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. 2025 Apr 7;246(5):1912–1921. doi: 10.1111/nph.70106

Mixotrophy in orchids: facts, questions, and perspectives

Marc‐André Selosse 1,2,3, † ,✉, Pierre‐Louis Alaux 1, † , Lara Deloche 1,4,5, Etienne Delannoy 4,5, Julita Minasiewicz 2, Spyros Tsiftsis 6, Tomas Figura 1,7, Florent Martos 1
PMCID: PMC12059541  PMID: 40195594

Summary

While orchids germinate thanks to carbon from their symbiotic fungi, variable carbon exchanges exist between adult orchids and their mycorrhizal fungi. Although some truly autotrophic orchids reward their fungi with carbon at adulthood, some species remain achlorophyllous and fully dependent on fungal carbon (mycoheterotrophy). Others are photosynthetic but also import fungal carbon: The so‐called mixotrophic (MX) orchids rely on fungi of diverse taxonomy and ecology. Here, we classify MX nutrition of orchids into three types. Type I mixotrophy associates with diverse Asco‐ and Basidiomycota that are either saprotrophic or ectomycorrhizal, entailing enrichment of the orchids in 2H, 13C, and 15N. The two other types associate with rhizoctonias, a polyphyletic assemblage of Basidiomycotas that is ancestrally mycorrhizal in orchids. Type II mixotrophy associates with rhizoctonias that secondarily evolved into saprotrophic or ectomycorrhizal ecology, and thus enrich the orchid in 2H, 13C, and 15N. Type III mixotrophy, which remains debated, associates with rhizoctonias that have retained their ancestral lifestyle, that is saprotrophic and/or endophytic in nonorchids, and only entail orchid enrichment in 2H and 15N. Based on a case study of achlorophyllous variants in Mediterranean Ophrys and on published data, we discuss the distinct nature and research perspectives of type III mixotrophy.

Keywords: isotopic enrichment, mycoheterotrophy, mycorrhiza, rhizoctonia, saprotrophy

Terrestrial orchids germinate with the help of fungi providing them with carbon, since their seeds are devoid of reserves (Rasmussen, 1995; van der Heijden et al., 2015; Dearnaley et al., 2016). This fungus‐mediated heterotrophic nutrition is called mycoheterotrophy. Most orchids later develop green organs and become photosynthetic, while the fungus colonizes the roots of the adult plant, in which it forms true mycorrhizal symbioses. However, various species from several independently arisen lineages remain nongreen and mycoheterotrophic (MH) at adulthood (Merckx, 2013). Moreover, two decades ago, some species were found to mix photosynthetic and MH nutrition: the mixotrophic (MX or partially MH) orchids (Selosse & Roy, 2009; Merckx, 2013).

Fungi associated with most orchid species, the so‐called rhizoctonias, belong to a polyphyletic aggregate of Basidiomycota, including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae (Dearnaley et al., 2012; Selosse et al., 2022). Strikingly, most MH orchids and the first MX orchids discovered mostly associate with nonrhizoctonia mycorrhizal partners, thus questioning whether rhizoctonias can feed MH adult plants (Merckx, 2013; Selosse et al., 2022). Yet, recent publications suggest that even some orchids associated with rhizoctonias could be MX (Selosse & Martos, 2014; Gebauer et al., 2016). In this article, we propose to delineate three types and ecological meanings of MX nutrition in orchids (Table 1). We also offer perspectives for future research on the evolution and physiology of these orchid–fungal interactions.

Table 1.

Three types of mixotrophy in orchids, numbered by order of discovery.

Type Fungal partner Isotopic enrichment Evolutionary origin Main habitat Albinos
I a Nonrhizoctonias, ectomycorrhizal 1 For 13C, 15N and 2H Derived: shift of fungal partner Forest Infrequent, but can be perennial
I b Nonrhizoctonias, saprotrophic 1
II a Rhizoctonias, ectomycorrhizal 1 For 13C, 15N and 2H Derived: shift of ecology of the fungal partner Forest Infrequent, but can be perennial
II b Rhizoctonias, saprotrophic 1
III AL rhizoctonias For 2H and15N Plesiomorphic: ancestral symbiosis Open places Rare, survival questionable
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Ancestral lifestyle (AL) in rhizoctonias qualifies their ancestral, poorly characterized saprotrophic and/or endophytic lifestyle resulting in limited 13C enrichment as compared to autotrophic plants (Selosse et al., 2022).

1

In some cases, a marginal, additional presence of AL rhizoctonias can be observed.

