Partial mycoheterotrophy (PMH) is a nutritional mode in which plants utilize organic matter, i.e. carbon, both from photosynthesis and a fungal source. The latter reverses the direction of plant-to-fungus carbon flow as usually assumed in mycorrhizal mutualisms. Based on significant enrichment in the heavy isotope ¹³C, a growing number of PMH orchid species have been identified. These PMH orchids are mostly associated with fungi simultaneously forming ectomycorrhizas with forest trees. In contrast, the much more common orchids that associate with rhizoctonia fungi, which are decomposers, have stable isotope profiles most often characterized by high ¹⁵N enrichment and high nitrogen concentrations but either an insignificant ¹³C enrichment or depletion relative to autotrophic plants. Using hydrogen stable isotope abundances recent investigations showed PMH in rhizoctonia-associated orchids growing under light-limited conditions. Hydrogen isotope abundances can be used as substitute for carbon isotope abundances in cases where autotrophic and heterotrophic carbon sources are insufficiently distinctive to indicate PMH.
To determine whether rhizoctonia-associated orchids growing in habitats with high irradiance feature PMH as a nutritional mode, we sampled 13 orchid species growing in montane meadows, four forest orchid species and 34 autotrophic reference species. We analysed δ²H, δ¹³C, δ¹⁵N and δ¹⁸O and determined nitrogen concentrations. Orchid mycorrhizal fungi were identified by DNA sequencing.
As expected, we found high enrichments in ²H, ¹³C, ¹⁵N and nitrogen concentrations in the ectomycorrhiza-associated forest orchids, and the rhizoctonia-associated Neottia cordata from a forest site was identified as PMH. Most orchids inhabiting sunny meadows lacked ¹³C enrichment or were even significantly depleted in ¹³C relative to autotrophic references. However, we infer PMH for the majority of these meadow orchids due to both significant ²H and ¹⁵N enrichment and high nitrogen concentrations. Pseudorchis albida was the sole autotrophic orchid in this study as it exhibited neither enrichment in any isotope nor a distinctive leaf nitrogen concentration.
Synthesis. Our findings demonstrate that partial mycoheterotrophy is a trophic continuum between the extreme endpoints of autotrophy and full mycoheterotrophy, ranging from marginal to pronounced. In rhizoctonia-associated orchids, partial mycoheterotrophy plays a far greater role than previously assumed, even in full light conditions.

1 INTRODUCTION

The Orchidaceae is usually referred to as the largest plant family with almost a tenth of described vascular plant species, that is c. 28,000 species in 736 genera (Chase et al., 2015; Christenhusz & Byng, 2016). Orchids have a world-wide distribution, and they occur in a variety of habitats only avoiding the polar regions and the driest deserts (Chase et al., 2015; Merckx et al., 2013). Regardless of their appearance as epiphytic, lithophytic, terrestrial or subterranean life-forms, all orchid species share the trait of producing large numbers of tiny, endospermless seeds containing only very small amounts of nutrients (Arditti & Ghani, 2000). Consequently, the trophic strategy of initial mycoheterotrophy—the colonization of dust-like seeds by mycorrhizal fungi and their provision of protocorms with nutrients until the photosynthetic seedling stage—is a characteristic feature of all Orchidaceae (Alexander & Hadley, 1985; Merckx et al., 2013; Rasmussen, 1995). Protocorms are the non-photosynthetic, fully mycoheterotrophic (FMH), pre-seedling stages formed after germination of dust seeds colonized by a mycorrhizal fungus. Some orchid species lack chlorophyll at maturity so they satisfy also in the adult stage all nutrient demands by exploiting their mycorrhizal fungi. Due to ongoing taxonomic work and recent discoveries (Suetsugu, 2016, 2017) the number of FMH orchid species is growing continuously from a minimum of 235 species (Merckx et al., 2013). All FMH Orchidaceae species belong to the subfamilies Vanilloideae, Orchidoideae and Epidendroideae and occur mostly in the tropics. They are associated with either ectomycorrhizal fungi or saprotrophic fungi that decompose litter or wood (Bidartondo, Burghardt, Gebauer, Bruns, & Read, 2004; Hynson, Preiss, Gebauer, & Bruns, 2009; Lee, Yang, & Gebauer, 2015; Martos et al., 2009; Ogura-Tsujita, Gebauer, Hashimoto, Umata, & Yukawa, 2009; Zimmer et al., 2007).

Fully mycoheterotrophic species can be identified by their achlorophyllous appearance and also by measuring the stable isotope natural abundances in their tissues. Fungi, being heterotrophic, are enriched in ¹³C as well as in ¹⁵N compared to their substrates and to autotrophic plants (Gebauer & Dietrich, 1993; Gleixner, Danier, Werner, & Schmidt, 1993; Trudell, Rygiewicz, & Edmonds, 2003). Mycoheterotrophic orchids either take up fungal material via hyphal lysis and/or transfer across intact membranes of fungus and orchid (Smith & Read, 2008). Following the systematic increase in the relative abundance of ¹³C and ¹⁵N at each trophic level in a food chain as originally described by DeNiro and Epstein (1978, 1981), orchid tissues consequently mirror the isotopic signature of their associated fungi (Gebauer & Meyer, 2003).

Stable isotope analysis together with the molecular identification of mycorrhizal fungi have become the standard tools for research on trophic strategies in plants, especially orchids (Leake & Cameron, 2010). Using stable isotope natural abundance analysis, a growing number of partially mycoheterotrophic (PMH) species have been identified (Gebauer & Meyer, 2003; Hynson, Schiebold, & Gebauer, 2016; Hynson et al., 2013) that simultaneously utilize both carbon from photosynthesis and a fungal source (PMH sensu Merckx, 2013). Isotopic enrichment in ¹³C of PMH orchids was considered until recently to be intermediate between autotrophic plants from the same microhabitats (Gebauer & Meyer, 2003) and FMH orchids that rely on their mycorrhizal fungi as sole nutrient source; ¹³C enrichment varies with light climate (Preiss, Adam, & Gebauer, 2010) and leaf chlorophyll concentration (Stöckel, Meyer, & Gebauer, 2011). There is evidence that FMH and PMH orchid species also meet their complete nitrogen demands through mycorrhizal fungi (Stöckel, Těšitelová, Jersáková, Bidartondo, & Gebauer, 2014) and as ¹⁵N enrichment varies with type of fungal symbiont, ¹⁵N enrichment of PMH orchids can even exceed the isotopic enrichment in ¹⁵N of FMH species (Schiebold, Bidartondo, Karasch, Gravendeel, & Gebauer, 2017).

