Chapter 25 Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks

Marc‐André Selosse1,2, Melissa Faust Bocayuva3, Maria Catarina Megumi Kasuya3, and Pierre‐Emmanuel Courty4,5 1 Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’Histoire naturelle, Paris, France 2 Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Poland 3 Department of Microbiology, Laboratory of Mycorrhizal Association, Universidade Federal de Viçosa, Brazil 4 Zurich‐Basel Plant Science Center, Department of Environmental Sciences, Botany, University of Basel, Switzerland 5 Department of Biology, University of Fribourg, Switzerland

25.1 Introduction without any known reward to the fungus (Figure 25.1a). Reported since the 19th In usual mycorrhizal associations, the fungus c­entury (see review in Bidartondo, 2005; exploits plant photosynthetic carbon (C) Selosse et al., 2011), they were once consid­ and provides mineral resources as a reward, ered as “saprophytic” but, since they use C such as nitrogen (N), phosphorous or water from their mycorrhizal fungi, Leake (1994) collected in the soil by its mycelium (van der proposed to call these plants “mycohetero­ Heijden et al., 2015). Since the exchanged trophs” (see Merckx, 2013 for review). resources are costly to each partner, any Indeed, their mycorrhizal fungi are, most of mutant investing less in providing nutrient the time, in turn mycorrhizal on other may expend its fitness at the expense of the nearby plants, although with some excep­ other partner. There is experimental support tions in tropical regions where saprotrophic that plant and fungi stop, or at least limit, fungi can be involved (e.g., Martos et al., interaction with partners that do not reward 2009; Ogura‐Tsujita et al., 2009). them (Werner and Kiers, 2015). Thus, by A variant of mycoheterotrophy occurs in difference, the interaction with the most plants that initiate their development as beneficial partners is favored, and mutualistic mycoheterotrophic seedlings before turning interactions are locally selected. green at adulthood (Merckx, 2013). This ini­ Such a partner choice, however, is clearly tial mycoheterotrophy is known in several avoided by mycoheterotrophic plants basal plant lineages disseminated by spores (Selosse and Rousset, 2011); these hetero­ (namely, Psilotum and Ophioglossum ferns, as trophic plants entirely rely on their mycor­ well as clubmosses; Read et al., 2000; Field rhizal fungi for mineral and C nutrition, et al., 2015), but also in plants that form

Molecular Mycorrhizal Symbiosis, First Edition. Edited by Francis Martin. © 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

451 452 Molecular mycorrhizal symbiosis

Mycoheterotrophic plant

C

High 13C Autotrophic and plant 15N content Enrichment in 13C

Mycorrhizal Fungus association Mycorrhizal association Depletion in 15N

(a) N

Mixotrophic plant

C

Photosyn- Intermediate thetates, 13 depleted Autotrophic C 13 and high in C plant 15N content Enrichment in 13C Mycorrhizal Fungus Mycorrhizal association association

Depletion in 15N

(b) N

Figure 25.1 Nutrition of mycohetero‐ and mixotrophic plants associated with ectomycorrhizal fungi that themselves associate with surrounding trees. In this model representing the usual situation in temperate ecosystems, N flow from soil (blue) and C flow from photosynthesis (orange) are displayed, and the red line indicates the interface between autotrophic host tree and ectomycorrhizal fungi where isotopic fractionation occurs (enrichment in 13C from host to fungus and depletion in 15N from fungus to host). (See insert for color representation of the figure.) minute seeds with extremely limited reserves trophic at adulthood (Figure 25.1b) – that is, and require fungal C to germinate as myco­ they obtain C from both their mycorrhizal heterotrophs, such as orchids (Rasmussen, fungi and their photosynthesis (Selosse and 1995; Dearnaley et al., 2012, 2015). Initial Roy, 2009; Hynson et al., 2013a). This nutri­ mycoheterotrophy may still fit into a tional strategy, where green adult plants mutualistic framework, since the fungus is obtain C from their mycorrhizal fungi and rewarded in C when plants are adult (“take its photosynthesis, was discovered in the last now, pay later” hypothesis; Field et al., 2015). decade (Gebauer and Meyer, 2003; Selosse Beyond these examples, adult green et al., 2004; Bidartondo et al., 2004; Julou plants from temperate regions that are et al., 2005). It is a kind of mixotrophy that i­nitially mycoheterotrophs were recently mixes autotrophy and heterotrophy and, discovered to remain partially mycohetero­ although orchids were instrumental in the Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 453

emergence of this concept, the phenomenon time (Tranchida‐Lombardo et al., 2010; Roy is now suspected – and partly demon­ et al., 2013) – up to 14 years for albinos strated – to be more widespread (Selosse and (Abadie et al., 2006). Although they tend to Roy, 2009). This chapter reviews the discovery perform less well than green individuals (see of mixotrophy in mycorrhizal plants, the below), some albinos do flower and fruit available data on mixotrophic physiology, and (Salmia, 1989; Julou et al., 2005; Tranchida‐ the evolutionary link between mixotrophy Lombardo et al., 2010). and full mycoheterotrophy. Albinos’ mycoheterotrophy is further cor­

