1 You are what you get from your fungi: carbon and nitrogen stable isotopes in Epipactis 2 orchids. 3 Julienne M.-I. Schiebold1, Martin I. Bidartondo2, 3, Peter Karasch4, Barbara Gravendeel5 & 4 Gerhard Gebauer1* 5 1Laboratory of Isotope Biogeochemistry, Bayreuth Center of Ecology and Environmental 6 Research (BayCEER), University of Bayreuth, 95440 Bayreuth, Germany 7 2Department of Life Sciences, Imperial College London, SW7 2AZ London, England 8 3Royal Botanic Gardens, Kew, TW9 3DS Richmond, Surrey, England 9 4Bavarian Mycological Society, Section Bavarian Forest, Ablegweg 9, 94227 Rabenstein, 10 Germany 11 5Naturalis Biodiversity Center, Leiden, Netherlands 12 *Author for correspondence: 13 Gerhard Gebauer, Email: [email protected], Tel: +49 921 552060 14 15 Total word count: 5654 16 Introduction: 1011 17 Materials and Methods: 2398 18 Results: 939 19 Discussion: 2918 20 Acknowledgements: 72 21 Number of figures: three figures, colour 22 Number of tables: three tables 23 Supporting information: three tables 24 1 25 Summary 26 Partially mycoheterotrophic plants are enriched in 13C and 15N compared to 27 autotrophic plants. Here, we hypothesise the identity of mycobiont clades found in 28 orchid roots is responsible for variation in 15N enrichment of leaf bulk tissue in 29 partially mycoheterotrophic orchids. 30 We used the genus Epipactis as a case study and measured carbon and nitrogen 31 isotope natural abundances of eight Epipactis species, fungal sporocarps of four Tuber 32 species and autotrophic reference plants. Fungal mycobionts were determined using 33 molecular methods. We compiled stable isotope data of six further Epipactis species 34 and 11 ectomycorrhizal and four saprotrophic basidiomycetes from the literature. 35 The 15N enrichment of Epipactis species varied between 3.2 ± 0.8 ‰ (E. gigantea; 36 rhizoctonia-associated) and 24.6 ± 1.6 ‰ (E. neglecta; associated with 37 ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10.7 ± 38 2.2 ‰) were significantly more enriched in 15N than ectomycorrhizal (5.2 ± 4.0 ‰) 39 and saprotrophic basidiomycetes (3.3 ± 2.1 ‰). 40 We suggest the observed gradient in 15N enrichment of Epipactis species is strongly 41 driven by 15N abundance in their mycobionts; i.e. 15N enrichment in Epipactis spp. 42 associated with rhizoctonias < 15N enrichment in Epipactis spp. with ectomycorrhizal 43 basidiomycetes < 15N enrichment in Epipactis spp. with ectomycorrhizal ascomycetes 44 and basidiomycetes < 15N enrichment in Epipactis spp. associated with 45 ectomycorrhizal ascomycetes. 46 47 Key words: ascomycetes, basidiomycetes, carbon, Epipactis, nitrogen, Orchidaceae, partial 48 mycoheterotrophy, stable isotopes 49 50 2 51 INTRODUCTION 52 Partial mycoheterotrophy is a trophic strategy of plants defined as a plant´s ability to obtain 53 carbon (C) simultaneously through photosynthesis and mycoheterotrophy via a fungal source 54 exhibiting all intermediate stages between the extreme trophic endpoints of autrotrophy and 55 mycoheterotrophy (Merckx, 2013). However, all so far known partially mycoheterotrophic 56 plants feature a change of trophic strategies during their development. In addition to all fully 57 mycoheterotrophic plants, all species in the Orchidaceae and the subfamily Pyroloideae in the 58 Ericaceae produce minute seeds that are characterised by an undifferentiated embryo and a 59 lack of endosperm. These "dust seeds" are dependent on colonisation by a mycorrhizal fungus 60 and supply of carbohydrates to facilitate growth of nonphotosynthetic protocorms in this 61 development stage termed initial mycoheterotrophy (Alexander & Hadely, 1985; Leake, 62 1994; Rasmussen, 1995; Rasmussen & Whigham, 1998; Merckx et al., 2013). At adulthood 63 these initially mycoheterotrophic plants either stay fully mycoheterotrophic (e.g. Neottia 64 nidus-avis) or they become (putatively) (I don't know if it's necessary to doubt autotrophy at 65 this point) autotrophic or partially mycoheterotrophic. With approximately 28,000 species in 66 736 genera the Orchidaceae is the largest angiosperm family with worldwide distribution 67 constituting almost a tenth of described vascular plant species (Chase et al., 2015; 68 Christenhusz & Byng, 2016) making initial mycoheterotrophy the most widespread fungi- 69 mediated trophic strategy. 70 71 Analysis of food-webs and clarification of trophic strategies with 13C and 15N stable isotope 72 abundance values have a long tradition in ecology (DeNiro & Epstein, 1978, 1981). DeNiro & 73 Epstein coined the term “you are what you eat – plus a few permil” (DeNiro & Epstein, 74 1976) to highlight the systematic increase in the relative abundance of 13C and 15N at each 75 trophic level of a food chain. In 2003, Gebauer & Meyer and Trudell et al. were the first to 76 employ stable isotope natural abundance analyses of C and N to distinguish the trophic level 77 of mycoheterotrophic orchids from surrounding autotrophic plants. 