Until recently, all identified PMH orchid species gaining nutrients from mixed sources were Epidendroideae exclusively associated with ectomycorrhizal fungi or with ectomycorrhizal fungi additionally to typical orchid mycorrhizal rhizoctonias, a polyphyletic group of basidiomycetes (Bidartondo et al., 2004). Nevertheless, the overwhelming majority of chlorophyllous orchid species is associated with rhizoctonia fungi (Dearnaley, Martos, & Selosse, 2012) and was thus assumed to be putatively autotrophic. However, the stable isotope profiles of rhizoctonia-associated orchids are most often characterized by conspicuously significant ¹⁵N enrichment and higher leaf total nitrogen concentrations compared to autotrophic plants, and either a lacking or modest but insignificant ¹³C enrichment, or even depletion relative to autotrophic references (Girlanda et al., 2011; Johansson, Mikusinska, Ekblad, & Eriksson, 2014; Liebel et al., 2010). Thus, the assumed autotrophy of rhizoctonia-associated orchids has been challenged and they have been called “cryptic mycoheterotrophs” (Hynson, 2016; Hynson et al., 2013). High N isotope abundances and high nitrogen concentrations might indicate that in addition to photosynthesis there is carbon gain from fungal sources, that is, nitrogen gain via organic compounds, and thus partial mycoheterotrophy (PMH).

In a comparison between ¹³C and ¹⁵N enrichment of FMH protocorms and mature individuals of the same species, Stöckel et al. (2014) found that achlorophyllous, FMH seedlings of rhizoctonia-associated orchids were far less enriched in ¹³C and ¹⁵N than protocorms of orchids that associate with ectomycorrhizal fungi. They proposed that especially the ¹³C enrichment measured in mature chlorophyllous orchids associated with saprotrophic rhizoctonia fungi might be too small to enable the detection of PMH, in contrast to a usually clear ¹³C enrichment of mature chlorophyllous orchids associated with ectomycorrhizal fungi. In other words, the carbon source isotope abundance of rhizoctonia-associated orchid species, namely rhizoctonia fungi, is too close to the isotope abundance of autotrophic plants to be distinguished (Gebauer, Preiss, & Gebauer, 2016). Stöckel et al. (2014) concluded that the routinely used δ¹³C ratios might not be sufficient to unequivocally identify PMH orchid species associated with rhizoctonia fungi. Recently, Gebauer et al. (2016) provided clear evidence that the trophic strategy of PMH is far more widespread in forest understorey orchids than previously assumed. They used δ²H in addition to the routinely employed δ¹³C and δ¹⁵N measurements following the reasoning that not only carbon and nitrogen but also hydrogen atoms are present in organic molecules that move from fungus to orchid during peloton digestion and/or by transfer across intact membranes. Consequently, δ²H values could thus also be used as indicators for mycoheterotrophic nutrition. The δ²H approach is based on the finding that secondary heterotrophic organic compounds (i.e. in our case compounds of fungal origin) are enriched in ²H compared to primary photosynthetic organic compounds (Yakir, 1992). This enrichment is substantiated in the increasing exchange of H atoms from hydroxyl groups in organic molecules with H atoms from surrounding tissue water molecules with increasing heterotrophy. The great value of H stable isotope abundances to identify carbon gain of heterotrophic origin was first elucidated in a study on the C and H stable isotope abundances of plant parasites and their hosts by Ziegler (1994). Holoparasites obtain all organic material from their hosts and thus mirror their δ¹³C values. However, as heterotrophic material is taken up from the host by the holoparasite, the holoparasite is always enriched in δ²H relative to its host. Thus, based on ²H enrichment, that study documents organic matter, i.e. carbon, gain by a parasite from its host independently of any ¹³C enrichment.

In Gebauer et al.'s (2016) study not only the ectomycorrhiza-associated FMH orchid species Neottia nidus-avis and PMH Cephalanthera damasonium, Cephalanthera rubra and Epipactis atrorubens were enriched in ²H simultaneously to ¹³C and ¹⁵N, serving as a proof of concept, but also four rhizoctonia-associated species (Cypripedium calceolus, Neottia ovata, Ophrys insectifera and Platanthera bifolia) were positioned in their ²H enrichment between autotrophic plants sampled as references and the FMH N. nidus-avis. Thus, justification was provided for substituting H for C stable isotope abundance analysis in cases where C stable isotope abundances of carbon sources are poorly differentiated (Gebauer et al., 2016) and mask the C flow between mycorrhizal fungi and orchids. Consequently, a significant ²H enrichment in plant tissue can serve as an indicator for flow of heterotrophic organic matter, i.e. carbon, from mycorrhizal fungi to plant and thus for PMH. Rhizoctonia-associated orchid species with C isotope abundances close to autotrophic plants can serve as a prime example. The above-mentioned eight orchid species were sampled in a closed-canopy beech forest with sparse herb understorey, and thus it is assumed that all orchids compensated lacking irradiance for efficient photosynthesis by increasing exploitation of their mycorrhizal fungi. The gain of organic matter via peloton digestion and/or by transfer through membranes is vital for all orchids in their early pre-photosynthetic stages and also for FMH orchids and ectomycorrhiza (ECM)-associated PMH orchids growing under light limitation. However, digestion and/or organic matter transfer might still be relevant for rhizoctonia-associated orchid species. Partial mycoheterotrophy could be a useful trait for chlorophyllous orchid species associated with rhizoctonia fungi to compete against other herbaceous species in open habitats and to help survive unfavourable conditions during adult dormant underground stages (Shefferson, 2009). Whether the overwhelming majority of rhizoctonia-associated orchid species growing in open habitats and thus under light-saturated conditions are PMH remains to be investigated.

