Introduction

Mycorrhizas represent a form of diffuse symbiosis, wherein a single plant simultaneously associates with multiple fungi and each fungus simultaneously associates with multiple plants (Smith and Read 2008). Mycorrhizas typically exhibit mutualistic relationships in which there is an exchange of plant-generated photosynthates and soil-derived mineral nutrients acquired by fungi (Smith and Read 2008). Most terrestrial plants, from liverworts to angiosperms, form such mutualistic arbuscular mycorrhizal (AM) symbioses with fungi belonging to Glomeromycotina.

However, many achlorophyllous (i.e., fully mycoheterotrophic) plants, including species belonging to families Burmanniaceae, Corsiaceae, Gentianaceae, Polygalaceae, Petrosaviaceae, Iridaceae, Thismiaceae, and Triuridaceae, associate with AM fungi, indicating that the transfer of C from AM fungi to plants should certainly occur (Leake 1994; Yamato 2001; Yamato et al. 2011; Bidartondo et al. 2002; Merckx and Bidartondo 2008; Merckx et al. 2010; Courty et al. 2011; Suetsugu et al. 2014; Gomes et al. 2017). Given that AM fungi are obligately biotrophic, these mycoheterotrophic plants indirectly obtain C from surrounding green plants through shared fungi (Bidartondo et al. 2002; Yamato et al. 2016; Gomes et al. 2017).

It is generally assumed that the transition from autotrophy to full mycoheterotrophy have occurred via an intermediate stage that is referred to as partial mycoheterotrophy or mixotrophy (Gebauer and Meyer 2003; Selosse and Roy 2009; Hynson et al. 2013; Lallemand et al. 2016). Indeed, partial mycoheterotrophy has been detected in many orchids and pyroloids that associate with ectomycorrhizal fungi. The abundance of natural stable isotopes has provided a useful means for assessing the degree of mycoheterotrophy in these taxa (Gebauer and Meyer 2003; Tedersoo et al. 2007; Hynson et al. 2013; Selosse et al. 2017b; Suetsugu et al. 2017, 2018, 2019, 2020), as the 13C and 15N abundances in ectomycorrhizal fungi are significantly higher than those in the surrounding plants (Gebauer and Meyer 2003; Hynson et al. 2013). For example, some green orchids that are closely related to mycoheterotrophic species show 13C and 15N abundances that are intermediate to that of autotrophic plants and full mycoheterotrophs, suggesting partial mycoheterotrophy (Selosse and Roy 2009; Hynson et al. 2013). Besides, deuterium (2H) abundance has been employed as an additional tool to identify partially mycoheterotrophic plants specifically in cases where 13C abundance could not be sufficiently used to do so (Gebauer et al. 2016; Schiebold et al. 2018; Schweiger et al. 2018, 2019). These studies have found that partially mycoheterotrophic species are widespread among forests and meadow orchids with the saprotrophic fungi, the rhizoctonia (Gebauer et al. 2016; Schiebold et al. 2018; Schweiger et al. 2019). Thus, while all the orchids and pyroloids produce minute seeds and rely on fungi for germination, adults of some species can still obtain C from these fungal partners in addition to that from photosynthesis.

