Changing Partners in the Dark: Isotopic and Molecular Evidence of Ectomycorrhizal Liaisons between Forest Orchids and Trees Author(s): Martin I. Bidartondo, Bastian Burghardt, Gerhard Gebauer, Thomas D. Bruns and David J. Read Source: Proceedings: Biological Sciences, Vol. 271, No. 1550 (Sep. 7, 2004), pp. 1799-1806 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/4142864 Accessed: 12-04-2015 21:09 UTC REFERENCES Linked references are available on JSTOR for this article: http://www.jstor.org/stable/4142864?seq=1&cid=pdf-reference#references_tab_contents You may need to log in to JSTOR to access the linked references. Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings: Biological Sciences. http://www.jstor.org This content downloaded from 169.229.32.36 on Sun, 12 Apr 2015 21:09:01 UTC All use subject to JSTOR Terms and Conditions Received 30 March 2004 174in THE ROYAL Accepted 17 May 2004 r1$EW'. SOCIETY Published online 30 July 2004 Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees Martin I. Bidartondol*, Bastian Burghardt2, Gerhard Gebauer2, Thomas D. Bruns' and David J. Read3 1Departmentof Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA 2Lehrstuhlfur Pflanzen6kologie, Universitdt Bayreuth, 95440 Bayreuth, Germany 3Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK In the mycorrhizal symbiosis, plants exchange photosynthates for mineral nutrients acquired by fungi from the soil. This mutualistic arrangement has been subverted by hundreds of mycorrhizal plant species that lack the ability to photosynthesize. The most numerous examples of this behaviour are found in the largest plant family, the Orchidaceae. Although non-photosynthetic orchid species are known to be highly specialized exploiters of the ectomycorrhizal symbiosis, photosynthetic orchids are thought to use free-living saprophytic or pathogenic fungal lineages. However, we present evidence that putatively photosynthetic orchids from five species that grow in the understorey of forests (i) form mycorrhizas with ectomycorrhizal fungi of forest trees and (ii) have stable-isotope signatures indicating distinctive pathways for nitrogen and carbon acquisition approaching those of non-photosynthetic orchids that associate with ectomycorrhizal fungi of forest trees. These findings represent a major shift in our understanding of both orchid ecology and evolution because they explain how orchids can thrive in low-irradiance niches and they show that a shift to exploiting ectomycorrhizal fungi precedes viable losses of photosynthetic ability in orchid lineages. Keywords: Epipactis; Cephalanthera; mycorrhizae; partial myco-heterotrophy; symbiosis; Tuber 1. INTRODUCTION Currently, there are two known mature-orchid nutritional The Orchidaceae is the largest and most diverse family of modes: (i) obligate autotrophy (i.e. photosynthetic) with plants on Earth. One of its most distinctive characteristics over 17 000 species; and (ii) obligate myco-heterotrophy is the production of minute seeds that contain only mini- (i.e. non-photosynthetic) with over 200 species. However, mal reserves of nutrients (Arditti & Ghani 2000). This it has been proposed recently that a third nutritional mode makes orchids dependent upon mycorrhizal fungi for the in which fungi subsidize the nutrition of putatively photo- provision of the resources necessary for germination and synthetic orchids ('partial myco-heterotrophy') accounts for growth, at least in the early stages of their development for a significant number of the forest-understorey species (Bernard 1909; Burgeff 1959). Such fungus-dependent currently considered obligate autotrophs (Gebauer & modes of nutrition, referred to as myco-heterotrophy, have Meyer 2003). evolved independently several times during plant evolu- Since early in the last century, it has been widely tion (Leake 1994). There is a widespread assumption that accepted that most orchid mycorrhizal fungi are saprophy- in the majority of orchid species, which are photosynthetic tic or pathogenic rhizoctonia-forming basidiomycete fungi that includes Cerato- in the adult phases of their lives, the ability to photo- (i.e. a polyphyletic assemblage Exidiales and synthesize will provide a release from the dependence on basidiales, Tulasnellales) (Bernard 1909; Roberts studies have shown that these fungi for carbon supplies (Smith & Read 1997). Nonethe- 1999). Experimental cultivable can sustain less, a