Here, we hypothesize that orchids in the subfamilies Epidendroideae and Orchidoideae forming mycorrhizas with rhizoctonia fungi and growing in habitats with high irradiance levels, such as montane meadows, feature PMH as nutritional mode. These plants may be predisposed to mycoheterotrophic nutrition due to initial mycoheterotrophy in the protocorm stage. We test our hypothesis by employing natural abundance analysis of δ²H, δ¹³C and δ¹⁵N and determination of nitrogen concentrations. In addition, we analysed δ¹⁸O to exclude a potential bias due to different transpiration by orchids and reference plants.

2 MATERIALS AND METHODS

2.1 Study design and site descriptions

We selected an area in the Northern Limestone Alps in the Eastern Alps of Europe with high orchid diversity and sampled a variety of orchid species to test for a general pattern. We measured multi-element stable isotope abundances (δ¹³C, δ¹⁵N, δ²H and δ¹⁸O) and identified mycorrhizal fungi with molecular methods. Orchid species with known trophic pathways such as full (N. nidus-avis (L.) Rich.) and PMH (C. rubra (L.) Rich. and Epipactis helleborine (L.) Crantz) associated with ectomycorrhizal fungi and autotrophic non-orchid plant species were sampled as references.

Seventeen orchid species were sampled at six sites in Austria and Germany during July in four consecutive years between 2012 and 2015. The major sampling area was the Marul Valley located in the biosphere park of the Great Walser Valley in the province of Vorarlberg in Western Austria between the village of Garfülla and the alp Laguz and nearby Laguz (N 47.1°–47.2°; E 9.8°–10°, 1,220–1,740 m a.s.l.). At Garfülla we sampled the FMH N. nidus-avis (L.) Rich. and the PMH C. rubra (L.) Rich. and E. helleborine (L.) Crantz (all members of the tribe Neottieae) in a nearby montane mixed deciduous and coniferous forest dominated by Fagus sylvatica L. and Picea abies (L.) H. Karst. Between Garfülla and alp Laguz, we sampled ten orchid species of the tribe Orchideae (Dactylorhiza majalis (Rchb.) P.F.Hunt & Summerh., Dactylorhiza incarnata (L.) Soó, Dactylorhiza viridis (L.) R.M.Bateman, Pridgeon & M.W.Chase, Gymnadenia conopsea (L.) R.Br., Gymnadenia nigra (L.) Rchb.f., Herminium monorchis (L.) R.Br., Neotinea ustulata (L.) R.M.Bateman, Pridgeon & M.W.Chase, P. bifolia (L.) Rich., P. albida (L.) Á.Löve & D.Löve and Traunsteinera globosa (L.) Rchb.) from open extensively mown or grazed montane meadows and Nardus grasslands (Tables S1 and S2). On the colline foothills of the Great Walser Valley in the Walgau region, we sampled Liparis loeselii (L.) Rich. (tribe Malaxideae) in a fen near the municipality of Thüringen and Spiranthes aestivalis (Poir.) Rich. (tribus Cranichideae) in a periodically wet Molinia meadow near Göfis (N 47.2°, E 9.7°–9.9°, 475 and 745 m a.s.l.). Still in the Northern Limestone Alps but in the Karwendel region in Southern Bavaria, Malaxis monophyllos (L.) Sw. was sampled at Schachen (N 47.4°; E 11.1°, 1,800 m a.s.l.) on a montane meadow. The Neottieae Neottia cordata (L.) Rich. was sampled in a coniferous forest dominated by P. abies near Fichtelsee in the low mountain range Fichtelgebirge in NE Bavaria (N 50.0°; E 11.9°, 760 m a.s.l.). Sampling followed the plot-wise sampling scheme by Gebauer and Meyer (2003); leaf samples from flowering individuals of all orchid species were taken in five replicates (resembling five 1 m² plots) together with three autotrophic reference plants under the same growth conditions and in the same microclimate. A broad range of non-orchid autotrophic references representing a variety of functional groups and growth forms including plants with arbuscular mycorrhizas, ectomycorrhizas or ericoid mycorrhizas and non-mycorrhizal species were sampled to depict the variability in autotrophic plants. Only leguminous, parasitic and carnivorous plants were excluded from sampling. Please refer to Gebauer and Meyer (2003) for further details of the sampling method. Sampling yielded a total of 85 samples from 17 orchid species (shoot samples of N. nidus-avis and leaf samples of all other orchid species) and 253 leaf samples from 34 neighbouring autotrophic reference species in 85 plots distributed over six sites (Tables S1 and S2). Nomenclature for orchid species and autotrophic references follows www.theplantlist.org (The Plant List, 2013).

2.2 Analysis of stable isotope abundance and nitrogen concentration

Leaf samples of the 17 orchid species (n = 85) and autotrophic references (n = 253) were washed with deionized water, dried to constant weight at 105°C, ground to a fine powder in a ball mill (Retsch Schwingmühle MM2, Haan, Germany) and stored in a desiccator fitted with silica gel until analysis. Relative C and N isotope natural abundances of the leaf samples were measured in dual element analysis mode with an elemental analyser (1108; Carlo Erba Instruments, Milano, Italy) coupled to a continuous flow isotope ratio mass spectrometer (delta S, Finnigan MAT, Bremen, Germany) via a ConFlo III open-split interface (Thermo Fisher Scientific, Bremen, Germany) as described in Bidartondo et al. (2004). Relative H and O isotope natural abundances of the leaf samples were measured with thermal conversion through pyrolysis (HTO, HEKAtech, Wegberg, Germany) coupled to a continuous flow isotope ratio mass spectrometer (delta V advantage; Thermo Fisher Scientific) via a ConFlo IV open-split interface (Thermo Fisher Scientific) as described in Gebauer et al. (2016). Due to memory bias each sample was analysed three times and the first two sample runs were skipped for reliable H isotope abundance determination. In order to minimize bias of post-sampling H atom exchange between organically bound hydroxyl groups in our samples and H₂O in ambient air (Yakir, 1992), we analysed samples of orchids and their respective reference plant samples together in identical sample batches. The O isotope abundances were measured to rule out a transpiration effect as a cause of differences in the H isotope abundance between orchids and non-orchid reference plants (Ziegler, 1988).