In contrast, evidence of partial mycoheterotrophy is largely lacking for AM plants (Giesemann et al. 2020), even though fully mycoheterotrophic plants from diverse lineages exploit AM fungi. Several studies utilizing 14C labeling showed that neighboring plants facilitate interplant C transfer by shared AM fungi, but the transferred C often remains in the roots of the recipient plants, likely within the fungal mycelium (Lerat et al. 2002; Pfeffer et al. 2004; Nakano-Hylander and Olsson 2007; Lekberg et al. 2010). In addition, natural stable isotopic approaches often provide inconclusive evidence for partial mycoheterotrophy in AM plants (Cameron and Bolin 2010; Merckx et al. 2010; Hynson et al. 2013; Bolin et al. 2017; Selosse et al. 2017b). It is known that AM fungal spores have 13C and 15N isotopic signatures similar to those of their host plants (Nakano et al. 1999; Merckx et al. 2010; Courty et al. 2011). Consequently, fully mycoheterotrophic plants can have isotopic signatures similar to those of the surrounding autotrophic plants (Merckx et al. 2010), while 13C, 15N, and 2H isotopes in many forest understory plants have been found to be enriched (Courty et al. 2011; Gomes et al. 2020). Courty et al. (2011) demonstrated that AM-forming fully mycoheterotrophic plants display 13C enrichment compared with that of surrounding understory autotrophic plants in tropical rainforests, the isotopic signature of both the AM fungi and the mycoheterotrophs was similar to that of canopy trees. The 13C enrichment in AM-forming mycoheterotrophs, thus, appears to reflect the δ13C values of canopy trees (plausible C sources of AM-forming mycoheterotrophs) that have higher δ13C values than understory plants due to differential stomatal opening, humidity, and photosynthetic rates (Gebauer and Schulze 1991; Courty et al. 2011; Selosse et al. 2017b). Therefore, the 13C signatures of AM-forming partial mycoheterotrophs should be much more similar to those of the surrounding autotrophic plants in open environments, such as fields or open-canopy forests (Hynson et al. 2013). Consistent with this assumption, most studies that have evaluated whether green plants that are phylogenetically close to mycoheterotrophic plants exploiting AM fungi have found negligible or non-existent isotopic differences between the candidate plants and surrounding autotrophs (Cameron and Bolin 2010; Merckx et al. 2010; Field et al. 2015; Bolin et al. 2017). The recent findings that some green understory plants that develop Paris-type AM show a significant enrichment in stable isotopes, compared with other autotrophic understory plants (Cameron and Bolin 2010; Giesemann et al. 2020), is possibly due to the same mechanism as those of AM-forming full mycoheterotrophs.

Significant 13C enrichment in mycoheterotrophic plants may be detected even in open habitats if both C3 and C4 plants associate with AM fungi, as C4 plants have significantly higher 13C abundances than C3 plants. This is owing to differences in isotopic fractionations related to primary CO2 fixation, a process that is catalyzed by Rubisco in C3 plants and PEPCase in C4 plants (O’Leary 1988; Těšitel et al. 2010, 2011). Considering that the 13C abundances in fungal spores mirror those in the surrounding plants that act as C donors (Nakano et al. 1999; Courty et al. 2011, 2015), assessment of 13C abundance can still provide an effective approach for detecting partial mycoheterotrophy in AM plants (Bolin et al. 2017). Indeed, Bolin et al. (2017) showed that the bluethread plants (Burmannia coelestis) were significantly enriched in 13C compared with that in the surrounding C3 reference plants in the population that also contains C4 plants. Therefore, even though B. coelestis can be cultivated without AM fungi or surrounding AM-forming plants (Merckx et al. 2010), this species could at least be facultatively partially mycoheterotrophic.

Here, we focused on the putatively partially mycoheterotrophic plant Pterygocalyx volubilis (Gentianaceae; Fig. 1) in fields that also contain C4 plants (Miscanthus sinensis and Microstegium vimineum). This gentian species has reduced stubby roots lacking root hairs that are the hallmarks of mycoheterotrophic plants (Leake 1994; Imhof et al. 2013). In addition, P. volubilis produces numerous minute dust seeds that are likely dependent on associations with AM fungi for their supply of C during germination (Murata 2003; Eriksson and Kainulainen 2011). Consistent with the previous observations (Fujiyoshi et al. 2005), our preliminary morphological investigations revealed that mycorrhizal colonization of P. volubilis is characterized by intracellular cell-to-cell spread and is categorized as a Paris-type AM that is a common morphological type of mycorrhizal associations in AM-forming mycoheterotrophs (Imhof et al. 2013). Therefore, we assume the likelihood of C transfer from C4 plants through shared AM networks. Based on these characteristics, partial dependence on fungal-derived C has been suspected (Murata 2003).