Type I mixotrophy is associated with nonrhizoctonia fungi

This nutrition of MX orchids relies on mycorrhizal association with nonrhizoctonias, either Basidiomycota or Ascomycota, which are either ectomycorrhizal on surrounding trees (type Ia, the most frequently described case; Selosse & Roy, 2009; Merckx, 2013) or leaf‐ or wood‐decaying saprotrophs (type Ib; Table 1; such as Cremastra or Calypso spp.; Suetsugu & Matsubayashi, 2021b; Yagame et al., 2021; Suetsugu et al., 2022). Few rhizoctonias can also occur on the roots (e.g. Julou et al., 2005; Abadie et al., 2006). The MX status of orchids with type I nutrition is supported by four main features.

  1. They are often closely phylogenetically related to fully MH species, sometimes even in the same genus, for example in the Neottieae tribe (Selosse & Roy, 2009) or within the genus Cremastra (Suetsugu et al., 2022).

  2. They tend to inhabit forests where canopy cover reduces light availability and limits photosynthesis (e.g. Julou et al., 2005). Moreover, some species have reduced photosynthetic surfaces and/or impaired photosynthetic abilities (e.g. Girlanda et al., 2006).

  3. Their isotopic content differs from that of autotrophic plants for 13C and 15N (Gebauer & Meyer, 2003; Julou et al., 2005), as well as 2H (Gebauer et al., 2016; Yagi et al., 2024). This aligns with the isotopic enrichments of their associated fungi, which can be estimated from fungal fruitbodies (Mayor et al., 2009) or from intracellular hyphal coils extracted from the mycorrhizas (e.g. Zahn et al., 2023). Whenever MH and autotrophic species co‐occur, the 13C isotopic content of these MX orchids is intermediate between that of MH and autotrophic species growing nearby, and the value of their relative enrichment allows calculation of the percentage of biomass derived from fungal and photosynthetic sources, using a linear two-source isotopic mixing model (Gebauer & Meyer, 2003). The fungal carbon contributions range from undetectable (May et al., 2020) to c. 90% (Hynson et al., 2013) and increase when plants receive less light (e.g. Gonneau et al., 2014). Furthermore, orchids with type I mixotrophy are often richer in total N and 15N isotope as compared to autotrophic plants, a feature that reflects the composition of their fungal food source (Abadie et al., 2006; Minasiewicz et al., 2023). The latter N‐related traits are indirectly linked to mixotrophy, and they do not always follow the 13C enrichment trends (e.g. Roy et al., 2013), since they also depend on other factors such as the identity of the mycorrhizal fungi (Schiebold et al., 2017).

  4. Albinos, that is fully achlorophyllous individuals, occur sometimes in these otherwise green species (Selosse et al., 2004). Their MH‐like isotopic enrichments demonstrate that they are fully supported by fungal resources (Julou et al., 2005; Abadie et al., 2006). In green individuals, isotopic enrichments of above‐ vs belowground organs (Roy et al., 2013) and allocation of 13C‐labeled photosynthates (Lallemand et al., 2019a) support different uses of carbon resources: fungal carbon supports belowground perennial rhizomes and early growth of the annual shoot, while later in the season, photosynthetic carbon supports aerial parts and fruiting. Thus, albinos survive well thanks to fungal carbon (Shefferson et al., 2016), but their shoots soon rely on autophagy (Lallemand et al., 2019b) and produce seeds more rarely and less abundantly due to restricted carbon supply (Roy et al., 2013), making the albino phenotype rare.

The observation of albinos in orchids with type I mixotrophy, even poorly fitted, supports the MX nutrition. Along with Features #1–#3, this suggests that such MX species may eventually evolve into purely MH species. However, more than just a loss of photosynthesis is required for a successful transition to MH nutrition (Jąkalski et al., 2021).

Type II mixotrophy is associated with rhizoctonia fungi of unusual ecology

Mixotrophy in orchids associated with rhizoctonias is more difficult to demonstrate using 13C content for two reasons. First, rhizoctonias do not form conspicuous fruitbodies, making it difficult to sample fungal material for isotopic analyses. Second, whenever fungal material was obtained by extracting hyphal coils from the mycorrhizas (Gomes et al., 2023; Zahn et al., 2023; Suetsugu et al., 2024a), its 13C content was only slightly enriched compared with that of autotrophic plants, in accordance with results from isotopic analyses of subterranean MH orchid seedlings (Stöckel et al., 2014). Thus, 13C analyses cannot detect or quantify fungal carbon in the biomass of rhizoctonia‐associated orchids.

