳13、氮15丰度。研究涵盖欧洲41个采样点，涉及不同草甸与森林生境，以及兰科植物的不同发育阶段。通过真菌ITS条形码测序（ITS barcoding）与电子显微镜技术分析，结果显示无论生境与发育阶段如何，两种鸟巢兰均主要与非外生菌根型的丝膜菌目（Sebacinales）B分支形成共生关系——该类群是光合自养兰科植物的丝核菌共生伙伴。仅少量检测到隶属于角担菌科（Ceratobasidiaceae）、蜡壳菌科（Tulasnellaceae）的丝核菌类群，以及外生菌根真菌。同位素丰度分析未检测到植株从外生菌根真菌中获取碳源，表明两种鸟巢兰的营养模式与共生丝核菌的光合自养兰科植物一致。结合近缘的部分或完全真菌异养物种（如堪察加鸟巢兰Neottia camtschatea、菌生鸟巢兰Neottia nidus-avis）与外生菌根型丝膜菌目A分支的共生关系，我们提出：鸟巢兰属对丝膜菌目真菌存在菌根偏好，且与非外生菌根型丝膜菌目B分支的共生关系应为该属的祖先状态。在同一真菌类群内，选择生态功能不同的菌根共生伙伴的这种转变，在兰科植物中极为罕见。此外，共生丝核菌的鸟巢兰属物种的发现，挑战了“鸟巢兰族演化出真菌异养的祖先预适应是转向外生菌根真菌”这一观点。