Fig. 1
figure 1

Pterygocalyx volubilisa flowering plant entwining a stem of Miscanthus sinensis.b Close-up view of a flower. c Reduced roots. d Dust seeds. Bar = 1 mm

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In this study, we evaluated the physiological ecology of P. volubilis. Specifically, we investigated whether the 13C signature of this AM-associated grassland gentian species differs sufficiently from that of the surrounding C3 and C4 plants, to determine whether P. volubilis is characterized by partial mycoheterotrophy. AM fungi are difficult to sample since they do not produce conspicuous organs such as fruitbodies, and sampling of extraradical mycelia in the field is technically unrealistic (Courty et al. 2011, 2015). This in turn hampers isotope analysis of AM fungi. We thus isolated AM fungal spores from soil surrounding the roots of P. volubilis individuals and determined the 13C and 15N abundances in these spores, by mass spectrometry optimized for microscale analysis (Ogawa et al. 2010). The 13C and 15N abundances in these spores obtained by microscale isotope analysis helped us to confirm the hypothesis that P. volubilis actually uses the carbon originating from AM fungi as a part of their energy sources.

Materials and methods

Study site and sampling

The field study was conducted in a warm and temperate grassland in Ikawa, Shizuoka Prefecture, Japan. The population contained ca. 30 flowering individuals of P. volubilis (Fig. 1). The samples for isotopic analysis were collected on 20 May 2017. The investigated population was dominated by Miscanthus sinensis. The C4 grasses including Miscanthus sinensis accounted for ca. 30% of total vegetation cover.

Six sampling plots of 1 m2 were selected in the investigated population. In each plot, we collected the leaves of a P. volubilis individual and an individual of each co-occurring C3 and C4 autotrophic reference species. To limit the influence of environmental factors on stable isotope ratios, such as atmospheric CO2 isotope composition, microscale light climate, and soil type (Gebauer and Schulze 1991), the leaves of reference plants were collected at the same height of the focal P. volubilis individual. This criterion led us to sample Miscanthus sinensis (C4 plant), Microstegium vimineum (C4 plant), Deutzia crenata (C3 plant) and Geranium thunbergii (C3 plant), since no other plants were present in more than two plots. In addition, we collected the soil around P. volubilis roots at each plot to obtain AM fungal spores.

Isotopic analysis of plants

The collected leaves were dried at 60 °C for 4 days and then cut into fine pieces using small scissors and a surgical knife. The abundances of stable C and N isotopes and total N concentrations of samples were measured at the Research Institute for Humanity and Nature (Kyoto, Japan) using a Delta XP mass spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) that are connected to a Flash EA 2000 elemental analyser (Thermo Fisher Scientific). The relative abundances of the stable isotopes were calculated as δ15N or δ13C = (Rsample/Rstandard − 1) × 1000 (‰), where Rsample represents the 13C/12C or 15N/14N ratio of the sample, respectively, and Rstandard represents the 13C/12C ratio of Vienna Pee Dee Belemnite (VPDB) or the 15N/14N ratio of atmospheric nitrogen. Both the isotope ratios of C and N were calibrated using the laboratory standards: glycine (δ13C = − 34.92‰) and l-threonine (δ13C = − 9.45‰) for C, and dl-alanine (δ15N = − 2.89‰) and l-alanine (δ15N = 22.71‰) for N, which are traceable back to international standards (Tayasu et al. 2011). The analytical standard deviations (SD) of these standards were 0.05‰ (δ13C, n = 8) for glycine, 0.05‰ (δ13C, n = 3) for l-threonine, 0.04‰ (δ15N, n = 6) for dl-alanine and 0.14‰ (δ15N, n = 17) for l-alanine. The total N concentrations in the leaf samples were calculated using sample weights and the volume of CO2 and N2 gas of the laboratory standards (Tayasu et al. 2011).