The 13C enrichment in rhizoctonias is much lower than that of ectomycorrhizal or saprotrophic Basidiomycota, likely due to their different nutritional strategy, which remains unclear. Indeed, the ecology of rhizoctonias outside orchid roots is largely hypothetical: they have clear abilities for saprotrophy, as evidenced by their in vitro growth (Rasmussen, 1995) and by analysis of their genomes (Miyauchi et al., 2020), but they turn out to be also endophytic in nonorchid roots (Selosse & Martos, 2014; evidence reviewed in Selosse et al., 2022). Moreover, their exact net carbon exchanges with orchids are still debated, as discussed later. For ease of reading, this poorly understood ecological niche, saprotrophic and/or endophytic, of rhizoctonias will hereafter be referred to as ‘ancestral lifestyle’ (AL) because it is likely plesiomorphic (= phylogenetically ancestral) in the different rhizoctonia lineages. This rhizoctonia ecology sometimes evolved into other, secondarily derived ecologies. Ectomycorrhizal associations arose at least twice in Tulasnellaceae (Tedersoo & Smith, 2013), twice in Ceratobasidiaceae, which often shifted to pure saprotrophy too (Veldre et al., 2013), and at least once in Serendipitaceae, which also evolved mycorrhizal associations with Ericaceae (Selosse et al., 2007; Weiß et al., 2016).

Such rhizoctonias with secondary ecology happen to support type II MX nutrition in orchids. Some of these MX species rely on ectomycorrhizal rhizoctonias (type IIa; Table 1), for example ectomycorrhizal Ceratobasidiaceae species in Platanthera minor (Yagame et al., 2012) or Apostasia nipponica (Suetsugu & Matsubayashi, 2021a). Others associate with purely saprotrophic rhizoctonias (type IIb; Table 1), for example Stigmatodactylus sikokianus with strictly saprotrophic Serendipitaceae showing 13C and 15N enrichment (Suetsugu et al., 2021a, 2024a). As for type I, these orchids can harbor a few AL rhizoctonias as well (Selosse et al., 2022). Although only a limited number of studies have reported them, MX species with type II nutrition exhibit the same four features as these with type I:

  1. Some species are phylogenetically close to MH species (e.g. within the genus Platanthera; Yagame et al., 2012) and, indeed, some purely MH orchids also rely on rhizoctonias with secondary ecology, such as Rhizanthella gardneri (Bougoure et al., 2010) or Chamaegastrodia shikokiana (Yagame et al., 2008) with ectomycorrhizal Ceratobasidiaceae.

  2. They have a forest ecology, entailing low illumination and limited photosynthetic ability.

  3. Their isotopic enrichments are similar to those of type I because the ecology of their rhizoctonias is similar (Yagame et al., 2012; Suetsugu et al., 2021a, 2021b, 2024a), and fungal contributions inferred from 13C enrichments can be high (> 50% for P. minor – Yagame et al., 2012; c. 90% for A. nipponica and S. sikokianus – Suetsugu & Matsubayashi, 2021a; Suetsugu et al., 2024a).

  4. Some species display albinos that can survive over several years (e.g. Suetsugu et al., 2019).

Affiliation of MX nutrition to type I or II may seem uncertain in some cases, Goodyera velutina (Suetsugu et al., 2019) and Cypripedium debile (Suetsugu et al., 2021b), which display a characteristic 13C enrichment, harboring few ectomycorrhizal nonrhizoctonias and mostly rhizoctonias from clades for which AL is not sure. In both species, albinos survive over several years and display marked 13C enrichment (Suetsugu et al., 2019, 2021a, 2021b, 2024b; Suetsugu & Matsubayashi, 2022), clearly affiliating their MX nutrition to type I or II.

Type III mixotrophy gains carbon from AL rhizoctonias

Mixotrophy of types I and II involves evolutionary shifts (Table 1), either a shift of fungal partners (type I) or a shift of the ecology of the involved rhizoctonias (type II). Yet, most orchids associate with AL rhizoctonias, which is the ancestral state in orchids (Wang et al., 2021; Selosse et al., 2022). It was initially suspected that at least some of these orchids could be MX, based on their small deviations in 13C content, slightly higher or lower than autotrophic plants (‘cryptic MH’ in Hynson et al., 2013; Selosse & Martos, 2014; Supporting Information Fig. S1). Most of the time, however, their 13C content falls in the range of autotrophic values (Liebel et al., 2010; Girlanda et al., 2011). This does not disprove MX nutrition per se, since AL rhizoctonias are not strongly enriched in 13C compared with autotrophs as mentioned previously.

A decisive step was recently taken when 2H enrichment as compared to autotrophic plants was observed, not only in type I and II orchids but also in some orchids mycorrhizal with AL rhizoctonias (Gebauer et al., 2016; Schiebold et al., 2017; Schweiger et al., 2018; Yagi et al., 2024). Such an enrichment could result from elevated transpiration, but it would be accompanied by a corresponding 18O enrichment, which does not occur. Instead, a gain of 2H‐enriched fungal resources was hypothesized to cause this. Fungal resources are likely flowing to 2H‐enriched orchids, as also indirectly supported by their high 15N and N content (e.g. Schweiger et al., 2018).