roborated by the absence of CO2 absorption in full light (Julou et al., 2005) and their 13C 25.2 Mixotrophy in orchids enrichment, which is similar to that of myco­ heterotrophic plants (Julou et al., 2005; Abadie 25.2.1 Isotopic and physiological et al., 2006; Roy et al., 2013). Albinos support evidences the likelihood of mixotrophy in their green The suspicion of mixotrophy in orchids first conspecifics. Accordingly, survival of albinos is came from two lines of observations, mainly also reported from green hemiparasitic plants on green species from the Neottieae such as Striga hermonthica, which are well tribe – isotopic anomalies and survival of known to use the C from parasitized plants’ achlorophyllous individuals. Mycohetero­ sap to support part of their C needs (Press trophic orchids associated with ectomycorrhi­ et al., 1991; Těšitel et al., 2010; and see below). zal fungi tend to be enriched in 13C and 15N, compared with autotrophic plants (Trudell 25.2.2 Mycorrhizal fungi that link et al., 2003), and this results from a high 13C mixotrophs to trees content of their fungi, and a N nutrition that Green orchids usually associate with rhizoc­ differs from that in autotrophic plants tonias, a polyphyletic group of Basidio­ (Hynson et al., 2013a; Figures 25.1a and mycetes that normally live as saprophytes or 25.2). Gebauer and Meyer (2003) discovered endophytes (namely Tulasnellaceae, Cerato­ that some green forest orchids display 13C and basidiaceae and some Sebacinales; Rasmussen, 15N abundances intermediate between those 1995; Dearnaley et al., 2012). However, of autotrophic plants and those of mycohet­ orchids displaying anomalies in 13C abun­ erotrophs associated with ectomycorrhizal dance also display another feature in this fungi, suggesting a partial mycoheterotrophy. respect: rhizoctonias are rare or absent in This feature was later confirmed in many their roots, and they instead associate with other studies (e.g., Bidartondo et al., 2004; fungi that normally form ectomycorrhizae Julou et al., 2005; Abadie et al., 2006; Tedersoo on forest trees, as do mycoheterotrophic et al., 2007; Figures 25.1b and 25.2). orchids (see Dearnaley et al., 2012 for review; Independently, achlorophyllous individ­ and Figure 25.1). uals, also called albinos (Figure 25.3), are Such associates were first discovered in anciently reported from the abovemen­ the genera Epipactis, Cephalanthera and tioned orchid species, especially Neottieae Limodorum from the Neottieae orchid tribe. species in the genera Epipactis (Salmia, 1989; Mixotrophic Epipactis species show a prefer­ Selosse et al., 2004) and Cephalanthera (Julou ence for ectomycorrhizal Pezizomycetes, et al., 2005; Abadie et al., 2006; Roy et al., mostly related to truffles, with many additional 2013). This phenotype remains stable over ectomycorrhizal fungi (Bidartondo et al., 454 Molecular mycorrhizal symbiosis

Autotrophic plants 15 δ N(% ) Mixotrophic plants Ea Mycoheterotrophic plants 8 Sg Ectomycorrhizal fungi Pc Sl Cp Saprotrophic fungi 4 Sc Tm Hm Tt Cu Hl 0 Ld Ta Hc Lo Os Pb Ga Ls

–4 Au

Ms Pa 13 δ C(% ) –8 –32 –30–28 –26 –24 –22

Figure 25.2 13C and 15N isotopic abundances in mixotrophic and mycoheterotrophic plants with these of co‐occurring plant and fungal species from an Estonian boreal forest (reproduced from Tedersoo et al., 2007; means ± SE or point for values without repetitions). Mixotrophic plants place between autotrophic and mycoheterotrophic plants, which are themselves close to ectomycorrhizal fungi (their food source in this case). Saprotrophic fungi are more enriched in 13C but less enriched in 15N than the previous ones. Autotrophic plants: Arctostaphylos uva‐ursi (Au), Melampyrum sylvaticum (Ms), Picea abies seedlings (Pa). Mixotrophic orchids (bold): Epipactis atrorubens (Ea), Listera ovata (Lo), Platanthera bifolia (Pb); mixotrophic pyroloids (bold): Chimaphila umbellata (Cu), Orthilia secunda (Os), Pyrola chlorantha (Pc). Mycoheterotrophic plants (bold, underlined): Hypopithys monotropa (Hm). Ectomycorrhizal fungi (italics, bold): Coltricia perennis (Cp), Helvella lacunosa (Hl), Helvella crispa (Hc), Sarcosphaera coronaria (Sc), Suillus granulatus (Sg), Suillus luteus (Sl), Thelephora terrestris (Tt), Tricholoma myomyces (Tm, the mycorrhizal fungus in the investigated H. hypopithys). Saprotrophic fungi (italics): Gymnopus acervatus (Ga), Lepista sordida (Ls), Limacella delicata var. glioderma (Ld), Tapinella atrotomentosa (Ta).

Figure 25.3 Albino and green plants in the mixotrophic Cephalanthera damasonium. This European orchid from the Neottieae tribe is normally green and relies on both photosynthesis and mycoheterotrophy (see shoots on the left) to adapt to low light levels of its forest environment. Some rare individuals are fully achlorophyllous, and survive as pure mycoheterotrophs (individual on the right). Photo by Laurent Berger. (See insert for color representation of the figure.) Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 455

2004; Selosse et al., 2004; Ogura‐Tsujita and disconnecting the fungus from its resources Yukawa, 2008; Ouanphanivanh et al., 2008; (= nearby tree roots) results in plant death. Shefferson et al., 2008; Liebel et al., 2010). Mixotrophic Cephalanthera species display a 25.2.3 repeated evolution large fungal spectrum, including of mixotrophy in orchids Cortinariaceae, Hymenogastraceae and mainly As described above for the genus Cymbidium Thelephoraceae (Bidartondo et al., 2004; (tribe Cymbidieae), mixotrophy is not limited Julou et al., 2005; Abadie et al., 2006; Matsuda to Neottieae. Genneria diphylla and Platanthera et al., 2008; Yamato and Iwase, 2008); minor (tribe Orchideae) display 13C enrich­ Mixotrophic Limodorum species specifically ment (Figure 25.2) and association with, associates with Russula (especially from the respectively, Russulales (Liebel et al., 2010) delica section; Girlanda et al., 2006; Liebel and a clade of Ceratobasidiaceae forming et al., 2010; Paduano et al., 2011; Bellino ectomycorrhizae (Yagame et al., 2012). An et al., 2014). Later, a similar feature was interesting debate exists over Corallorhiza tri- reported in Asian Cymbidium species from the fida (tribe Maxillarieae), a pale greenish spe­ Cymbidieae tribe: the green C. lancifolium and cies in an otherwise fully mycoheterotrophic C. goeringii, which have 13C abundances inter­ genus. It is sometime considered as myco­ mediate between autotro­phic species and the heterotrophic, feeding on ectomycorrhizal mycoheterotrophic C. macrorhizon and Thelephora (e.g., McKendrick et al., 2000; C. aberrans (Motomura et al., 2010) and associ­ Cameron et al., 2009), but it is mixotrophic, ate with a mix of rhizoctonias and ectomycor­ based on its 13C natural abundance (with rhizal taxa (Russulaceae, Thelephoraceae 25% of its C originating from photosynthesis; and Sebacinaceae; Ogura‐Tsujita et al., 2012). Zimmer et al., 2008; see next section). Indeed, In all, the presence of ectomycorrhizal whereas plastidial genomes show deletions in fungi and the 13C enrichment in mixotrophic other Corallorhiza species, they look intact in orchids suggests that surrounding trees are C. trifida (Freudenstein and Doyle, 1994). To the ultimate C source of mixotrophic orchids our knowledge, the sole available evidence (Figure 25.1), although this still requires for mixotrophy in tropical orchids, based on direct demonstration (e.g., by labeling isotopic data, is for the Cheirostylis montana experiments). It is even considered that from Thailand (tribe Cranichideae; Roy et al., availability of ectomycorrhizal fungi could 2009), but mixotrophy may await discovery be a limitation for mixotrophic orchids in more terrestrial tropical orchids. (Liebel et al., 2010). The necessary link to surrounding trees gives a posteriori explana­ tions for experiences carried out by Sadovsky 25.3 the use (1965) at a time when European protection of photosynthetic and fungal laws allowed destructive manipulation of C sources in mixotrophic orchids. In repeated attempts to transplant orchids various orchids, he listed some species that did not survive the process. The list 25.3.1 Impaired photosynthesis interestingly mixes full mycoheterotrophs in mixotrophic orchids (i.e., Neottia nidus‐avis) and mixotrophic Investigations on photosynthetic apparatus species (Cephalanthera, Limodorum and and in situ gas exchanges also support