Isolation of AM fungal spores

Spores of AM fungi were isolated from soil using a method similar to those described in Brundrett et al. (1996) and Courty et al. (2011). The soil of all the plots were pooled, mixed, and sifted (with a 4-mm sieve) before analysis. Each of four subsamples of 10 g soil was soaked and mixed in a substantial volume of tap water, and passed through 500-, 125-, and 53-μm sieves. The contents of the 125- and 53-μm sieves were resuspended in distilled water and centrifuged at 2000 rpm for 5 min. The resulting pellet was again resuspended with 15 mL of distilled water and layered onto 15-mL LUDOX HS-40 (colloidal silica with a density of 1.3 g/mL, Sigma-Aldrich) solution. After centrifugation at 2000 rpm for 5 min, opaque band containing spores was collected with a pipette, passed through a 53-mm sieve, and washed with distilled water. Spores were collected from the material on the sieve transferred to a Petri dish under a dissecting microscope. Approximately 100 spores were dried at 60 °C for 24 h.

Isotopic analysis of AM fungal spores

Our spore samples were too small in volume (< 0.1 mg) to measure using a standard EA-IRMS system. For this reason, δ13C and δ15N values of combined spores (10–12 μg dry weight, n = 5) were measured at the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), using a Flash EA 1112 coupled to a Deltaplus XP IRMS (Thermo Fisher Scientific, Bremen, Germany) instrument, which is optimized for microscale isotope analysis (Ogawa et al. 2010). The total N concentrations and isotope ratios of C and N were calibrated using the laboratory standards: l-tyrosine (δ13C = − 20.83‰, δ15N = 8.74‰), dl-alanine (δ13C = − 25.36‰, δ15N = − 2.89‰) and glycine (δ13C = − 34.92‰, δ15N = 2.18‰) (Tayasu et al. 2011). The analytical precisions of the method determined by repeated analysis of l-tyrosine standards are ± 0.07‰ (1σ, n = 9) for δ13C and ± 0.18‰ (1σ, n = 9) for δ15N.

Statistics

After confirming that the datasets were normally distributed using Bartlett’s test, differences in the δ15N values were analyzed using one-way ANOVA, followed by Fisher’s multiple comparison test. Since the δ13C values and the total N concentrations values were not normally distributed, a Kruskal–Wallis nonparametric test and a sequential Mann–Whitney U test were used.

In addition, enrichment factors (ε) were calculated from the δ values of each plant group based on the definition by Gebauer and Meyer (2003): ε = δS − δREF, where δS represents a single δ13C value of a P. volubilis individual and δREF represents the mean value of all autotrophic C3 reference plants from a specific sampling plot (Preiss and Gebauer 2008). When we calculated the enrichment factors (ε) of AM spores, we used a simplified site-wise calculation, since spores could not be investigated plot-wise, due to their minute status (Hynson et al. 2013; Preiss et al. 2010). The percentage of C derived from fungi (% Cdf) in P. volubilis was estimated using the linear two-source mixing model: % Cdf = (εPC/εSC) × 100, where εPC represents the enrichment factor of P. volubilis individuals, and εSC is the mean enrichment factor of AM spores as the endpoint. This model is based on the previous knowledge that AM-forming fully mycoheterotrophs and AM fungal spores have similar 13C isotopic signatures (Courty et al. 2011).

Results

The δ13C values of P. volubilis (− 30.8 ± 1.3‰; mean ± SD) were significantly higher than those of autotrophic C3 reference plants (− 32.5 ± 1.5‰; P < 0.05; Fig. 2, Table S1). The δ13C values of AM fungal spores (− 25.9 ± 3.0‰) were significantly higher than those of not only C3 plants but also P. volubilis (P < 0.001 in both comparisons). Using the mean ε13C values of AM fungal spores as the fully mycoheterotrophic endpoint, the percentage of C derived from AM fungi was estimated as 23.0 ± 15.3% in P. volubilis.

