Spatial repartition and genetic relationship of green and albino individuals in mixed populations of Cephalanthera orchids

Several green orchids of the Neottieae tribe acquire organic carbon both from their mycorrhizal fungi and from photosynthesis. This strategy may represent an intermediate evolutionary step towards mycoheterotrophy of some non‐photosynthetic (albino) orchids. Mixed populations of green and albino individuals possibly represent a transient evolutionary stage offering opportunities to understand the evolution of mycoheterotrophy. In order to understand the emergence of albinos, we investigated patterns of spatial and genetic relationships among green and albino individuals in three mixed populations of Cephalanthera damasonium and one of C. longifolia using spatial repartition and Amplified fragment length polymorphism (AFLP) markers. Two of these populations were monitored over two consecutive flowering seasons. In spatial repartition analyses, albino individuals did not aggregate more than green individuals. Genetic analyses revealed that, in all sampled populations, albino individuals did not represent a unique lineage, and that albinos were often closer related to green individuals than to other albinos from the same population. Genetic and spatial comparison of genets from the 2‐year monitoring revealed that: (i) albinos had lower survival than green individuals; (ii) accordingly, albinos detected in the first year did not correspond to the those sampled in the second year; and (iii) with one possible exception, all examined albinos did not belong to any green genet from the same and/or from the previous year, and vice versa. Our results support a scenario of repeated insurgence of the albino phenotypes within the populations, but unsuccessful transition between the two contrasting phenotypes. Future studies should try to unravel the genetic and ecological basis of the two phenotypes.     

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 analysed 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 nongreen (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 nonectomycorrhizal 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 nonectomycorrhizal 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.