Epipactis spp.), suggesting that, in both cases, m­ixotrophic nutrition. CO2 exchanges in 456 Molecular mycorrhizal symbiosis

Cephalantera damasonium revealed that albi­ differs. Gas exchange measurements and nos were full heterotrophs, while green CO2 labeling provide snapshot information individuals exhibited a normal photosyn­ about photosynthetic activity, while stable thetic response to light (Julou et al., 2005; isotope natural abundance data integrate Roy et al., 2013; Gonneau et al., 2014). After the sources of C gain over the entire life 13 in situ CO2 labeling, C. damasonium showed history­ of a tissue. This difference may sub‐normal, slightly reduced CO2 assimila­ explain the diverging views on C. trifida, as tion (Cameron et al., 2009) while, in con­ discussed above. trast, C. trifida showed nearly no C A main use of stable isotopes is to quan­ assimilation, close to the mycoheterotrophic tify heterotrophy level (Figure 25.1) thanks control (N. nidus‐avis). This was congruent to a linear mixing model (Phillips and Gregg, with chlorophyll content and fluorescence 2001). Briefly, the 13C abundance in mixo­ values (a proxy for proper assembly of trophs is considered as a mix of a proportion photosystems) that were respectively sub‐ x of fungal C and (1 – x) of photosynthetic C. normal, versus very reduced for photosyn­ Reference values for auto‐ and mycohetero­ thesis in these two species (Julou et al., 2005; trophic biomass can be derived from 13C Zimmer et al., 2008; Cameron et al., 2009). abundance of surrounding autotrophs and Intrinsic photosynthetic limitations also mycoheterotrophs, although some interspe­ exist in Limodorum abortivum, where photo­ cific variability may exist between 13C abun­ synthesis does not compensate respiration, dances of co‐occurring MH species (Zimmer even in full light (Girlanda et al., 2006). et al., 2007; Motomura et al., 2010; Gonneau Investigating green, variegated and et al., 2014), so that albinos (Figure 25.2) are albino individuals of C. damasonium, Stöckel ideal conspecific references for mycohetero­ et al. (2011) found an inverse linear rela­ trophic biomass. Not unexpectedly, such tionship between leaf chlorophyll concen­ analyses revealed a continuum from auto­ trations and the proportion of photosynthetic trophy to full mycoheterotrophy (Hynson C in leaves. In some C. damasonium popula­ et al., 2013a). tions, there is evidence that low light con­ Isotopic approaches also revealed the ditions force the plant to live near the plasticity of mixotrophy. First, from one site compensation point (where photosynthesis to another, for the well‐studied C. damaso- counterbalances respiration; Julou et al., nium, heterotrophy level ranges from 20% 2005). Thus, both intrinsic and environmental to 85% (Gebauer and Meyer, 2003). A com­ factors drive mixotrophy, depending on parison in various light environments species and sites. showed that low light levels result in stronger mycoheterotrophy, while high irra­ 25.3.2 Environmental diances drive the plant more towards auto­ and developmental plasticity trophy, both in C. damasonium (Preiss et al., of mixotrophy 2010) and in Epipactis fibri (Gonneau et al., Measurement of photosynthetic activity 2014). In Epipactis helleborine, shading exper­ approaches and stable isotope natural iments increased the proportion of fungal C abundances are powerful tools for investi­ in shoot biomass (Gonneau et al., 2014). gating mixotrophic metabolism. However, Since we have no quantified C budget for the information gained from these techniques mixotrophs, however, we ignore whether Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 457

13C enrichment in more shaded plants fruits mostly rely on photosynthetic C reflects a compensation for reduced photo­ (Gonneau et al., 2014; Figure 25.4). Indeed, synthesis by increased recovery of fungal C, the requirement of fruits for photosynthesis or simply a lower dilution of a fixed amount may explain the albinos’ lower fitness (see of fungal C in a lower input of photosyn­ below). This model also gives a new raison thetic C. Indeed, many deficiencies observed d’être to the dormancy often observed in in albinos suggested a C deficit (Roy et al., these perennial plants, since “dormant” 2013) whose basal metabolism is three times r­hizomes can indeed receive fungal C lower than that of green individuals (Julou (Shefferson, 2009; Gonneau et al., 2014; et al., 2005), so that fungal C may not fully Shefferson et al., 2016; Figure 25.4). In a compensate for photosynthesis. Second, rare experiment manipulating the fungus, in shoot mixotrophy may vary over the season, situ fungicide application on Limodorum abor- at least under high irradiances; in Neottieae, tivum at flowering time did not change the heterotrophy level increases from 80% to fruit set (Bellino et al., 2014). Interestingly, 100% at shoot emergence from soil to about concentrations of photosynthetic pigments 20% at fruiting time (Roy et al., 2013; were enhanced in fruits, and 13C abundances Gonneau et al., 2014). At this time, the pho­ suggested that photosynthesis was indeed tosynthesis of expanded leaves, whose spe­ enhanced as a compensation. cific efficiency is enhanced along the growth Mixotrophy in orchids is, thus, a flexible season (Roy et al., 2013), takes over the C nutritional mode, driven by light and furniture. developmental stage. However, all of the Third, 13C abundances support that dif­ abovementioned data were obtained from ferent organs display variable amount of Neottieae species, and replicate analyses in fungal C. While rhizomes, young emerging other phylogenetic frameworks are required shoots and roots mainly rely on fungal C, to challenge the current model (Figure 25.4).