Fig. 2
figure 2

Values of δ13C and δ15N (mean ± 1 SD) in the leaves of Pterygocalyx volubilis (Pv, gray triangle), neighboring C3 (open circle) and C4 (filled circle) autotrophic plants, and AM spores (gray square)

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The δ15N values of AM fungal spores (− 1.1 ± 1.0‰) were significantly higher than that of autotrophic reference plants (− 3.0 ± 1.1‰; P < 0.01), while there was no significant difference in the δ15N values between AM fungal spores and P. volubilis (− 1.7 ± 1.5‰; P = 0.41). The δ15N values of P. volubilis were also significantly higher than those of autotrophic reference plants (P < 0.05). The total N concentrations in P. volubilis (3.6 ± 0.9 mmol/g) were also significantly higher than those in reference plants (2.4 ± 0.6 mmol/g; P < 0.01). In addition, the N concentrations of AM fungal spores (5.6 ± 1.6 mmol/g) were significantly higher than those of not only autotrophic plants but also P. volubilis (P < 0.01 in both comparisons).

Discussion

Partial mycoheterotrophy in Pterygocalyx volubilis

We found that the δ13C values of both AM fungal spores and P. volubilis were significantly enriched compared with those in co-occurring C3 plants. Slightly higher δ13C values have been reported for AM fungal spores than for the AM roots of C3 plants (the C donors) (mean difference 0.72‰, maximum difference 4.1‰) (Courty et al. 2015). Nonetheless, this cannot explain the difference in δ13C values between the C3 plants and AM fungal spores observed in this study (mean difference 6.6‰, maximum difference 10.2‰). This provides evidence that the large degree of enrichment that we observed in the AM spores reflects the influence of the nearby C4 plants, and that the mixed C isotope signature of the AM fungi originates from both C3 and C4 hosts. Consequently, our stable isotope results strongly suggest that C had been transferred from C4 plants to P. volubilis via epiparasitism since the δ13C values of P. volubilis were intermediate between those of AM fungal spores and co-occurring C3 plant. The δ13C values of P. volubilis provide the evidence for partial mycoheterotrophy in P. volubilis. In addition, we found that the δ15N values of both AM fungal spores and P. volubilis were significantly higher than those in the surrounding plants. The intermediate δ15N values of P. volubilis between those of autotrophic reference plants and AM fungal spores provide further evidence for partial mycoheterotrophy in P. volubilis.

Using the enrichment factors of AM spores as a fully mycoheterotrophic endpoint, we estimated that P. volubilis obtained 23 ± 15% of its total C demand from its fungal partners. Approximately 20% of organic matter derived from a fungal source falls within the lower range of the values previously reported for partially mycoheterotrophic species (Gebauer and Meyer 2003; Bidartondo et al. 2004; Julou et al. 2005; Abadie et al. 2006; Stöckel et al. 2011, 2014; Suetsugu et al. 2017; Schweiger et al. 2018). However, our estimation may not be precise due to several limitations. First, AM fungal spores may have δ13C values different from those of AM hyphae (the active transport pathways), suggesting that it is difficult to use AM spores to accurately estimate the fungal contribution to mycoheterotrophic plants. However, Walder et al. (2013) have shown similar δ13C values in AM spores and hyphal networks in microcosms containing both C3 and C4 species. In addition, since it is technically difficult to recover clean hyphae without losing any cytoplasm by breaking them (Courty et al. 2011), the isotopic abundances of spores are the best proxies for those of AM hyphae since one can obtain spores from the natural field (Courty et al. 2011; Walder et al. 2013). Second, it remains unknown whether these spores are mycobionts that link P. volubilis to the surrounding vegetation. However, apart from highly specialized interaction found in fully mycoheterotrophic plants, green plants including putatively partially mycoheterotrophic ones are associated with very diverse AM mycobionts (Merckx et al. 2010; Suetsugu et al. 2014; Yamato et al. 2014; Gomes et al. 2017). In fact, approximately 80% of all angiosperms are associated with AM fungi that contain less than 300 species (Öpik et al. 2013). Given that these spores include multiple morphological species such as Septoglomus and Rhizophagus, these spores will include at least some mycobionts in P. volubilis and the surrounding vegetation.