large proportion of green, and hence putatively pho- easily fungi below-ground develop- ment of some orchids carbon from soil tosynthetic, orchids grow in such deeply shaded forest by transferring matter to & Read habitats that carbon gains from photosynthesis are likely to organic developing seedlings (Smith Interest in the that other functional be minimal. In fact, complete loss of photosynthetic ability, 1997). possibility of be with coupled with obligate myco-heterotrophy into the adult groups fungi might symbiotic partners photo- orchids has been aroused by two sets of phase, may have evolved at least 20 times in the Orchida- synthetic recently observations: (i) molecular ecological analy- ceae (Molvray et al. 2000). Because terrestrial orchids independent ses have shown that several wholly non-photosynthetic include some of the most vulnerable components of plant orchid form orchid mycorrhizas with hardly culti- communities worldwide (Batty et al. 2002), it is of pressing species vable that form with concern to determine how these orchids are sustained fungi simultaneously ectomycorrhizas the roots of neighbouring trees (Taylor & Bruns 1997; throughout their life cycle under natural conditions. Selosse et al. 2002); and (ii) mass-spectrometric analyses of wholly non-photosynthetic orchids, monotropes and orchids & *Author and address for correspondence: Imperial College London and putatively photosynthetic (Gebauer Meyer 2003; Royal Botanic Gardens, Kew TW9 3DS, UK ([email protected]). Trudell et al. 2003) of forest habitats have revealed that Proc. R. Soc. Lond. B (2004) 271, 1799-1806 1799 0 2004 The Royal Society doi: 10. 1098/rspb.2004.2807 This content downloaded from 169.229.32.36 on Sun, 12 Apr 2015 21:09:01 UTC All use subject to JSTOR Terms and Conditions 1800 M. I. Bidartondo and others Orchids use ectomycorrhizalfungi their tissues carry nitrogen and carbon stable-isotope sig- (L = 4), and four species characteristicof open environments,E. natures indicative of alternative pathways for the acqui- atrorubens (Hoffm. ex Bernh.) Besser (L = 6), Platanthera chlor- sition of these elements. In fact, Gebauer and Meyer antha (Cust.) Rchb. p. (L = 6), E. palustris (L.) Crantz (L = 8) postulated that putatively photosynthetic forest orchids are and Dactylorhizamajalis s.l. (L = 8). For the current status of connected both to typical orchid mycorrhizal fungi (i.e. the Neottiaeae phylogenetics (including Epipactisand Cephalanthera) rhizoctonia-forming fungi) 'and to basidiomycetes forming see Bateman et al. (2004). ectomycorrhizas with trees' (p. 221). We combine molecular and mass-spectrometric approa- (c) Mass-spectroscopy analysis ches and apply them to the same individual plants of eight Following a sampling methodology described elsewhere orchid species growing in their natural plant communities (Gebauer & Meyer 2003), in July 2003 we collected, from each of at four sites in Germany. These species represent three the four sites, four replicate leaves of between one and four photo- functional groups: photosynthetic orchids of open habitats, synthetic orchid species and leaves of accompanying non-orchid putatively photosynthetic orchids of shaded habitats and ground vegetation (for species lists see electronic Appendix A). wholly non-photosynthetic orchids. We show that the latter On the three forest sites, the non-orchid plants encompassed four two groups are colonized by fungi that are ectomycorrhizal functional types: ectomycorrhizalplants (ECM), plants forming associates of trees and that their shoot nitrogen and carbon arbuscular mycorrhizas or non-mycorrhizal plants (AM/NM), signatures are sufficiently distinct from those of orchids and leguminous plants potentially living in symbiosis with colonized by rhizoctonia-forming fungi to indicate reliance nitrogen-fixing bacteria and forming arbuscular mycorrhizas upon alternative nutritional pathways. We also surveyed (AM/FIX). At the non-forested wetland site, the non-orchid the mycorrhizal fungi of three additional orchid species at plants included AM/NM and AM/FIX plants only. In addition, 12 locations outside Germany and confirmed that associa- we sampled one wholly myco-heterotrophic orchid species from tions between putatively photosynthetic orchids of shaded forest site 1. Soil samples from the uppermost 5 cm were collected habitats and ectomycorrhizal fungi are widespread and adjacent to each orchid plant.