One should note that uncertainties persist in interpreting 2H enrichments, which have complex origins. Indeed, 2H fractionation varies during the synthesis of organic compounds (Holloway‐Phillips et al., 2022; Baan et al., 2023a; Lehmann et al., 2024). This variation can be significant even among autotrophic species grown at the same location (e.g. Chikaraishi et al., 2004; He et al., 2020; Holloway‐Phillips et al., 2022; Schuler et al., 2023; Baan et al., 2023b). Thus, disentangling the 2H enrichment of mycoheterotrophy from species‐specific biochemical fractionation remains somewhat challenging. Consequently, caution is still required when interpreting 2H enrichment as sole evidence of gain of fungal resources.

Mixotrophic orchids associated with AL rhizoctonias and displaying 2H enrichment represent our type III mixotrophy (Table 2). On the one hand, their carbon gain is somewhat expected since AL rhizoctonias do feed orchids at germination so that there is a physiological mechanism for the plant to harvest and utilize fungal C. On the other hand, type III orchids do not really fit three of the four features presented by type I and II orchids: Indeed, while isotopic enrichments for 2H and often 15N (Feature #3) are shared, the other features (Features #1, #2, and #4) differ somewhat, as follows.

  1. They are often not closely related to fully MH species (e.g. in the genera Orchis or Ophrys). Moreover, as stated in the Introduction section, we do not know any fully MH orchids associated with AL rhizoctonias. Accordingly, linear two‐source isotopic mixing models of 2H enrichments estimate that, among cases studied so far, the fungal contribution reaches at most 20% (Schweiger et al., 2018), that is much less than the gains for type I and II mixotrophs so that the available carbon may be insufficient to reach full MH nutrition.

  2. Their ecological niche, mostly in open habitats, exerts less selective pressure for nonphotosynthetic carbon sources than the forest ecology of most type I and II species. One should note, however, that few species with type III mixotrophy in meadows that also thrive in ectomycorrhizal forests may shift to type I mixotrophy: Ophrys insectifera (Schweiger et al., 2018) and Neottia ovata (Wang et al., 2021) not only associate with AL rhizoctonias but also harbor ectomycorrhizal fungi when growing in forest environments, where alteration of their isotope signatures (Schweiger et al., 2018) suggests that these partners can contribute to their resources.

  3. Albinos are much rarer in orchids with type III mixotrophy, and more generally in orchids mycorrhizal with AL rhizoctonias, than in species with type I and II mixotrophy. To the best of our knowledge, no study has been published about such albinos. To document their characteristics, we analyzed three meadow Ophrys populations associated with rhizoctonias, whose 13C and 15N enrichments supported type III mixotrophy, and which displayed a total of 15 albinos (Box 1; Fig. 1). This case study did not evidence long‐term survival of albinos, which did not form bulbs with reserves for the next year. They display an etiolated phenotype and represent a fatal shift to albinism, for unknown reasons, rather than a perennial phenotype. Isotopic analyses cannot rule out that they survive on reserves accumulated in bulbs during the previous year, in which they were probably green (Box 1), rather than on fungal resources. Our results are consistent with a poor ability of AL rhizoctonias to support MH growth, and the limited carbon transfer from AL rhizoctonias reported previously.

Table 2.

Comparison of albinos vs green individuals in three Ophrys populations from meadows (see raw data in Supporting Information Tables S1 and S2).

1. Samos Island (Greece) 2. Pas de l'Escalette (France) 3. Cirque de Navacelles (France)
Geocodes 37°39′51″N, 26°50′38″E 43°54′03″N, 03°30′43″E 43°49′53″N, 03°39′50″E
Ophrys species Ophrys pelinaea Ophrys litigiosa Ophrys apifera
and sampling April 23, 2015 May 5, 2016 May 28, 2016
Albinos/green 1 4/20 5/14 6/18
With new bulb 2 , 3 0/19* 0/ni 0/18*
Flower number 3 3.75 ± 0.96/3.85 ± 1.23 ns 2.80 ± 0.84/3.07 ± 0.92 ns 4.33 ± 1.63/4.44 ± 1.65 ns
Height (cm) 3 30.35 ± 4.79/21.90 ± 3.99* 21.00 ± 3.61/19.49 ± 3.84 ns 48.83 ± 2.64/36.00 ± 6.29*
Leaf δ13C (‰) 4 −30.60 ± 0.10/−31.61 ± 0.09* −30.03 ± 0.42/−31.86 ± 0.44* −26.30 ± 0.48/−28.72 ± 0.25*
New bulb δ13C −29.80 ± 0.28‰ (n = 4) −30.22 ± 0.16 (n = 6) ni
Leaf δ15N (‰) 4 −2.46 ± 0.46/−2.13 ± 0.27 ns −0.36 ± 0.27/−1.60 ± 0.05* −0.05 ± 0.15/v1.01 ± 0.17*
New bulb δ15N −1.91 ± 0.92 (n = 4) −1.32 ± 0.21 (n = 6) ni
Leaf %N 1.38 ± 0.03/2.47 ± 0.07* 4.12 ± 0.34/2.37 ± 0.28* 4.34 ± 0.31/2.67 ± 0.04*
New bulb %N 1.10 ± 0.22 (n = 4) 4.25 ± 0.34 (n = 6) ni
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ni, not investigated; ns, not significant. Numbers and means (±SD) for albinos are italicized while those for green individuals are not.