AUTOTROPHY (PLANT Green PHOTOSYNTHESIS) leaf Fruit

Fitness Fungal carbon by seeds (at sprouting and dormancy?) Stem Photosynthates (at owering & fruiting) Fungal C (at fruiting) Rhizome Roots Fitness by survivals MYCOHETEROTROPHY (FUNGAL CARBON) FUNGUS

Figure 25.4 A model of mixotrophic orchids use of fungal C (black) versus photosynthates (grey), in different seasons. This model, based on data from Neottieae orchids, is modified from Gonneau et al. (2014). 458 Molecular mycorrhizal symbiosis

There are some caveats with isotopic Roy, 2009; Liebel et al., 2010; Roy et al., methods. First, they do not take into account 2013), a trend also featuring mycohetero­ catabolism that may not mix the two C trophs (Trudell et al., 2003; Figure 25.2). sources in a similar ratio (although in C. It suggests a particular pathway of N acquisi­ damasonium at least, 13C abundances do not tion in mixo‐ and mycoheterotrophs that significantly differ in biomass and catabolism; differs from the usual one in autotrophs. Roy et al., 2013). Second, the contribution of Mixotrophs tend to have a lower 15N abun­ 3 recycled, C‐depleted respiratory CO2 may dance and N/C ratio than mycohetero­ be relatively more important in mixotrophs, trophs, again supporting here a trophic due to a lower total need for CO2. Third, strategy intermediate between auto‐ and mixotrophic species are likely to have lower mycoheterotrophy. photosynthesis rates, resulting in better Some tropical mycoheterotrophic 13 equilibration of CO2 concentration between orchids associate with saprotrophic fungi environmental air and stomatal chamber such as Coprinus and Psathyrella (Ogura‐ and, thus, increased 13C discrimination Tsujita et al., 2009; Martos et al., 2009; (= lower 13C abundance in photosynthetic C). Selosse et al., 2010): is there some evidence The two latter factors could entail lower 13C of mixotrophy associated with such fungi? abundances in photosynthetic C than in Since saprotrophic fungi should provide a s­urrounding autotrophs, leading to overesti­ high 13C, low 15N organic matter (Figure 25.2; mation of autotrophy level when using the Martos et al., 2009), isotopic analyses could later as references. As a more general result, test for this. Recently, the green orchid several species with “normal” 13C abundance Cremastora appendiculata (tribe Cypripedieae), may be “cryptic” mixotrophs, undetectable living in dark and wet forests and asso­ by isotopic methods. ciated with saprotrophic Coprinellus spp. (Psathyrellaceae), was claimed to be mixo­ 25.3.3 are ectomycorrhizal trophic (Yagame et al., 2013), but direct fungi the only C providers demonstration remains pending until iso­ of mixotrophic orchid? topic data is available. More generally, one The C source of mixotrophic orchids is, thus, may recommend more investigations of the mycorrhizal network linking auto­ tropical ecosystems, where saprotrophs‐ trophic hosts to mycoheterotrophs, although associated orchids are likely to be more one may deplore the absence of labeling f­requent (Selosse et al., 2010). experiments that directly demonstrate the C The question of a potential mixotrophy is transfers from autotrophic plant to mixo­ still unclear for the vast majority of rhizocto­ trophic orchids (see, however, McKendrick nias‐associated orchids which, classically, et al., 2000) for the possibly mixotrophic are considered as autotrophs that reward C. trifida). However, two indirect biochemical their fungus at adulthood (Rasmussen, evidences support that mixotrophs feed on 1995; Cameron et al., 2008; Field et al., ectomycorrhizal fungi: both 15N abundance 2015). Indeed, although they support myco­ and N/C ratio are high in mixotrophic heterotrophic germination of most orchids, orchids, compared with surrounding auto­ rhizoctonias have never so far been found trophs (Gebauer and Meyer, 2003; Abadie to be mycorrhizal in adult mycohetero­ et al., 2006; Tedersoo et al., 2007; Selosse and trophic orchids (with the exception of some Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 459