Measured relative isotope abundances are denoted as δ values that were calculated according to the following equation: δ¹³C, δ¹⁵N, δ²H or δ¹⁸O = (R_sample/R_standard − 1) × 1000 (‰), where R_sample and R_standard are the ratios of heavy to light isotope of the samples and the respective standard. Standard gases (Riessner, Lichtenfels, Germany) were calibrated with respect to international standards (CO₂ vs. PDB, N₂ vs. N₂ in air, H₂ and CO vs. SMOW) with the reference substances ANU sucrose and NBS19 for the C isotopes, N1 and N2 for the N isotopes, CH7, V-SMOW and SLAP for H isotopes and IAEA601 and IAEA602 for the O isotopes, all provided by the IAEA (International Atomic Energy Agency, Vienna, Austria). Reproducibility and accuracy of the C and N isotope abundance measurements were routinely controlled by measuring the laboratory standard acetanilide (Gebauer & Schulze, 1991). In relative C and N isotope natural abundance analyses, acetanilide was routinely analysed with variable sample weight at least six times within each batch of 50 samples. The maximum variation in δ¹³C and δ¹⁵N both within and between batches was always below 0.2‰. For relative H and O isotope natural abundance analyses, benzoic acid was routinely analysed with variable sample weight at least six times within each batch of 40 samples. The maximum variation in δ²H and δ¹⁸O both within and between batches was always below 4‰ for δ²H and 0.6‰ for δ¹⁸O.

Total nitrogen concentrations in leaf and shoot samples were calculated from sample weights and peak areas using a six-point calibration curve per sample run based on measurements of the laboratory standard acetanilide with a known nitrogen concentration of 10.36% (Gebauer & Schulze, 1991).

2.3 Molecular identification of mycorrhizal fungi

Mycorrhizal fungi of some of the orchid species under investigation were already known form previous studies. Nonphotosynthetic N. nidus-avis shows high fungal specificity for Sebacina (ectomycorrhizal clade A) (McKendrick, Leake, Taylor, & Read, 2002; Selosse, Weiß, Jany, & Tillier, 2002). Cephalanthera rubra associates with Leptodontidium, Phialophora and Tomentella (Bidartondo et al., 2004). The mycorrhizal communities of E. helleborine have been studied thoroughly by several authors and are diverse as they consist of ectomycorrhizal ascomycetes and basidiomycetes and also typical orchid mycorrhizal rhizoctonia fungi; for example Ceratobasidium, sebacinoids, Tuber (Bidartondo et al., 2004), Helvella, Wilcoxina (Ogura-Tsujita & Yukawa, 2008), Inocybe, Thelephora (Tĕšitelová, Tĕšitel, Jersáková, Ríhová, & Selosse, 2012) and Tricholoma and Russula (Jacquemyn, Waud, Lievens, & Brys, 2016). The meadow orchid G. conopsea forms typical orchid mycorrhizae with Tulasnella, Ceratobasidium, Thanatephorus and Sebacina, but also with members of the Pezizales (Stark, Babik, & Durka, 2009). Mycorrhizal communities of P. bifolia are dominated by Ceratobasidiaceae, but also Tulasnellaceae and Sebacinales (Esposito, Jacquemyn, Waud, & Tyteca, 2016). Pseudorchis albida has a diverse fungal community dominated by Tulasnella (Jersáková, Malinová, Jeřábková, & Dötterl, 2011; Kohout, Těšitelová, Roy, Vohník, & Jersáková, 2013).

To check whether the prerequisite of orchid mycorrhizas with rhizoctonia was met in the remaining meadow orchids, we identified their mycorrhizal fungi by molecular methods. Of the sampled orchid species, L. loeselii, M. monophyllos, N. cordata, S. aestivalis, D. incarnata, D. majalis, D. viridis, G. nigra, H. monorchis, N. ustulata and T. globosa, two roots per sampled orchid individual were cut, rinsed with deionized water, placed in CTAB buffer (cetyltrimethylammonium bromide) and stored at −18°C until further analysis. Root cross-sections were checked for presence and status of fungal pelotons in the cortex cells. Two to six root sections per orchid individual were selected for genomic DNA extraction and purification with the GeneClean III Kit (Q-BioGene, Carlsbad, CA, USA). The nuclear ribosomal internal transcribed spacer (ITS) region was amplified with the fungal-specific primer combinations ITS1F/ITS4 and ITS1/ITS4-Tul (Bidartondo & Duckett, 2010). All positive PCR products were purified with ExoProStart (GE Healthcare, Buckinghamshire, UK) and sequenced bidirectionally with an ABI3730 Genetic Analyser using the BigDye 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and absolute ethanol/EDTA precipitation. All DNA sequences were checked and visually aligned with Geneious version 7.4.1 (http://www.geneious.com, Kearse et al., 2012) and compared to GenBank using BLAST (http://blast.ncbi.nlm.nih.gov). All unique DNA sequences have been submitted to GenBank (accession numbers: KY271858–KY271875).

2.4 Calculations and statistics

To enable comparisons of C, N, H and O stable isotope abundances between the 17 orchid species sampled at six different sites we used an isotope enrichment factor approach to normalize the data. Normalized enrichment factors (ε) were calculated from measured δ values as ε = δ_S − δ_REF, where δ_S is a single δ¹³C, δ¹⁵N, δ²H or δ¹⁸O value of an orchid individual or an autotrophic reference plant and δ_REF is the mean value of all autotrophic reference plants by plot (Preiss & Gebauer, 2008). The δ¹³C, δ¹⁵N, δ²H and δ¹⁸O values, enrichment factors ε¹³C, ε¹⁵N, ε²H and ε¹⁸O and nitrogen concentrations of 17 orchid species and autotrophic references are available in Tables S1 and S2.

We tested for pairwise differences in the isotopic enrichment factors ε¹³C, ε¹⁵N, ε²H and ε¹⁸O and nitrogen concentration between the sampled orchid species and all autotrophic reference plants using a nonparametric Mann–Whitney U test after significant nonparametric Kruskal–Wallis H test. To test for differences between orchids and autotrophic references in nitrogen concentrations, the orchids were grouped according to type of fungal partner and degree of mycoheterotrophy (FMH ECM: fully mycoheterotrophic orchids associated with ECM fungi; PMH ECM: partially mycoheterotrophic orchids associated with ECM fungi; rhizoctonia: orchids associated with rhizoctonia fungi; autotrophic references). The p-values were adjusted using the correction after Benjamini and Hochberg (1995). For statistical analyses we used r version 3.1.2 (R Development Core Team, 2014) with a significance level of α = 0.05.