78 79 Today, stable isotope analysis together with the molecular identification of fungal partners 80 have become the standard tools for research on trophic strategies in plants, especially orchids 81 (Leake & Cameron, 2010). Since the first discovery of partially mycoheterotrophic orchids 82 (Gebauer & Meyer, 2003), the number of species identified as following a mixed type of 83 trophic strategy has grown continuously (Hynson et al., 2013, 2016; Gebauer et al., 2016). 84 One of the well-studied orchid genera in terms of stable isotopes and molecular identification 3 85 of orchid mycobionts is the genus Epipactis ZINN (Bidartondo et al., 2004; Tedersoo et al., 86 2007; Hynson et al., 2016). Epipactis is a genus of terrestrial orchids comprising 70 taxa (91 87 including hybrids) (‘The Plant List’, 2013) with mainly Eurasian distribution. Epipactis 88 gigantea is the only species in the genus native to North America and Epipactis helleborine is 89 naturalised there. All Epipactis species are rhizomatous and summergreen and they occur in 90 various habitats ranging from open wet meadows to closed-canopy dry forests (Rasmussen, 91 1995). Partial mycoheterotrophy of several Epipactis species associated with ectomycorrhizal 92 mycobionts (E. atrorubens, E. distans, E. fibri and E. helleborine) has been elucidated using 93 stable isotope natural abundances of C and N. They all turned out to be significantly enriched 94 in both 13C and 15N (Hynson et al., 2016). Orchid mycobionts of the Epipactis species in the 95 above-mentioned studies were ascomycetes and basidiomycetes simultaneously 96 ectomycorrhizal with neighboring forest trees and in some cases additionally basidiomycetes 97 belonging to the polyphyletic rhizoctonia group well known as forming orchid mycorrhizas 98 have also been detected (Gebauer & Meyer, 2003; Bidartondo et al., 2004; Abadie et al., 99 2006; Tedersoo et al., 2007; Selosse & Roy, 2009; Liebel et al., 2010; Gonneau et al., 2014). 100 Epipactis gigantea and E. palustris, the only two Epipactis species colonising open habitats 101 and exhibiting exclusively an association with rhizoctonias, showed no 13C and only minor 102 15N enrichment (Bidartondo et al., 2004; Zimmer et al., 2007). 103 104 The definition of trophic strategies in vascular plants is restricted to an exploitation of C and 105 places mycoheterotrophy into direct contrast to autotrophy. The proportions of C gained by 106 partially mycoheterotrophic orchid species from fungi have been quantified by a linear two- 107 source mixing-model approach (Gebauer & Meyer, 2003; Preiss & Gebauer, 2008; Hynson et 108 al., 2013). Variations in percental C gain of partially mycoheterotrophic orchids from the 109 fungal source are driven by plant species identity placing e.g. the leafless Corallorhiza trifida 110 closely towards fully mycoheterotrophic orchids (Zimmer et al., 2008; Cameron et al., 2009) 111 and by physiological and environmental variables such as leaf chlorophyll concentration 112 (Stöckel et al., 2011) and light climate of their microhabitats (Preiss et al., 2010). Carbon gain 113 in the orchid species Cephalanthera damasonium, for example, can range from 33% in an 114 open pine forest to about 85% in a dark beech forest (Gebauer, 2005; Hynson et al., 2013). 115 116 By far less clear is the explanation of variations in 15N enrichment found for fully, partially 117 and initially mycoheterotrophic plants, but also for putatively autotrophic species (Gebauer & 118 Meyer, 2003; Abadie et al., 2006; Tedersoo et al., 2007; Preiss & Gebauer, 2008, Selosse & 4 119 Roy, 2009; Liebel et al., 2010, Hynson et al., 2013). This 15N enrichment was found to be not 120 linearly related to the degree of heterotrophic C gain (Leake & Cameron, 2010; Merckx et al., 121 2013). Using the linear two-source mixing-model approach to obtain quantitative information 122 of the proportions of N gained by partially mycoheterotrophic orchid species from the fungal 123 source, some species even exhibited an apparent N gain above 100% (Hynson et al., 2013). 124 Reasons for this pattern remained unresolved and could just be explained by lacking coverage 125 of variability in 15N signatures of the chosen fully mycoheterotrophic endpoint due to 126 different fungal partners (Preiss & Gebauer, 2008; Hynson et al., 2013). 127 128 Here, we hypothesise that the type of mycobionts in the roots of orchid species (i.e. 129 ectomycorrhizal basidiomycetes, ectomycorrhizal ascomycetes or basidiomycetes of the 130 rhizoctonia group) is responsible for the differences