Despite several limitations for precise estimation in the degree of mycoheterotrophy, the assumed relatively low dependence of P. volubilis on fungal-derived C may be linked to the high levels of irradiation that typify this plant’s grassland habitats. Accordingly, we speculate that the partially mycoheterotrophic nutritional mode of P. volubilis may have evolved as a byproduct of the acquisition of minerals, such as N, rather than C, as suggested for some partially mycoheterotrophic plants (Selosse and Roy 2009; Selosse et al. 2017a). The difference in both 15N natural abundance and N concentrations between P. volubilis and autotrophic reference plants could provide some evidence to support this hypothesis (see also next section).

Evolution of partial mycoheterotrophy in open grasslands

It is of interest to note that, while most of the partially and fully mycoheterotrophic plants examined to date grow in forests with dense overstories that produce deep shade (Merckx et al. 2013a), P. volubilis, in contrast, grows in open habitats exposed to high irradiance levels. It has been suggested that an evolutionary switch from autotrophy to full mycoheterotrophy may be accompanied by a shift towards growth in more shaded habitats (Merckx et al. 2013a). However, several recent studies have also shown that some rhizoctonia-associated orchids that grow in open habitats are partially mycoheterotrophic (Girlanda et al. 2011; Schiebold et al. 2018). These studies suggest that the C demand cannot be the sole driver of this nutritional strategy. In a broader context, mixotrophy may have frequently evolved in not only mycoheterotrophic but also parasitic and carnivorous plants that grow in nutrient-poor open habitats to meet the mineral demands of these plants (Selosse and Roy 2009; Selosse et al. 2017a).

In this regard, although mixotrophy is generally considered in the context of C acquisition (Press 1989), mixotrophy sensu lato has long been recognized in hemiparasitic and carnivorous plants, which often grow in open grasslands (Selosse and Roy 2009; Selosse et al. 2017a). Hemiparasitic and carnivorous plants typically obtain their requisite non-C nutrients by parasitizing other plants or digesting animals (Selosse and Roy 2009; Selosse et al. 2017a), and their efficiency in this respect contributes to the success of these plants in nutrient-poor environments (Quested 2008). In addition, certain hemiparasitic and carnivorous plants gain a proportion of their organic C from their host plants (Klink et al. 2019; Press et al. 1987; Schulze et al. 1991). This suggests a scenario wherein C flow occurs due to meeting mineral needs (Hynson et al. 2013; Selosse and Roy 2009), with the resulting mixotrophy in turn facilitating the emergence of further C dependence.

Accordingly, partial mycoheterotrophy might be expected to evolve under light-saturated conditions to compensate for deficiencies associated with growth in mineral nutrient-poor environments. Indeed, P. volubilis is often found growing in nutrient-poor grasslands. In this respect, it is notable that both δ15N values and N concentrations in P. volubilis were significantly higher than those in the surrounding vegetation. In contrast, a previous study has demonstrated that both values of AM-forming fully mycoheterotrophs are not always higher than those of co-occurring autotrophic plants (Courty et al. 2011; Gomes et al. 2020; Merckx et al. 2010), while mycoheterotrophic plants associated with ectomycorrhizal fungi are always characterized by higher δ15N values and N concentrations than those of autotrophic plants (Gebauer and Meyer 2003; Julou et al. 2005; Stöckel et al. 2011). The discrepancy of δ15N values and N concentrations among AM-forming mycoheterotrophs including P. volubilis may suggest that AM-forming mycoheterotrophs utilize nitrogen from different sources, such as from their associated fungal partners or from the substrate by themselves, as discussed by Gomes et al. (2020).

Evolution of partial mycoheterotrophy in a lineage without fully mycoheterotrophic relatives

Similar to the majority of angiosperms, members of Gentianaceae family are primarily autotrophic plants that form AM associations (Leake 1994). Nonetheless, a small percentage (less than 2%) of Gentianaceae species, such as those in the genera Voyria and Voyriella, are fully mycoheterotrophic (Cameron and Bolin 2010). Phylogenetic analyses have shown that full mycoheterotrophy has appeared independently at four different times during the evolutionary history of Gentianaceae: once in both Voyria and Voyriella, once in Exochaenium, and at least once in Exacum. However, these genera are only distantly related to P. volubilis (Merckx et al. 2013b).