3 RESULTS

3.1 Stable isotope abundances

Mean enrichment in ¹³C of all orchid species in this study varied between −2.9‰ ± 0.9‰ (H. monorchis) and 5.7‰ ± 0.8‰ (N. nidus-avis; Figure 1a, Table S1). Post hoc pairwise Mann–Whitney U tests after a significant Kruskal–Wallis test (χ² = 114.672, df = 17, p < .001) showed that only N. nidus-avis (p_adj = .014), C. rubra (p_adj = .029), E. helleborine (p_adj = .029) and N. cordata (p_adj = .016) were significantly enriched relative to autotrophic references in ¹³C. All other species were either significantly depleted relative to autotrophic references or not distinguishable from them (Table 1).

Details are in the caption following the image — **Figure 1**
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Mean enrichment factors (a) ε²H and ε¹³C ± 1 SD and (b) ε¹⁵N and ε¹³C ± 1 SD of 17 orchid species sampled in the Northern Limestone Alps in Austria and Germany and the Fichtelgebirge in NE Bavaria/Germany in July of the four consecutive years 2012–2015. One fully mycoheterotrophic orchid species associated with ectomycorrhizal fungi (FMH ECM: blue squares; *Neottia nidus-avis*), two partially mycoheterotrophic orchid species associated with ectomycorrhizal fungi (PMH ECM: red squares; *Cephalanthera rubra* and *Epipactis helleborine*) and 14 orchid species associated with rhizoctonia fungi (rhizoctonia: yellow circles; *Dama* = *Dactylorhiza majalis*, *Mamo* = *Malaxis monophyllos*, *Psal* = *Pseudorchis albida*, *Trgl* = *Traunsteinera globosa*; n = 5 for each of the 17 orchid species; for a full species list see Table S1). The green box represents mean enrichment factors ± 1 SD for the autotrophic reference plants that were sampled together with the orchid species (n = 253, see Table S2) whereas mean ε values of reference plants are zero by definition

Table 1. Results from post hoc pairwise comparisons for the enrichment factors ε¹³C, ε¹⁵N and ε²H between 17 orchid species and autotrophic references using the Mann–Whitney U test after Kruskal–Wallis tests. The p-values were adjusted using the correction after Benjamini and Hochberg (1995). Significant differences between orchids and their autotrophic references are marked in bold. n = 5 for each of the 17 orchid species and n = 253 for autotrophic reference plants

Species	ε¹³C		ε¹⁵N		ε²H
Species	U	p _adjust	U	p _adjust	U	p _adjust
Neottia nidus-avis	1,265	.014	1,265	.006	1,265	.007
Cephalanthera rubra	1,133.5	.029	1,261	.006	1,138	.032
Epipactis helleborine	1,055	.029	1,260	.006	1,260	.007
Liparis loeselii	37	.016 a	1,124	.026	1,265	.007
Malaxis monophyllos	330.5	.111	1,094	.026	381	.214
Neottia cordata	1,208.5	.016	1,181.5	.015	1,213	.011
Spiranthes aestivalis	166	.029 a	1,229	.007	1,249	.007
Dactylorhiza incarnata	193	.029 a	1,069	.026	1,166	.024
Dactylorhiza majalis	303.5	.081	1,095	.026	912	.159
Dactylorhiza viridis	60	.016 a	1,061.5	.026	1,108.5	.032
Gymnadenia conopsea	110.5	.029 a	1,247	.006	1,062	.032
Gymnadenia nigra	775	.486	993.5	.056	1,180	.020
Herminium monorchis	12.5	.014 a	1,035	.032	1,230	.009
Neotinea ustulata	187.5	.029 a	1,214	.008	1,086	.032
Platanthera bifolia	584	.820	1,250	.006	1,024	.046
Pseudorchis albida	167	.029 a	730	.641	950	.111
Traunsteinera globosa	374.5	.182	1,231	.007	571.5	.781
	χ² = 114.674, df = 17, p < .001		χ² = 140.992, df = 17, p < .001		χ² = 125.817, df = 17, p < .001

^a Significantly depleted relative to autotrophic references.

All species except for G. nigra (p_adj = .056) and P. albida (p_adj = .641) were significantly enriched in ¹⁵N relative to autotrophic references as shown by post hoc pairwise Mann–Whitney U tests after a significant Kruskal–Wallis test (χ² = 140.992, df = 17, p < .001, Table 1). Mean enrichment of the orchid species ranged between 0.3‰ ± 1.4‰ (P. albida) and 16.5‰ ± 1.6‰ (N. nidus-avis) in ¹⁵N (Figure 1a, Table S1).

Mean enrichment in ²H varied between −5.6‰ ± 7.1‰ (M. monophyllos) and 45.5‰ ± 7.0‰ (N. nidus-avis; Figure 1b, Table S1). The only species that were not enriched in ²H relative to autotrophic references, according to post hoc pairwise Mann–Whitney U tests after a significant Kruskal–Wallis test (χ² = 125.817, df = 17, p < .001), were M. monophyllos (p_adj = .214), D. majalis (p_adj = .159), P. albida (p_adj = .111) and T. globosa (p_adj = .781). All other orchid species were significantly enriched relative to autotrophic references (Table 1).

Although the Kruskal–Wallis test to assess differences between orchid species and autotrophic references in ε¹⁸O was significant (χ² = 62.123, df = 17, p < .001), no significant differences between orchid species and autotrophic references could be detected after adjustment of p-values (Table S3). The ε¹⁸O values varied between −2.4‰ ± 0.5‰ (G. conopsea) and 2.3‰ ± 2.6‰ (E. helleborine) and thus ranged around the mean of autotrophic references (−0.2‰ ± 1.7‰; Figure S1, Tables S1 and S2).