*

Significant (P < 0.05; see notes for statistics).

1

Number of albinos observed and number of green individuals studied (among a larger number) – this is the number of replicates for the values below.

2

Presence, for each phenotype, of the newly formed bulb with reserves for the growth in year n + 1.

3

Significance according to the Tukey's HSD test.

4

Significance according to Student's t‐test; Fig. S1.

Box 1. Albinos in orchids with ancestral lifestyle (AL) rhizoctonias: case studies of three Mediterranean Ophrys species.

Ophrys species are perennial orchids that form a subterranean bulb with reserves for next year, every year after the emergence of their first leaf (Rasmussen, 1995). With the exception of Ophrys insectifera (which also lives in forest conditions with type I mixotrophy; Schweiger et al., 2018), Ophrys species mostly grow in open places (Rasmussen, 1995; Girlanda et al., 2011) where they display no 13C enrichment (Liebel et al., 2010; Gomes et al., 2023) but are enriched in 15N and/or 2H (Girlanda et al., 2011; Gebauer et al., 2016). Moreover, Ophrys species from meadows mostly associate with ancestral lifestyle rhizoctonias (Tulasnellaceae and Ceratobasidiaceae; Rasmussen, 1995; Schatz et al., 2010; Liebel et al., 2010; Girlanda et al., 2011), although other fungi can be present (Gomes et al., 2023). We thus consider meadow Ophrys species as potential models for type III mixotrophy.

Here, we investigate meadow populations of three Ophrys species displaying in all 15 albinos (Tables 2, S1; Fig. 1a,b), with the closest ectomycorrhizal trees located > 45 m away. Fungal typing performed by PCR on the roots of the investigated individuals (n = 4 per phenotype and population) provided positive results for Basidiomycota (using general primers ITS86‐F and ITS4; 79% positive PCR) and Tulasnellaceae (using 5.8S‐OF and ITS4‐Tul; 83% positive PCR; all primers and molecular methods as in Petrolli et al., 2022). Underground observations (Table S1) and samplings were performed by lateral digging to allow survival, and whole plants were only harvested for isotopic analyses.

All examined individuals had bulbs from the previous year (Fig. 1c), suggesting that they had already sprouted in previous years. However, while all green individuals initiated any new bulb with reserves for the following year (but one in Samos), no albino displayed a new bulb (Table 2). Thus, they may turn albino before death, in agreement with the fact that no albino survival was observed over 2010–2011 (n = 2 albinos), 2011–2012 (n = 6), and 2013–2014 (n = 2) at Navacelles and 2013–2014 (n = 2) at Escalette (M.‐A. Selosse, pers. obs.). The absence of a new bulb is at odds with the fact that underground part type I mixotrophs, albino or green, are mainly sustained by fungal C, and not by photosynthesis (Roy et al., 2013; Gonneau et al., 2014; Lallemand et al., 2019a).

Shoots had similar flower numbers in both phenotypes, but albinos tended to be taller than green individuals in all populations (Table 2), significantly in Samos (1.38× taller; P < 0.001) and Navacelles (1.36×, P < 0.0001). This contrasts with albinos of type I mixotrophs, which tend to be smaller than green individuals (Julou et al., 2005; Abadie et al., 2006; Roy et al., 2013). This is reminiscent of shade‐induced stem elongation, a response well‐known in monocots (Liu et al., 2016), which may be induced by impaired light perception and/or reduced carbon nutrition in Ophrys, while it does not affect albinos of type I mixotrophs.

Isotopic analyses were carried out as in Yagame et al. (2021) (Table S2; Fig. S1). The 13C enrichments of albinos and green individuals did not significantly differ from surrounding autotrophic plants (Fig. S1), except for green Ophrys pelinaea at Samos which, together with the other orchid Orchis anatolica, was significantly depleted in 13C. Thus, Ophrys mycorrhizal rhizoctonias are likely AL (i.e. not ectomycorrhizal nor truly saprotrophic). In the three populations, albinos were 13C‐enriched compared with green individuals (Table 2), from 1 to 2.4‰. On the one hand, such an enrichment is expected if albinos gain carbon from slightly 13C‐enriched rhizoctonias (Stöckel et al., 2014; Gomes et al., 2023; Zahn et al., 2023). On the other hand, new bulbs were enriched in 13C (Table 2), not significantly differently from albino leaves at Escalette (P = 0.75 according to the Tukey's HSD test), but significantly more at Samos (P < 0.0001). Indeed, heterotrophic organs are enriched in 13C (Cernusak et al., 2009) so that the use of bulb reserves from previous years by albinos may explain the 13C enrichment. We cannot distinguish whether albinos grow on fungal and/or bulb carbon.