rare rhizoctonia lineages that secondarily (Tello et al., 2014; see Selosse and Martos, evolved ectomycorrhizal abilities; e.g., 2014, for a discussion). In fact, mycohetero­ Yagame et al., 2012). trophic germinating orchid seedlings do not Yet, many rhizoctonias‐associated strongly differ from autotrophic adults for orchids display higher 15N abundance and 13C abundance (Stöckel et al., 2014) so, lim­ N/C ratio than surrounding autotrophs (e.g., ited amounts of C obtained from rhizocto­ Girlanda et al., 2011), and even slight enrich­ nias would not drastically modify orchids’ ments in 13C, such as Barlia robertsiana isotopic signature. (tribe Ophrydeae), Habenaria tridactylites, Elaborating on the newly‐found ecology Anacamptis laxiflora and Orchis purpurea (tribe of rhizoctonias and their supposed isotopic Orchideae) (Liebel et al., 2010; Girlanda abundances, Selosse and Martos (2014) pro­ et al., 2011). Does this mean a low level of posed that some rhizoctonias‐associated mycoheterotrophy in at least some rhizocto­ orchids could be mixotrophic. While the nias‐associated species? Indeed, 13C abun­ impact would be most often not noticeable dances of rhizoctonias have hitherto been on 13C abundances, it may explain why unknown, mainly because their fruit bodies some species display slightly higher (see are inconspicuous or absent, so that their above) or lower (in some Orchideae such as biomass is difficult to sample. A first expla­ Goodyera spp.; Hynson et al., 2009a; Liebel nation may be a low C input from the rare et al., 2010; Hynson et al., 2013a; Johansson ectomycorrhizal fungi that sporadically et al., 2015) 13C abundances than in sur­ colonize rhizoctonias‐associated orchids, rounding autotrophs. Under this view, all as observed in the genera Cypripedium adult green orchids would be predisposed to (Shefferson et al., 2005, 2007), Gymnadenia mixotrophy by their mycoheterotrophic (Stark et al., 2009), Orchis (Lievens et al., germination. Labeling experiment are now 2010) or Cryptostylis (Sommer et al., 2012). awaited to support this speculation for A second, more speculative explanation rhizoctonias‐associated orchids; since these arose recently. orchids transfer C to their fungus in some It turns out that rhizoctonias are also cases (Cameron et al., 2008), the net C transfer endophytes in roots of non‐orchid plants. also requires to be established (Selosse and Beyond a large repertoire of secreted Martos, 2014). enzymes allowing saprotrophy, their genome bears traits typical for biotrophy, such as small, secreted effector‐like proteins, 25.4 Mixotrophy in Ericaceae and regression of some metabolic pathways (Zuccaro et al., 2011; Kohler et al., 2015). 25.4.1 pyroloids, ericaceous Environmental data now confirm the plants with a link endophytic­ nature of some Tulasnellaceae to mycoheterotrophy (Girlanda et al., 2011), many Cerato­ Orchids were main players in the discovery basidiaceae (Veldre et al., 2013) and most of mixotrophy, but this nutrition is also pre­ Sebacinales (Selosse et al., 2009; Weiß et al., sent in pyroloids, species from the Pyroleae 2011). There is some evidence that 13C sub‐tribe of Ericaceae. Pyroloids are common, abundance in endophytes is very variable, small, shade‐tolerant perennial shrubs from but often close to that of autotrophic plants temperate, alpine and boreal forests of the 460 Molecular mycorrhizal symbiosis

Northern hemisphere. They are candidate specific association with ectomycorrhizal mixotrophs for three reasons. First, a close fungi supports early mycoheterotrophic phylogenetic relationship links Pyroleae development (Hashimoto et al., 2012; with two other mycoheterotrophic tribes, Hynson et al., 2013b; Johansson et al., 2015). the Monotropeae and the Pterosporeae (all Finally, 14C labeling showed transfer from members of the Monotropoideae; Kron autotrophs to pyroloids, in dual pot cultures et al., 2002; Tedersoo et al., 2007; Matsuda of Larix kaempferi seedlings and Pyrola incar- et al., 2012), although the latter lineages nata from Japan (Hashimoto et al., 2005). represent an independent emergence of Sadly, however, this nice experiment is only heterotrophy­ (Liu et al., 2011; Braukmann published as a colloquium abstract. and Stefanovic, 2012; Lallemand et al., 2016). The pyroloid Pyrola aphylla itself is 25.4.2 Mixotrophy and its even mycoheterotrophic (Zimmer et al., plasticity in pyroloids 2007; Hynson and Bruns, 2009), although Investigation of mixotrophy by stable iso­ its recognition as a separate species, a species topes provided variable results. A first complex (Jolles and Wolfe, 2012), or simply investigation in two boreal Estonian forests an albino variant of the green P. picta remains revealed that some pyroloids had higher 13C unclear (Haber, 1987). Second, as in orchids, and 15N abundances and N/C than surround­ seeds are very small, and pyroloids undergo ing autotrophs, including autotrophic plants a mycoheterotrophic subterranean germina­ from the Ericaceae family (Tedersoo et al., tion (Christoph, 1921; Lihnell, 1942) into a 2007; Figures 25.1 and 25.2). Based on a subterranean root, supported by mycorrhi­ linear mixing model (using the Monotropeae zal fungi (Hashimoto et al., 2012). Hypopytis monotropa as baseline), 10–68% of Last, as with many other basal Ericaceae C was of fungal origin in Orthilia secunda, (Selosse et al., 2007), pyroloids form associa­ Pyrola chlorantha, P. rotundifolia and tions with ectomycorrhizal fungi, whose Chimaphila umbellata. The latter species did involvement has long been somewhat hidden not differ from autotrophs on one of the by the specific (“ectendomycorrhizal”) struc­ investigated sites, and heterotrophy level ture of the mycorrhizae. Indeed, the fungus did not correlate among sites, indicating not only forms a sheath around the pyroloids’ environmental variability, and possibly dif­ root, but also penetrates into cortical root ferent regulation for this parameter among cells, which contain hyphal coils (Khan, 1972; species. In a second set of investigations in Robertson and Robertson, 1985; Vincenot more southern European sites and in et al., 2008). Yet, barcoding of the fungal taxa California (Zimmer et al., 2007; Hynson involved revealed typical ectomycorrhizal et al., 2009b), 15N abundance and N/C values fungi that also colonize surrounding tree roots were intermediate between autotrophic and (Robertson and Robertson 1985; Tedersoo mycoheterotrophic plants for O. secunda, et al., 2007; Zimmer et al., 2007; Vincenot C. umbellata, P. chlorantha, P. minor and P. picta. et al., 2008; Hynson and Bruns, 2009; Matsuda However, based on 13C abundance, only et al., 2012), thus allowing a mycelial link to P. chlorantha showed significant C gain at a s­urrounding autotrophic trees. single low irradiance site. Recent investigations on germinating More recently, O. secunda and P. chlorantha pyroloids confirmed that a more or less from Sweden were confirmed mixotrophic, Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 461