Here, we provide the first stable isotope abundance data for M. monophyllos, N. cordata, S. aestivalis, D. incarnata, D. viridis, G. nigra and H. monorchis and additionally the first H isotope data for D. majalis, G. conopsea, N. ustulata and P. albida (Figure 1, Table 1). Similar to the forest orchid species N. nidus-avis, C. rubra and E. helleborine, N. cordata is significantly enriched in ¹³C, ¹⁵N and ²H.

3.2 Nitrogen concentrations

Nitrogen concentrations between the three groups of orchids (FMH ECM: fully mycoheterotrophic orchid species associated with ectomycorrhizal fungi, PMH ECM: partially mycoheterotrophic orchid species associated with ectomycorrhizal fungi, rhizoctonia: orchid species associated with rhizoctonia fungi) and autotrophic references were significantly different (χ² = 62.686, df = 3, p < .001; Figure 2). Pairwise comparisons showed that total nitrogen concentrations were highest in PMH ECM (2.64 ± 0.22 mmol/g_dw) and FMH ECM (2.48 ± 0.35 mmol/g_dw) and were not significantly different (p_adj = .582). Nitrogen concentrations in the leaves of orchids associated with rhizoctonia fungi (1.76 ± 0.35 mmol/g_dw) were significantly lower (p_adj < .001) than in the leaves of orchids associated with ECM fungi. Nitrogen concentrations were lowest in the leaves of autotrophic references (1.48 ± 0.48 mmol/g_dw; p_adj < .001; Figure 2, Tables S1 and S2).

3.3 Molecular identification of mycorrhizal fungi

Cortex cells in the orchid roots of all 11 investigated orchid species, except M. monophyllos, contained pelotons apparent as dense coils of fungal hyphae. The colonization level ranged between 10% and 100% of cortex cells filled with pelotons. Colonization was conspiciously poor in L. loeselii where pelotons were only visible in 10% of the cortex cells in the roots of two of the five sampled individuals. Yet, for the majority of species of open meadow habitats we investigated, and N. cordata sampled in a coniferous forest, associations with rhizoctonia fungi were found (Table 2). Associations with basidiomycetes matching DNA sequences of the Ceratobasidiaceae genera Thantephorus and Ceratobasidium and the Tulasnellaceae Tulasnella were most frequent (Table 2). The orchid mycorrhizal basidiomycete Sebacina (Sebacinaceae) was only detected in one individual of D. majalis. Potentially ectomycorrhizal fungi of the ascomycete order Helotiales and the genus Peziza (Ascomycota) were only found in a root of one individual of the orchid species L. loeselii and H. monorchis. An Ascomycota species of unknown mycorrhizal type was detected in two individuals of D. majalis. The dark septate endophyte Phialocephala was found in the roots of two individuals of D. majalis, whereas the dark septate endophyte Leptodontidium orchidicola (Ascomycota) was more frequent and found in the roots of L. loeselii, M. monophyllos and G. nigra (Table 2).

Table 2. Orchid mycorrhizal fungi identified from roots of 11 orchid species from six sites in Austria and Germany (ECM = fungi forming ectomycorrhizas; printed in bold). n: number of orchid individuals in which a fungus was detected. Pelotons in the cortex cells were visible in all species except for Malaxis monophyllos

Species	Mycorrhizal fungi (n)	Type of mycorrhizal fungi	Best match sequence/accession number (GenBank)	Identity (%)	Max score
Liparis loeselii	Leptodontidium orchidicola (1)	Dark septate endophyte	KF646097.1	99	1,123
Liparis loeselii	Helotiales (1)	ECM	JX001621.1	96	837
M. monophyllos	L. orchidicola (2)	Dark septate endophyte	KF646097.1	98	1,096
M. monophyllos	Tulasnella sp. (3)	Rhizoctonia	JF926510.1	94	832
Neottia cordata	Tulasnella sp. (2)	Rhizoctonia	AB369933.1	98	1,175
Spiranthes aestivalis	Tulasnella calospora (1)	Rhizoctonia	GU166403.1	98	1,120
Spiranthes aestivalis	Thanatephorus fusisporus (2)	Rhizoctonia	HQ441575.1	95	1,051
Dactylorhiza incarnata	Ceratobasidium sp. (2)	Rhizoctonia	EU218894.1	99	1,230
Dactylorhiza incarnata	Tulasnella sp. (2)	Rhizoctonia	AB369931.1	95	1,033
Dactylorhiza majalis	Phialocephala sp. (2)	Dark septate endophyte	KF156325.1	97	1,402
	Ascomycota sp. (2)	NA	GU566289.1	99	1,146
	Tulasnella sp. (4)	Rhizoctonia	AB369933.1	95	1,031
	Tulasnella sp. (1)	Rhizoctonia	JN655633.1	99	1,020
	Sebacina sp. (1)	Rhizoctonia	AB831798.1	98	1,002
Dactylorhiza viridis	Ceratobasidium sp. (5)	Rhizoctonia	EU218894.1	99	1,230
Gymnadenia nigra	Ceratobasidium sp. (1)	Rhizoctonia	EU218894.1	99	1,201
	L. orchidicola (1)	Dark septate endophyte	AF486133.1	99	1,158
	Tulasnella sp. (3)	Rhizoctonia	AB369931.1	94	968
Herminium monorchis	Peziza sp. (1)	ECM	AF491609.1	99	1,059
Herminium monorchis	Ceratobasidiaceae (2)	Rhizoctonia	KC243940.1	97	1,007
Neotinea ustulata	Tulasnella sp. (1)	Rhizoctonia	KF537641.1	98	1,116
	Thanatephorus (5)	Rhizoctonia	AB712278.1	97	1,022
	Ceratobasidiaceae (1)	Rhizoctonia	HM141034.1	97	1,022
Traunsteinera globosa	Ceratobasidium sp. (4)	Rhizoctonia	EU218894.1	99	1,157