Considering 15N, Ophrys species were significantly enriched compared with autotrophic controls (but N‐fixing species; Table S1; Fig. S1), suggesting that they display type III mixotrophy. Compared with green individuals, albinos of Escalette and Navacelles were significantly enriched in 15N by c. 1.7‰ and in total N by more than 60% (Table 2), which could mean a higher use of fungal resources (see main text). Yet, in Samos, an opposite trend was observed (albeit not significant; Table 2). Whenever new bulbs were examined (Table 2), their 15N content was always indistinguishable from that of the green leaves of the year that support them (Escalette: P = 0.18; Samos P = 0.88). Bulb N content was close to that of albino leaves (P > 0.05 for both site), but their 15N enrichment was either similar (at Samos; P = 0.45) or depleted (at Escalette; P < 0.001). Nitrogen parameters provide no clear support for increased use of fungal resources in albinos.

Unfortunately, no 2H enrichment was measured since the method was not used in orchid research at the time of analyses and evidence for type III mixotrophy in these Ophrys ssp. remains here limited to 15N enrichment. Moreover, measuring 2H enrichment would not distinguish between fungal and bulb resources in albinos, as both are likely enriched in 2H. To conclude, the observed Ophrys albinos are unlikely to survive to the next year, their higher shoots contrasts with the phenotype of albinos in type I mixotrophs, and their isotopic enrichments are congruent with reserves either from their old bulb or from the fungus.

Fig. 1.

Fig. 1

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Albino of Ophrys pelinaea pictured at Samos in 2015. (a) Albino shoot and flowers. (b) Green and albino leaves. (c) Underground part of a green individual with remains of the bulb of next year (red dotted circle) and remain of the old bulb (red arrow). Bars, 1 cm.

The type III exploitation of fungal carbon thus differs from that of types I and II, on the plant side and/or on the AL rhizoctonia side.

How type III challenges our assumptions on mixotrophy?

Strikingly, the few available studies of simplified in vitro design have reported net carbon transfer from orchids to their mycorrhizal AL rhizoctonias. First, Serapias strictiflora displayed similar 13C enrichment when linked to a Tulasnellaceae cultivated either on substrate devoid of organic compounds or on dead maize roots, which are naturally enriched in 13C (Látalová & Baláž, 2010), so that no carbon flux from the fungus to the plant occurred. Yet, in the second condition, the fungus displayed 13C enrichment intermediate between its dead maize substrate and the orchid, suggesting that the plant provided 69% of its carbon needs. In a second experiment, carbon labeling of Goodyera repens and its Ceratobasidiaceae partner showed reciprocal carbon transfer, with a net resulting flow to the fungus (Cameron et al., 2006, 2008). In a third experiment in which another Ceratobasidiaceae species connected adult Dactylorhiza fuchsii with developing MH seedlings, labeling of adults revealed a transfer of carbon to the fungus and to the seedlings (Read et al., 2024). Unfortunately, 2H enrichments were not estimated in these experiments (indeed, the first two were designed before this tool was introduced into orchid research), but such a measurement is clearly required in future similar research.

That said, how can we reconcile these in vitro data with the existence of type III mixotrophy in orchids associated with AL rhizoctonias? Of course, it can be argued that the orchids studied in vitro do not display type III mixotrophy and/or that the experimental conditions did not favor a carbon flow from AL rhizoctonias. But, most importantly, 2H enrichment of orchids is not inconsistent with a net carbon flow to the fungus (by net flow, we mean the difference between the orchid‐to‐fungus and fungus‐to‐orchids raw flows). We propose an interpretation of these seemingly contradictory data by considering that isotopic content demonstrates a raw, but not a net flow from fungus to orchid. Thus, if the orchid provides more carbon on its side, as seen in the experiments of Cameron et al. (2006, 2008), the net flow occurs in the direction of the AL rhizoctonias. This would explain the absence (1) of MH species based on such fungi and (2) of perennial albinos in type III mixotrophy (Box 1). One should not mistake the main carbon source in this exchange: Just because a baker gives change to a customer does not mean that the customer depends on the baker for their money.

We expand here the hypothesis of ‘C‐exchangers’ proposed by Lallemand et al. (2018) for Ericaceae from the Pyroleae tribe. This group displays a range of situations, including MH species (Hynson et al., 2009), MX species with type I nutrition (Matsuda et al., 2012; Sakae et al., 2024), and species receiving carbon from their fungi but devoid of albino variants and unable to use more fungal carbon in the shade (Lallemand et al., 2018). For the latter type, Lallemand et al. (2018) suggested that they gain fungal carbon due to their specific mycorrhizal mechanisms and reward the fungi with even more photosynthetic carbon. Such C‐exchangers transfer significant amounts of carbon in both directions so that the plant displays isotopic enrichment, but no net dependence on fungal carbon.