and pyroloid seedlings displayed enrich­ for mixotrophic orchids. Moreover, some ments in 13C, as expected for mycohetero­ pyroloids often have long, metric under­ trophs (Johansson et al., 2015). Given the ground rhizomes, so that the biomass of a broad range of irradiances in investigated given aerial shoot may mobilize resources sites, light availability was unlikely to be acquired by distant other shoots, perhaps the only driver of the heterotrophy level submitted to other conditions and different (Zimmer et al., 2007). Alternatively, it was level of heterotrophy. Interestingly, P. japon- suggested that the C gain was not solely ica, in which 13C abundances respond to based on C needs, but could arise as a “side‐ light levels, consists of isolated shoots, and product” of the nitrogen and phosphorus does not form rhizomes. nutrition (Selosse and Roy, 2009), as is Further supporting physiological varia­ known in some mixotrophic algae (see tions among pyroloids, a phylogenetic anal­ below); indeed, a specific strategy to obtain ysis by Matsuda et al. (2012) suggested that fungal N in pyroloids could explain their the Pyrola + Orthilia clade may have evolved unusual 15N abundance and their disap­ mixotrophic abilities, as further supported pearance after anthropogenic N deposition by the inclusion of the mycoheterotrophic (Allen et al., 2007), and some fungal C may P. aphylla, while the Moneses + Chimaphila move along with the nitrogen. sister clade would be less prone to mixotrophy However, light level was driving the het­ (Matsuda et al., 2012). erotrophy level in two cases. For Pyrola A C gain from fungi may explain some japonica, a Japanese mixotrophic pyroloid particular ecophysiological features of pyro­ (whose mycorrhizae are devoid of fungal loids. First, P. incarnata has few reserves in mantles), 13C abundance negatively corre­ winter (Isogai et al., 2003), which could be lated with light availability (Matsuda et al., compensated if fungal C contribute to devel­ 2012), suggesting higher heterotrophy lev­ opment of new organs in spring, as described els in shaded conditions. The species prefer­ above for Neottieae orchids. Indeed, in entially associates with Russula spp., and the P. japonica, the temporal spatial patterns of rate of mycorrhizal colonization and Russula distribution of hyphal coils and starch sug­ frequency was higher in shaded conditions, gests that carbon reserves may have been perhaps compensating for low light levels. acquired after interaction of the root with In an experimental shading of C. umbellata fungi. Second, a low capacity for adjusting and P. picta (the only experimental studies vegetative growth and leaf area after shad­ hitherto carried out on mixotrophic pyro­ ing was observed in P. rotundifolia (Hunt and loids; Hynson et al., 2012), leaf sugars reflect­ Hope‐Simpson, 1990), which could also be ing the immediate C source supported a due to a buffering effect of fungal C. Finally, decrease of fungal C contribution over the the sensitivity to forest disturbances (logging, growth season in the first species, but not burning; Zimmer et al., 2007) may be due to the second. a response to factors affecting mycorrhizal The variability of response to light level networks. thus suggests that the regulation of mixotro­ However, at least some green pyroloids phy is variable among pyroloid species, so can be outplanted (e.g., Hunt and Hope‐ that only some species change C nutrition in Simpson, 1990), in sharp contrast with accordance to light availability as reported mixotrophic orchids. It can be speculated 462 Molecular mycorrhizal symbiosis that the use of fungal C may be more facul­ trophic – a mode of nutrition that allowed tative in pyroloids than in orchids, allowing the acquisition of plastids from ingested transient disconnections from mycorrhizal algae (Maruyama and Kim, 2013). network under some environmental Mixotrophy is ecologically relevant in c­onditions (especially in well‐illuminated oceans, where planktonic algae achieve up glasshouses). This may also resolve the dis­ to 95% of the bacterivory in the superficial crepancies between studies reporting varia­ layer (Zubkov and Tarran, 2008; Moorthi ble levels of heterotrophy (Tedersoo et al., et al., 2009). 2007; Zimmer et al., 2007; Hynson et al., By contrast, mixotrophy in land plant 2009b; see above), if one assumes that dif­ evolved secondarily, from a fully autotrophic ferent populations display variable levels of ancestor, and its ecological relevance is still dependency to fungal C and mycorrhizal to be estimated in terrestrial ecosystems (see network. Section 25.6 – Perspectives). Mixotrophy is However, there is, hitherto, no analysis mainly found in plants displaying close of photosynthetic apparatus and perfor­ interactions with surrounding organisms. mances in situ for pyroloids. Moreover, the Beyond mixotrophy on fungal C, making existence of a differential allocation of pho­ use of the widespread mycorrhizal symbio­ tosynthetic versus fungal resources in the sis, mixotrophy also occurs in hemiparasitic different plant organs, as described for plants (Press and Graves, 1995; Těšitel et al., mixotrophic orchids, deserves further studies 2010). The later are green plants that obtain to establish physiological comparisons of mineral nutrient and, sometimes, carbon by mixotrophy between pyroloids and orchids. parasitizing other plants. For example, the normally green Striga hermonthica can sur­ vive as albino (Press et al., 1991). Těšitel et al. 25.5 Evolution (2010; and see references therein) supported of mixotrophy that hosts provide 20–80% of the biomass in and mycoheterotrophy root hemiparasites from the Olacaceae and Orobanchaceae families, and 50–80% of the 25.5.1 the repetitive evolution biomass in stem hemiparasites from the of mixotrophy in autotrophic Loranthaceae. eukaryotes Mixotrophic lineages add to a long list of 25.5.2 Is mixotrophy autotrophic taxa displaying partial hetero­ a predisposition to the evolution trophy. Mixotrophic strategies are, indeed, of full mycoheterotrophy? ecologically and mechanistically very Mixotrophy is often viewed as an intermedi­ diverse, as exemplified below. Many inde­ ate step towards the evolution of full myco­ pendent phyla of planktonic eukaryotes heterotrophy (Bidartondo et al., 2004; are mixotrophic (Flynn et al., 2013), either Selosse et al., 2004). Mycoheterotrophic spe­ by uptake of dissolved organic matter cies are nested within mixotrophic lineages (Kamjunke and Tittel, 2009), or by phago­ in Neottiae (Abadie et al., 2006; Selosse and trophy on unicellular preys (Jones, 2000). Roy, 2009) and in the genus Cymbidium The latter process is a plesiomorphic con­ (Motomura et al., 2010; Ogura‐Tsujita et al., dition, since ancestors of plastid‐bearing 2012). The question is a bit more complex taxa are considered to have been phago­ for pyroloids; while mycoheterotrophic Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 463