Green photosynthetic orchids and pyroloids that are closely related to mycoheterotrophic orchids tend to be partially mycoheterotrophic (Hynson et al. 2013; Selosse and Roy 2009). Given that the evolution from autotrophy to full mycoheterotrophy is a gradual process, both initial and partial mycoheterotrophy can be considered intermediate steps in this process. In this respect, the evolution of initial mycoheterotrophy is the critical step for the release from full autotrophy (Eriksson and Kainulainen 2011; Jacquemyn and Merckx 2019). Therefore, it can be speculated that partial mycoheterotrophy may have evolved in plant groups that contain examples of initial mycoheterotrophy but do not include any fully mycoheterotrophic species. Partial mycoheterotrophy in P. volubilis would be one such example, given that species of Gentianaceae with dust seeds have been suspected to be initially mycoheterotrophic (Eriksson and Kainulainen 2011; Murata 2003). Similarly, species of Obolaria (Gentianaceae), which do not have fully mycoheterotrophic relatives, have been suspected of being partial mycoheterotrophs (Cameron and Bolin 2010; Leake 1994). Indeed, Cameron and Bolin (2010) found that Obolaria virginica specimens they examined were significantly enriched in 13C compared with that of the surrounding vegetation. Considering that this species is a forest understory plant, C transfer from canopy leaves to Obolaria individuals is strongly suggested.

Furthermore, it is highly likely that many partially mycoheterotrophic species remain undiscovered, in view of the fact that AM associations have been shown to be the most common type of mycorrhizal association (Leake 1994; Merckx et al. 2013a). It is worth noting that P. volubilis also forms Paris-type AM association. Only very recently, Giesemann et al. (2020) showed that 2H abundance can be used to identify partially mycoheterotrophic AM plants specifically in cases where 13C abundance does not provide unequivocal evidence of such. They also suggested that partial mycoheterotrophy could potentially be widespread among ca. 100,000 plant species that develop Paris-type AM associations since (i) all AM-forming full mycoheterotrophic plants reported so far show Paris-type AM associations, and (ii) the seemingly autotrophic plant Paris quadrifolia without any known (even initially) mycoheterotrophic relatives appears to obtain nearly 50% carbon from its fungal partners. The daring hypothesis by Giesemann et al. (2020) warrants further investigation.

Conclusions

The present study is one of the few studies to provide convincing evidence that partial mycoheterotrophy can arise in plants forming associations with AM fungi (Giesemann et al. 2020). To our knowledge, this study is the first to report 13C and 15N abundances in both an AM-forming partially mycoheterotrophic plant and the spores of surrounding AM fungi. Nevertheless, the fact that, to date, there have been relatively few descriptions of partial mycoheterotrophy in AM plants may not necessarily be a reflection of the rarity of such associations. Instead it rather suggests the difficulty in demonstrating such relationships based on 13C and 15N abundance analyses, which have facilitated the discovery of mixotrophy in orchids and pyroloids (Selosse et al. 2017b). The transfer of C from AM fungi to putative partially mycoheterotrophic plants is difficult to detect based on 13C abundance, particularly under conditions where the surrounding vegetation primarily comprises species characterized by C3 photosynthesis.

Nonetheless, determining the δ13C can represent a powerful approach for detecting partial mycoheterotrophy in plants that form associations with AM fungi when these plants co-occur with C4 plants, as significantly more enriched δ13C values are expected in mycoheterotrophic plants under these circumstances than when C3 plants predominate in the surrounding vegetation, as previously suggested (Bolin et al. 2017). In addition, when the δ15N values are higher than those in the surrounding vegetation, the δ15N values could be an additional tool to detect partially mycoheterotrophic plants (Giesemann et al. 2020). The findings of the present study and Giesemann et al. (2020) led us to consider that partial mycoheterotrophy with AM associations (particularly Paris-type ones) could be considerably more common than hitherto believed.