Data from: 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.植物对真菌碳的依赖（真菌异养，mycoheterotrophy）经历了多次独立演化。在兰科植物中，该特性与菌根从丝核菌属（rhizoctonia）向外生菌根真菌（ectomycorrhizal fungi）的转变，以及植株自然丰度下较高的碳13（13C）和氮15（15N）同位素丰度密切相关。部分真菌异养物种的绿色近缘类群呈现出相似的同位素特征，但其中大多数尚未被研究，这使得我们对真菌异养演化路径的认知仍存在模糊之处。我们针对包含绿色与非绿色（真菌异养）物种的鸟巢兰属（Neottia）内的2个绿色物种——卵叶鸟巢兰（Neottia ovata）和心叶鸟巢兰（N. cordata，隶属于鸟巢兰族（Neottieae）），分析了其菌根共生关系与生物量的碳13、氮15同位素丰度。本研究覆盖欧洲41个样地，涵盖不同的草甸与森林生境，以及兰花的不同发育阶段。通过真菌ITS条形码（ITS barcoding）测序与电子显微镜（electron microscopy）观察，我们发现两种鸟巢兰均主要与非外生菌根型的蜡壳耳目B分支（Sebacinales Clade B）形成共生关系——该类群是绿色兰科植物的丝核菌共生伙伴——且这一共生模式不受生境与生长阶段的影响。此外仅少量检测到来自角担菌科（Ceratobasidiaceae）、胶膜菌科（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其从外生菌根真菌中获取碳的证据，表明与丝核菌共生的绿色兰科植物仍采用常规营养策略。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、无叶鸟巢兰N. nidus-avis）与蜡壳耳目A分支（Sebacinales Clade A）外生菌根真菌的共生关系，我们提出：鸟巢兰属对蜡壳耳目类真菌存在共生偏好，且与非外生菌根型蜡壳耳目B分支的共生关系可能为该属的祖先型共生模式。这种在同一真菌类群内选择生态功能迥异的菌根共生伙伴的共生偏好转变，在兰科植物中较为罕见。此外，存在与丝核菌共生的鸟巢兰物种这一事实，对‘鸟巢兰族（Neottieae）中转向外生菌根真菌是真菌异养演化的祖先预适应’这一假说提出了挑战。