4 DISCUSSION

All rhizoctonia-associated meadow orchid species lack ¹³C enrichment or even display a significant depletion in ¹³C relative to autotrophic references (Table 2). However, most of these species are significantly enriched in both ¹⁵N and ²H relative to autotrophic species and are thus PMH (Table 1). Average nitrogen concentrations of rhizoctonia-associated orchid species are significantly higher than those of autotrophic references which suggests transfer of fungal material in colonized orchid cells. Pseudorchis albida is the sole species in this study that shows neither enrichment in any of ¹³C, ¹⁵N or ²H nor has a distinctive nitrogen concentration that would differentiate this meadow orchid from autotrophic species (Figure 2). Consequently, P. albida remains the only species among the investigated orchids that should be categorized as apparently autotrophic (Table 1). Dactylorhiza majalis also lacks ²H enrichment but shows a significant enrichment in ¹⁵N relative to autotrophic references, though its leaf total nitrogen concentration is not higher than that of autotrophic references (Tables S1 and S2). If at all, D. majalis is only marginally PMH. A lacking ²H enrichment, but a pronounced ¹⁵N enrichment additionally to significant higher total leaf nitrogen concentrations, characterizes both M. monophyllos and T. globosa; thus, these two meadow species are slightly PMH. Gymnadenia nigra is significantly enriched in ²H, indicating a mycoheterotrophic nutrient gain, but it lacks ¹⁵N enrichment and has only a marginally higher nitrogen concentration.

Partial mycoheterotrophy is much clearer for the remaining meadow orchid species, L. loeselii, S. aestivalis, D. incarnata, D. viridis, G. conopsea, H. monorchis, N. ustulata and P. bifolia, sharing the trophic strategy of PMH as expressed by both significantly higher ¹⁵N and ²H enrichment relative to autotrophic references and on average significantly higher nitrogen concentrations (1.76 ± 0.35 vs. 1.48 ± 0.48 mmol/g_dw). It is worth noting that the measured nitrogen concentrations lie in the same range as previously reported by Liebel et al. (2010) as 1.67 ± 0.44 mmol/g_dw for non-neottioid orchids and 1.40 ± 0.53 mmol/g_dw for autotrophic reference species. Liparis loeselii shows a similar depletion in ¹³C as L. hawaiensis sampled on the tropical island of Oahu by Hynson (2016). However, L. loeselii had a higher ¹⁵N enrichment and lower nitrogen concentration than both L. hawaiensis and L. nervosa, the latter sampled in a warm-temperate evergreen broad-leaved forest in Japan by Motomura, Selosse, Martos, Kagawa, and Yukawa (2010). Spiranthes aestivalis shows ¹⁵N enrichment and ¹³C depletion similar to the closely related S. spiralis on an open meadow habitat in Italy evaluated by Liebel et al. (2010). The ¹⁵N enrichment and ¹³C depletion in D. incarnata and D. viridis are similar to the enrichment factors measured in D. majalis in a similar habitat, but contrasting D. incarnata and D. viridis are significantly enriched in ²H. Both Gymnadenia species in this study share a similar ²H enrichment, but only G. conopsea exhibits significant ¹⁵N enrichment typical of PMH, and closely related G. nigra appears only slightly PMH. However, significant enrichment in ¹⁵N and ²H relative to autotrophic references as observed in H. monorchis and N. ustulata seem to be common for most rhizoctonia-associated meadow orchids in the Orchidoideae. Summarizing, P. albida, D. majalis and P. bifolia exhibit similar ¹⁵N and ¹³C enrichments as reported in previous studies (Bidartondo et al., 2004; Gebauer & Meyer, 2003; Johansson et al., 2014; Stöckel et al., 2014; Tedersoo, Pellet, Kõljalg, & Selosse, 2007). Additionally, ²H enrichment in the rhizoctonia-associated orchids C. calceolus, N. ovata, O. insectifera and P. bifolia determined in an earlier study by Gebauer et al. (2016) ranged between 20.4 and 41.3‰ and was thus much higher than measured here (−5.6‰ to 27.9‰).

For most rhizoctonia-associated meadow orchids we observed depletion in ¹³C, enriched ¹⁵N and ²H values, and no ¹⁸O enrichment (Figure 1 and Figure S1; Table S3). We conclude that the majority of rhizoctonia-associated meadow orchid species is PMH because ²H enrichment can be used as indicator for the gain of C in the form of heterotrophic organic matter and thus as substitute for ¹³C enrichment in C sources that are insufficiently distinguished in their C isotope abundances. A higher transpiration by orchids compared to autotrophic plants as driver of ²H enrichment can be ruled out here, as the orchids should then be depleted in ²H simultaneously to both depletions in ¹³C and ¹⁸O (Cernusak, Pate, & Farquhar, 2004; Gebauer et al., 2016; Ziegler, 1996), a pattern not observed here.

In this study, we furthermore confirm full mycoheterotrophy for the achlorophyllous forest orchid N. nidus-avis that forms mycorrhizas with Sebacina A (McKendrick et al., 2002; Selosse et al., 2002), according to its high enrichment in ¹³C and ¹⁵N, as reported in previous studies (Bidartondo et al., 2004; Gebauer & Meyer, 2003; Preiss et al., 2010; Stöckel et al., 2014), and also based on a high enrichment in ²H (Gebauer et al., 2016; Figure 1). However, it is unclear why we find a less pronounced ²H enrichment (45.5‰ ± 7.0‰) compared to the results of Gebauer et al. (2016) (69.1‰ ± 5.5‰). Still, it is evident that achlorophyllous N. nidus-avis gains its entire N, C and (C-H bound) H from a fungal source and ¹³C, ¹⁵N and ²H enrichments are highest compared to all other green-leaved species under study. We also reaffirm PMH for C. rubra and E. helleborine (Abadie et al., 2006; Bidartondo et al., 2004; Johansson et al., 2014; Preiss et al., 2010; Schiebold, Bidartondo, Karasch et al., 2017), two forest species that associate with a wide spectrum of ectomycorrhizal and rhizoctonia fungi (Bidartondo et al., 2004; Jacquemyn et al., 2016; Ogura-Tsujita & Yukawa, 2008; Tĕšitelová et al., 2012), due to these species’ significant enrichment in ¹³C and ¹⁵N relative to autotrophic references but also due to their intermediate ²H enrichment (Figure 1). The ²H enrichment in C. rubra was higher in a previous study by Gebauer et al. (2016) (29.1‰ ± 4.6‰) than in this study (10.2‰ ± 3.2‰) but this might be due to the different light climate at the sampling sites. Here, we sampled C. rubra individuals in a more open mixed deciduous and coniferous forest while the previous study was in a closed-canopy beech forest. In addition, the lower ¹³C enrichment in our study (1.2‰ ± 0.3‰ vs. 2.9‰ ± 1.8‰) supports this reasoning because ectomycorrhiza-associated PMH orchids are known to increase exploitation of fungal carbon as indicated by increasing ¹³C enrichment with decreasing irradiance (Preiss et al., 2010). The ²H enrichment in E. helleborine was in a similar range as previously measured in closely related E. atrorubens (22.1‰ ± 4.8‰ vs. 18.8‰ ± 6.0‰). Nitrogen concentrations in all three orchid species predominantly or exclusively associated with ECM fungi (2.64 ± 0.22 mmol/g_dw for C. rubra and E. helleborine, and 2.48 ± 0.35 mmol/g_dw for N. nidus-avis) confirm the overall picture that mean total nitrogen concentrations in Orchidaceae partnering with ECM fungi are generally twice as high as in autotrophic non-legume plants regardless of their degree of mycoheterotrophy (Hynson et al., 2016). It is noteworthy that the reason for similar nitrogen concentrations in PMH and FMH orchids associated with ECM fungi is likely to due to sampling. While leaf material was used to determine nitrogen concentrations of autotrophic references and of orchid species associated with ECM and rhizoctonia fungi, shoot material was used to analyse nitrogen concentrations of FMH species associated with ECM fungi (N. nidus-avis). Shoot material is known to generally have lower nitrogen concentrations than leaf material (Gebauer, Rehder, & Wollenweber, 1988).