P. aphylla clearly evolved within a mixo­ both the plastid and the nucleus. However, trophic clade (Liu et al., 2011; Braukmann the question of reversions from mixotrophy and Stefanovic, 2012; Matsuda et al., 2012), to full autotrophy remains pending, and the story is less clear when considering the would not be unexpected if photosynthesis relationship of pyroloids to the two myco­ genes were not lost or modified in heterotrophic basal Ericaceae tribes, m­ixotrophs. We call for more phylogenetic Monotropeae and Pterosporeae. They were analysis of some tribes where this may once considered to all belong to single sub‐ have occurred, such as the Neottieae. Here, family (the Monotropoideae; Kron et al., two groups are autotrophic, as shown by 2002; Tedersoo et al., 2007), in which myco­ survival upon transplantation (Sadovsky, heterotrophy could have arisen from mixo­ 1965), 13C abundances and association trophic ancestors. However, the monophyly mainly to rhizoctonias: the Epipactis palustris‐ of Monotropoideae is now challenged (Liu gigantea clade (Bidartondo et al., 2004; et al., 2011; Braukmann and Stefanovic, Zimmer et al., 2007; Illyés et al., 2009) and 2012), and the existence of mixotrophic some basal green Neottia species (= the for­ relatives to Monotropeae and Pterosporeae mer genus Listera; Oja et al., 2015; Těšitelová is now uncertain. et al., 2015). Although Neottieae phylogeny Undoubtedly, in both orchids and pyro­ and evolution of nutritional traits remain loids, the mycoheterotrophic germination of poorly resolved, these groups are unlikely to seedlings (very probably an ancestral trait in be basal (Pridgeon et al., 2008; Roy et al., these taxa) was linked to the acquisition of 2009). Thus, either their autotrophy is mixotrophy. One main question is whether ancestral (and mixotrophy arose several mixotrophic plants without mycohetero­ times in Neottieae), or the Neottieae trophic germination exist (and the answer ancestor was already mixotrophic, and they may come from other mixotrophic taxa, see represent reversion to autotrophy. More Section 25.6 – Perspectives). resolved phylogenies, but also analyses of One ecological process may have repeat­ plastid integrity and functioning in mixo­ edly selected for increasing heterotrophy trophs, is now required. levels in mixotrophs. As herbs or small More generally, mixotrophs should now shrubs, mixotrophs typically grow in young enter the area of genomics, transcriptomics forests with loose canopy. Such places tend and metabolomics, with special attention to to become darker over times, with canopy albinos (see below), to better understand closing due to ongoing plant ecological (i) their metabolism, (ii) how they evolved s­uccession. Thus, mixotrophs undergo from autotrophs, and (iii) how they open the increasing shading over their lifespan, which door to the evolution of mycoheterotrophy. selects for more light‐independent C supply (Selosse and Roy, 2009). Indeed, such a 25.5.3 the difficult evolutionary selective pressure may explain the conver­ transition from mixotrophy gent evolution to full mycoheterotrophy to full mycoheterotrophy observed at least four times within the The transition between mixotrophy and Neottieae (Roy et al., 2009). mycoheterotrophy was investigated in Full mycoheterotrophy was never shown Neottieae, where albinos (Figure 25.3) can to be evolutionary reversible, maybe due to be viewed as intermediate steps. Yet, albinos irreversible loss of photosynthetic genes in are often unfit (Salmia, 1989; but see Julou 464 Molecular mycorrhizal symbiosis et al., 2005) and rare in natural populations mycoheterotrophs within similar genetic (Tranchida‐Lombardo et al., 2010). This backgrounds. Moreover, they can be used as prompted some works to understand why ecological equivalents to mutants in genetic they failed the transition. Roy et al. (2013) studies; their dysfunctions suggest a contrario carried out a detailed phenotypic comparison what makes mycoheterotrophy successful. between albinos and co‐occurring green Elaborating on this, Roy et al. (2013) sug­ individuals. Albinos displayed: gested that a successful mycoheterotrophy (i) more frequent shoot drying at fruiting, would require at least: possibly due to stomatal dysfunctions; (i) degeneration of leaves and stomata that (ii) lower basal metabolism (as seen from are no longer necessary but enhance respiration in the dark); water loss, especially in albinos, due to (iii) increased sensitivity to pathogens and altered regulation of water exchanges herbivores; in stomata that lack functional plastids; (iv) higher dormancy and maladapted (ii) optimization of the temporal pattern sprouting and, probably due to the of fungal colonization and/or reserve p­revious differences; formation to ensure C resources at time (v) fewer seeds per fruits, with lower ger­ of fruiting; mination capacity (Roy et al., 2013). (iii) restriction of shoot sprouting to It was later supported (see above) that, at only years of reproduction, since purely time of fruiting, the low fungal colonization vegetative shoots, which provide C to and the requirement of photosynthesis for mixotrophs, are useless in mycohetero­ aerial organs (Figure 25.4) may be causal trophs; and for the abovementioned trends in albinos (iv) new defenses against pathogens and (Bellino et al., 2014; Gonneau et al., 2014). herbivores, because of the abolition of Whatever the cause, albinos have a 103‐fold mimetism with surrounding vegetation reduction of their fitness, and thus fail a after chlorophyll loss, and of the s­uccessful transition to mycoheterotrophy a­ppetence of N‐rich mycoheterotrophic because some traits inherited from their tissues. recent green ancestors are maladaptive (Roy Successful transition to mycoheterotro­ et al., 2013). There is evidence that, in some phy is thus likely to require progressive and populations, their fitness is higher (e.g., joint evolution of many traits, whereas a Julou et al., 2005), so that some environ­ sudden loss of photosynthesis in mixotrophs ments may support them better (Roy et al., leads to unfit plants. Two major predictions 2013; Shefferson et al., 2016). However, can be proposed. First, if the final transition they remain rare everywhere, and the tran­ to mycoheterotrophy occurs in an albino sition to heterotrophy is more complex than individual, it probably belongs to a mixotro­ simple loss of function in a mixotroph. phy species that already evolved advanced Albinos represent snapshots of failed independence to light and many other associ­ transitions from mixotrophy to mycohetero­ ated traits. Second, this supports an evolu­ trophy. Although the determinism of their tionary metastability of mixotrophy – a strong albinism, genetic or environmental, remains dependence to light (especially for seeds pro­ unknown, albinos offer fascinating models duction; Figure 25.4) prevents the conver­ for comparing the physiology of mixo‐ and sion of mixotrophs into full heterotroph. Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 465