This interpretation is open to falsification and experimental testing. It does not exclude that some type III orchids get net carbon gain from the interaction, but this remains to be demonstrated to assess whether type III mixotrophy is distinct or not from types I and II in which the MX orchids receive a net gain.

If type III nutrition corresponds to that of such C‐exchangers, is it truly a mixotrophy? This is a matter of definition. On the one hand, it fits the definition of Merckx (2013), referring to ‘the ability of a plant to obtain carbon simultaneously through autotrophy and mycoheterotrophy’, and indeed type III nutrition has been considered MX since the very first evidence of 2H enrichment (‘partially MH’ in Gebauer et al., 2016). Thus, it may seem too late to adopt any alternative. On the other hand, one would have preferred to keep the word mixotrophy strictly for orchids having a net carbon gain from their fungi, that is a carbon budget requiring the fungus, although this is less easy to demonstrate. This option would have caused less overlap with autotrophy. Indeed, C‐exchangers cover their net energetic needs thanks to photosynthesis and are net carbon donors: They can thus be considered autotrophic. It remains unclear whether type III orchids are C‐exchangers or low level, but net carbon receivers – and both cases may even exist (Fig. 2). However, their C‐exchanger status would explain some features of type III mixotrophy, including the discrepancies with several in vitro experiments and the instability of their albino phenotype (Box 1).

Fig. 2.

Fig. 2

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Evolutionary pathway linking mixotrophic nutrition types I to III, according to Table 1, and mycoheterotrophic nutrition in orchids. Ancestral lifestyle (AL) rhizoctonia: rhizoctonias with ancestral lifestyle, that is saprotrophic and/or endophytic (see main text). Continuous arrows in gray display the most likely evolutions between the nutrition types, which occurred repeatedly, while thinner dotted arrows display possible, uncertain evolutionary pathways; some species may even display two different types depending on the ecological conditions. Yet, there are no necessary trends, so species can stay at any stage, revert to a previous one or proceed further. Although we cannot rule out direct evolution to types I and II, type III may represent a predisposition for it.

To summarize, 2H enrichments do not provide direct evidence that type III orchids are not autotrophic, at least based on current knowledge. This also applies to the discovery of 2H enrichments in arbuscular mycorrhizal (AM) plants: Some AM plants with the so‐called Paris‐type mycorrhizas display isotopic 2H enrichments, which were claimed to provide evidence for carbon gain from AM fungi (Giesemann et al., 2020, 2021; Gomes et al., 2023). AM symbiosis is the main mycorrhizal association in land ecosystems (van der Heijden et al., 2015), and AM fungi are shared by many neighboring plants (common mycorrhizal networks; Selosse et al., 2006; van der Heijden et al., 2015; Magkourilou et al., 2024). Whether the fungus gains or provides carbon is of major importance for, respectively, the mutualistic or parasitic outcome of the mycorrhizal association. Indeed, the existence of common mycorrhizal networks would become highly unstable if many plants acted as carbon sinks. If 2H‐enriched AM plants are C‐exchangers, then they do not entail any cost to the network nor to the plants linked by shared fungi. Here again, more research is urgently required to understand the exact carbon budget and impact on the fungus of 2H‐enriched AM plants.

Be it proven that orchids (and nonorchid plants) with typ III mixotrophy are C‐exchangers, and thus mostly feed on their photosynthesis, the debate on whether or not to qualify them as ‘mixotrophs’ should be re‐opened. Until then, the use of the term ‘type III mixotrophy’ in the future literature would encapsulate their specific relation to their mycorrhizal fungi and the questions raised previously.

Research perspectives

In the future, type III mixotrophy evidently deserves more investigation, some of which will be valuable for AM research too. First, the ecology of AL rhizoctonias needs to be clarified, between saprotrophy and/or endophytism in nonorchid roots, or even other trophic niches. It should be clarified in a phylogenetic framework for rhizoctonia families since extant attempts (Veldre et al., 2013 for Ceratobasidiaceae; Weiss et al., 2016 for Serendipitaceae) provide too preliminary a view of the ecological diversifications. Moreover, these taxa should be more carefully surveyed in the environment, especially the Tulasnellaceae that escape the universal primers used for barcoding of fungal communities (Vogt‐Schilb et al., 2020; Petrolli et al., 2022). Thanks to the available genomes (e.g. Miyauchi et al., 2020), gene expression in their diverse environmental microsites, from soil or dead organic matter to orchid mycorrhizas and nonorchid roots, could be analyzed.

