Chlorophyllous and Achlorophyllous Specimens of Epipactis microphylla (Neottieae, Orchidaceae) Are Associated with Ectomycorrhizal Septomycetes, including Truffles M.-A. Selosse 1 , A. Faccio 2 , G. Scappaticci 3 and P. Bonfante 2 (1) UMR CNRS 7138 ‘‘Syste ´matique, Adaptation et Evolution’’ and Service de Syste ´matique Mole ´culaire (IFR CNRS 101), Muse ´um National d’Histoire Naturelle, 43, rue Cuvier, 75005 Paris, France (2) Dipartimento di Biologia Vegetale dell’Universita `, Istituto per la Protezione delle Piante—CNR, Viale Mattioli, 25, 10125 Torino, Italy (3) Socie ´te ´ Franc ¸aise d’Orchidophilie, Les Combeaux, Quartier Les Rouvie `res, 26220 Dieulefit, France Received: 7 August 2003 / Accepted: 29 October 2003 / Online publication: 27 April 2004 Abstract Mycoheterotrophic species (i.e., achlorophyllous plants obtaining carbon from their mycorrhizal fungi) arose many times in evolution of the Neottieae, an orchid tribe growing in forests. Moreover, chlorophyllous Neottieae species show naturally occurring achlorophyllous indi- viduals. We investigated the fungal associates of such a member of the Neottieae, Epipactis microphylla, to un- derstand whether their mycorrhizal fungi predispose the Neottieae to mycoheterotrophy. Root symbionts were identified by sequencing the fungal ITS of 18 individuals from three orchid populations, including achlorophyl- lous and young, subterranean individuals. No rhizocto- nias (the usual orchid symbionts) were recovered, but 78% of investigated root pieces were colonized by Tuber spp. Other Pezizales and some Basidiomycetes were also found. Using electron microscopy, we demonstrated for the first time that ascomycetes, especially truffles, form typical orchid mycorrhizae. All identified fungi (but one) belonged to taxa forming ectomycorrhizae on tree roots, and four of them were even shown to colonize sur- rounding trees. This is reminiscent of mycoheterotrophic orchid species that also associate with ectomycorrhizal fungi, although with higher specificity. Subterranean and achlorophyllous E. microphylla individuals thus likely rely on tree photosynthates, and a partial mycoheterotrophy in individuals plants can be predicted. We hypothesize that replacement of rhizoctonias by ectomycorrhizal symbionts in Neottieae entails a predisposition to achlorophylly. Introduction Mycorrhizal fungi form mutualistic associations on plants roots, where they usually exchange plant photo- synthates for soil-collected mineral nutrients [45]. However, a considerable variability exists among the various mycorrhizal interactions, and some plants may even recover carbon from their fungal partners. Because of the low specificity of mycorrhizal fungi, coexisting plants can share identical fungal symbionts. Such links have been demonstrated to allow carbon transfer between plants, both in vitro [16] and in field conditions [44]. The relevance of this carbon flux at the physiological and ecological levels is still debated [15, 35]. However, some species, namely the mycoheterotrophic plants (MHPs), exclusively rely on this flux to achieve their growth and reproduction. MHPs are achlorophyllous plants with reduced root systems that obtain both mineral nutrients and carbon from their fungal partners [23]. Molecular methods have showed that MHP species have a strong mycorrhizal specificity to narrow fungal clades, which in most cases form mycorrhizae with surrounding green plants [49]. Carbon labeling has demonstrated that the latter plants constitute the ultimate source of carbon for both the MHP and the fungus [7, 8, 24]. Although the effect on the fitness of the carbon-furnishing plant and the fungus is still unclear, MHPs are therefore considered as ‘‘my- corrhizal cheaters’’ [6]. Species phylogenetically close to MHPs are good candidates for a partial mycoheterotro- phy, i.e., for relying not only on their photosynthesis, but also partly on a carbon flux from the fungus [18]. In addition, the study of such species, likely representing a state ancestral to mycoheterotrophy, can clarify how MHPs arose in plant evolution. As a preliminary step, Correspondence to: M.-A. Selosse; E-mail: ma.selosse@wanadoo.fr 416 DOI: 10.1007/s00248-003-2034-3 d Volume 47, 416–426 (2004) d Ó Springer-Verlag New York, LLC 2004