Ecologically, this may limit the emergence of Indeed, works investigating candidate pure C sinks in mycorrhizal networks. Glomeromycetes‐associated mixotrophs found that isotopic differences with auto­ trophs are low, or even absent, except some­ 25.6 Perspectives times for 15N (Cameron and Bolin, 2010; Merckx et al., 2010; Field et al., 2015), so 25.6.1 Finding more mixotrophic that no firm conclusion can be derived from lineages: the pending question these studies. The green fern Ophioglossum of arbuscular mycorrhizal vulgatum, whose spores undergo mycohet­ mixotrophs erotrophic germination, was submitted to an The existence of mixotrophy based on experimental tracing of nutrient flow, and arbuscular mycorrhizal fungi (Glomero­ was shown to provide C to the Glomero­ mycetes) is still unclear, although the domi­ mycetes associated to its root (Field et al., nance of this mycorrhizal symbiosis in many 2015), but the direction of net transfer was ecosystems would make it ecologically not established. Finally, green species of the r­elevant. Candidate green species exist in Burmanniaceae family, suspected of mixo­ several taxa that also display mycohetero­ trophy because the family abounds in trophic lineages, such as the Burmanniaceae, m­ycoheterotrophic species, can be cultivated Gentianaceae, Dioscoreales, Polygalaceae, alone (Merckx et al., 2010), suggesting that Iridaceae, Pandanales or Petrosaviaceae their mixotrophy on Glomeromycetes is not (Selosse and Roy, 2009). They should be necessary. especially (but not only) searched for among Thus, there is no conclusive work on the species with very small seeds, which existence of mixotrophy in Glomeromycetes‐ likely undergo the same mycoheterotrophic associated plants. This add up to a contro­ g­ermination as pyroloids and orchids. versy about the ability of Glomeromycetes Unfortunately, 13C and 15N abundances to transfer C to host plants. On the one that allowed discovery and study of mixo­ hand, some isotopic labeling demonstrated trophy in orchids and pyroloids are of lim­ C transfer between plants by way of ited help when considering Glomeromycetes. Glomeromycetes – for instance, between Mycoheterotrophic plants associated with understorey herbs and overstorey tree (Lerat Glomeromycetes have isotopic signature et al., 2002). However, on the other hand, in similar to that of surrounding autotrophs many studies, the transferred C stays in the (Merckx et al., 2010; Courty et al., 2011). roots of receiving plants, most likely in the This is probably because: fungus (Fitter et al., 1999; Lekberg et al., (i) the N nutrition in these mycohetero­ 2010). However, Glomeromycetes‐associated trophs follows pathways similar to mycoheterotrophs show that the transfer of autotrophs (indeed, the N contents are C from fungi to plant is definitively possible. also similar; Merckx et al., 2010; Courty Thus, the hunt for Glomeromycetes‐associ­ et al., 2011), and ated mixotrophs still deserves attention, and (ii) Glomeromycetes do strongly differ in experiments should go beyond fungal C 13C abundances from the autotrophs on labeling, in order to establish the direction which they are mycorrhizal (Courty of net transfer and the net contribution of et al., 2015). fungal C to the plant C budget. 466 Molecular mycorrhizal symbiosis

25.6.2 elucidating the parasitic fungi, insufficient to launch the arms race nature of mixotrophy and the emergence of specificity. Finally, the perplexing question of the The question of the impact on partners is impact of mixotrophy on mycorrhizal also extremely relevant at community level; fungi, and beyond them on C‐donating how mixotrophs modify surrounding plant surrounding plants, remains unclear. Indeed, and fungal communities is still not known. this applies to mycoheterotrophs as well. This open question is relevant, at least in Although such plants are often presented as boreal forest, where mixotrophs can domi­ epiparasites or cheaters on the mycorrhizal nate the understorey (Tedersoo et al., 2007), symbiosis (Douglas, 2008), we are not aware and is not assessed for full mycohetero­ of rigorous demonstration for this (Selosse trophs either. It is well recognized that and Roy, 2009); vitamins, hormones, or hemiparasitic plants exert both deleterious shelter effects could, at least theoretically, and indirectly facilitative effects on the sur­ compensate for the C costs. rounding plants (Watson, 2009), contribut­ Investigations comparing fitness of C‐ ing to the shaping of plant communities. donating plants and fungi with, or without, Depending on whether their targets are par­ C‐receiving plants, are crucially lacking, asitized or rewarded, and whether these probably because the estimation of fitness, ­targets are dominant or rare species, the especially for the fungi, is difficult. If parasit­ outcome on fungal and plant biodiversity of ism occurs, the mechanisms that maintain mixotrophs can be different – they may the interaction while a poorly rewarding favor or disfavor dominant species. It may host, leading to interaction abortion on be argued that the rather small mixotrophs the fungal side (Kiers et al., 2011), require have limited impact on large adult trees extreme attention, because mixo‐ and myco­ surrounding them. However, the way they heterotrophs may abuse the usual pathway influence the regenerating seedlings, for partners’ reconnaissance and dialog. which are likely to be more sensitive to C One feature potentially linked to para­ loss, can alter the canopy community after sitism – specificity – deserves more atten­ some time. tion. While mycoheterotrophic are often Mixo‐ and mycoheterotrophic plants specific in their fungal associations, despite offer a fascinating, newly and fully open exceptions, mixotrophs are much less often research area, demonstrating the power of specific (for comparisons in similar phylo­ mycorrhizal networks in shaping plant evo­ genetic background, see Ogura‐Tsujita et al., lution. After the first decade, where barcod­ 2012; Yamato et al., 2014; Těšitelová et al., ing of the fungi and stable isotope analyses 2015). The specificity of mycoheterotrophs flourished, we hope that the study of mixo­ can be viewed as a hallmark of parasit­ trophic nutrition will expand by integrating ism – that is, the result of an evolutionary new tools issuing from –omic technologies, arms race between the parasitic plant and to unravel more of the physiological process, its fungal prey, entailing specialization to on the one hand, and to allow the emer­ avoid fungal defenses. Under this hypothe­ gence of an integrative ecological view of sis, the lower specificity of mixotrophs community biology and biochemical cycles would suggest a lower parasitic load on on the other hand. Chapter 25: Mixotrophy in mycorrhizal plants: Extracting carbon from mycorrhizal networks 467

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