Meadow orchid species associated with rhizoctonia fungi appear predisposed to a varying degree of PMH nutrition due to their obligate gain of organic matter via peloton digestion and/or by transfer of compounds across membranes in their early pre-photosynthetic stages. For the most efficient carbon gain, switching fungal partners from rhizoctonia to ECM fungi (Bidartondo et al., 2004) or saprotrophic non-rhizoctonia fungi (Lee et al., 2015; Ogura-Tsujita et al., 2009) seems to be necessary, and also to sustain full mycoheterotrophy. As far as we know, no FMH orchid species specializes on rhizoctonia. We propose that PMH in rhizoctonia-associated chlorophyllous orchid species is a useful trait to improve competitive success in open habitats and helps survive unfavourable conditions during periods of dormancy. Nevertheless, PMH sensu Merckx (2013) in rhizoctonia-associated orchids does not exclude the occurrence of bi-directional carbon fluxes as shown for the orchid Goodyera repens (Cameron, Johnson, Read, & Leake, 2008).

5 CONCLUSIONS

Our study demonstrates that PMH is a trophic continuum between the extreme endpoints of autotrophy and full mycoheterotrophy. We infer PMH for the meadow orchid species L. loeselii, S. aestivalis, D. incarnata, D. viridis, G. conopsea, H. monorchis, N. ustulata and P. bifolia, as expressed by both significantly higher ²H and ¹⁵N enrichment relative to autotrophic references and on average significantly higher nitrogen concentrations, even though these traits are less pronounced than in the PMH forest species C. rubra and E. helleborine. Of the meadow orchid species associating with rhizoctonia fungi, P. albida is the sole apparently autotrophic orchid species in this study as it shows neither enrichment in ¹³C, ¹⁵N or ²H, nor a distinctive nitrogen concentration. Dactylorhiza majalis, M. monophyllos and T. globosa are only marginally PMH as they all lack ²H enrichment but are characterized by pronounced ¹⁵N enrichment and significantly higher leaf total nitrogen concentrations. Gymnadenia nigra is on a similar position of this continuum as it is distinctive from autotrophic plants in its ²H enrichment.

Our findings support the hypothesis that orchids in the subfamilies Epidendroideae and Orchidoideae forming orchid mycorrhizas with rhizoctonia fungi and growing in habitats with high irradiance, such as montane meadows, feature PMH as nutritional mode. Significant ¹⁵N enrichment and higher total nitrogen concentrations in cryptic mycoheterotrophs can be used as indicators for PMH in rhizoctonia-associated orchid species as recently suggested by Hynson (2016). We conclude that PMH in rhizoctonia-associated orchid species, as elucidated here by pronounced ²H enrichment, plays a far greater role in orchids of open habitats than expected. The implications of our findings for the ecology, evolution and conservation of orchids, mycorrhizal fungi and co-occurring plants, and the function of their ecosystems, now deserve investigation.

ACKNOWLEDGEMENTS

This work was supported by the German Research Foundation DFG (GE565/7-2). The authors thank Christine Tiroch and Petra Eckert (BayCEER – Laboratory of Isotope Biogeochemistry) for skilfull technical assistance with stable isotope abundance measurements. We thank Pedro Gerstberger, Florian Fraaß and Andreas Beiser for information about the locations of the sampled orchid populations and Heiko Liebel for sampling M. monophyllos. Some orchid species were sampled during students’ field courses in 2012 and 2015 and with support of student field assistants. We also thank the Regierung von Oberfranken, the Regierung von Oberbayern (Bavaria, Germany) and the Bezirkshauptmannschaft Bludenz (Vorarlberg, Austria) for authorization to collect orchid samples.

AUTHORS’ CONTRIBUTIONS

J.S. and G.G. had the idea for this investigation; J.S. collected the plant samples and conducted the molecular analysis of mycorrhizal fungi; J.S., A.M. and F.L. prepared the samples for stable isotope analysis and performed the data analysis; M.I.B. supervised the molecular analysis of mycorrhizal fungi; G.G. supervised the sample isotope abundance analysis; J.S. drafted the manuscript. All co-authors contributed critically to the drafts of the manuscript and gave final approval for publication.

DATA ACCESSIBILITY

Data available from the Dryad Digital Repository https://doi.org/10.5061/dryad.3nf8b (Schiebold, Bidartondo, Lenhard et al., 2017) and GenBank (accession numbers: KY271858–KY271875).

Supporting Information

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Citing Literature

Volume106, Issue1

January 2018

Pages 168-178

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Exploiting mycorrhizas in broad daylight: Partial mycoheterotrophy is a common nutritional strategy in meadow orchids

Abstract

1 INTRODUCTION