Second, a budget of the orchid–fungus carbon exchange should be achieved in type III mixotrophy. With better knowledge of AL ecology at hand, reciprocal labeling of orchids and fungus in situ could test the relevance and generalize the conclusions of the in vitro experiments mentioned previously (Cameron et al., 2008; Látalová & Baláž, 2010; Read et al., 2024). By any means of investigation, a full carbon budget of some model orchids with type III mixotrophy is required. Finally, detection of the use of fungal carbon in type III mixotrophy urgently needs a more routinely tractable, more direct and less expensive proxy than 2H enrichment, which cannot be obtained in most laboratories (unlike 13C enrichment for types I and II): Perhaps, thanks to comparative transcriptomic and metabolomic research (as used for MH orchids; Jąkalski et al., 2021), some metabolites or specific genes can be found as fingerprints.

Finally, experiments and studies have largely focused on terrestrial and temperate orchids, although most species are tropical. Among these, 80% are epiphytic. Interestingly, some of the latter have a crassulacean acid metabolism (CAM) photosynthesis adapting them to the dry epiphytic conditions (Givnish et al., 2015): CAM metabolism entails a higher 13C enrichment than in (sometimes coexisting) orchids performing C3 photosynthesis. This feature offers a natural labeling to investigate the carbon flux from CAM orchids to their AL mycorrhizal rhizoctonias.

Outlook

The three MX nutrition types presented in Table 1 represent distinguishable stages from evolutionary pathways between full autotrophy and MH life in orchids, which may apply in other plant families (Merckx, 2013). In orchids, the identity of the mycorrhizal fungus itself displays stages in this pathway, as examined by Selosse et al. (2022). For both MX nutrition and mycorrhizal fungi, shifts (Fig. 2) are multiple, at any taxonomic level, as expected from permanent evolution of the symbiosis. Plastic species that shift mixotrophy from type III in meadows to type I in forest environments, such as O. insectifera or N. ovata (Schweiger et al., 2018; Wang et al., 2023), potentially represent ongoing evolutionary transitions between types. We also know some species devoid of isotopic enrichment that thus receive nothing from their AL rhizoctonias, such as Pseudorchis albida (Schiebold et al., 2017): whether such true autotrophs are ancestral and/or due to reversions remains unclear (Fig. 2).

Orchids exhibit a fascinating evolutionary versatility in their fungal associates and mycorrhizal exchanges. This makes them excellent models not only for studying the diversity of full or partial MH nutrition but also for examining the dynamics of plant–fungus mutualism at the border of exploitation.

Competing interests

None declared.

Author contributions

M‐AS and P‐LA wrote the paper. M‐AS and ST designed the study of Box 1 (sampled with TF and analyzed with the help of LD). All other authors, including ED, JM and FM, contributed to preliminary discussions of typology, and they edited and revised the manuscript. M‐AS and P‐LA contributed equally to this work.

Disclaimer

The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.

Supporting information

Fig. S1 Mean δ13C and δ15N values of plants sampled at Samos, Escalette, and Navacelle.

Table S1 Phenotypic raw data for investigated Ophrys species.

Table S2 Stable isotopic (13C and 15N) and nitrogen content raw data for investigated Ophrys species.

Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

NPH-246-1912-s001.pdf (606.7KB, pdf)

Acknowledgements

We acknowledge the Fédération France Orchidées for help in locating albino Ophrys in France, and Jan and Liesbeth Essinkand for access to the Samos site. The presented research was supported by the ORCHIDOMICS grant funded by Narodowe Centrum Nauki (National Science Center, Poland) grant no. [2015/18/A/NZ8/00149]. We are grateful to ‘EDF Division de l'Ingénierie du Parc et de l'Environnement (DIPDE)’ and EDF R&D as well as to Institut Universitaire de France for additional financial support. We thank the staff of the Service de Spectrométrie de Masse Isotopique of the Muséum national d'Histoire naturelle (SSMIM), namely Denis Fiorillo for his help with stable isotope analyses. We apologize to all authors of papers not cited due to space limitation. We acknowledge the editor Maarja Öpik, two anonymous referees, and Dr. Kenji Suetsugu for their helpful comments on an earlier version of this manuscript, as well as David Marsh for English corrections.

Data availability

All raw data for Box 1 report (isotopic and phenotypic measurements) are available in Supporting Information (respectively, in Tables S2 and S1).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Mean δ13C and δ15N values of plants sampled at Samos, Escalette, and Navacelle.

Table S1 Phenotypic raw data for investigated Ophrys species.

Table S2 Stable isotopic (13C and 15N) and nitrogen content raw data for investigated Ophrys species.

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NPH-246-1912-s001.pdf (606.7KB, pdf)

Data Availability Statement

All raw data for Box 1 report (isotopic and phenotypic measurements) are available in Supporting Information (respectively, in Tables S2 and S1).

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