Spotlight Do chlorophyllous orchids heterotrophically use mycorrhizal fungal carbon? 1 2 Marc-Andre´ Selosse and Florent Martos 1 Muse´ um national d’Histoire naturelle, De´ partement Syste´ matique et Evolution (UMR 7205 ISYEB), CP 50, 45 rue Buffon, 75005 Paris, France 2 School of Life Sciences, University of KwaZulu-Natal, Private Bag X01 Scottsville, Pietermaritzburg 3209, South Africa The roots of orchids associate with mycorrhizal fungi, intermediate between those of achlorophyllous orchids the rhizoctonias, which are considered to exchange min- and autotrophic plants [3], which is expected because they eral nutrients against plant carbon. The recent discovery mix both nutritional strategies. that rhizoctonias grow endophytically in non-orchid Beyond these exceptions, however, the vast majority of plants raises the possibility that they provide carbon orchids associate with rhizoctonias and, in adulthood, dis- 13 to orchids, explaining why some orchids differ in isoto- play a C abundance almost similar to that of autotrophic pic abundances from autotrophic plants. plants (Figure 1) [1,3], in spite of some small deviations (see below). It is thus currently assumed that rhizoctonia-asso- Orchids display several evolutionary innovations, including ciated orchids are autotrophic in adulthood, and do not use a mycorrhizal symbiosis with particular fungal lineages. rhizoctonias C after germination (e.g., [4]). These fungi, hitherto considered to be soil saprotrophs, are But these assumptions are challenged by two observa- collectively called the rhizoctonias [1]. One characteristic tions. First, rhizoctonia-associated orchids often display 15 feature of this symbiosis is symbiotic germination, where unusually high nitrogen (N) and N content compared to rhizoctonias colonize the reserveless orchid seeds and pro- other autotrophic plants [3,5]. These features are observed vide carbon (C) to the seedlings that grow heterotrophically to a greater extent in the fully or partially heterotrophic [1]. After the development of green leaves, adult orchids orchids that obtain organic matter from their mycorrhizal acquire autotrophy, and mycorrhizal fungi are thought to fungi [3,4], likely because (i) their pathway for gaining N receive C as a reward for providing mineral nutrients [2], as differs from that in autotrophic plants and (ii) the fungi have 15 in all other mycorrhizal symbioses [1]. However, recent high N and N contents. A gain of fungal organic matter in works suggest the reverse mechanism might also take rhizoctonia-associated orchids could thus explain their high 15 place, and that fungal C could actually be provided by the N and N content. Second, a recent paper reports that fungi to adult chlorophyllous orchids. seedlings of two rhizoctonia-associated orchids, which het- erotrophically recover C from their rhizoctonias, display 13 Orchids that use and abuse mycorrhizal fungal C lower C abundances than mycoheterotrophic orchids, al- A dependence on fungal C in adult orchids has been shown though these abundances are somewhat above those of for a small number of forest species that are partially or fully autotrophic plants [5]. Thus, C transferred from rhizocto- photosynthetic and display two particular features. First, nias to orchid protocorms is isotopically closer to C acquired their mycorrhizal partners have changed from the usual through photosynthesis, and small C transfers may be rhizoctonias to other fungal lineages: these orchids now difficult to detect. It is still ignored whether this reflects a associate with fungi, mostly from Basidiomycota, that either specific fractionation during rhizoctonia–to–orchid transfer, form mycorrhizae on surrounding trees (the so-called ecto- or if, as for fully or partially heterotrophic orchids mentioned 13 mycorrhizal fungi) [3] or live as litter- or wood-decaying previously, this simply reflects the C abundance of the saprotrophs [4]. Second, these orchids display much higher fungal source without fractionation. We speculate that the 13 C abundance than the C3 plants that surround them. The second explanation is likely, because an overlooked ecologi- 13 achlorophyllous orchids that obtain their entire C from their cal niche of rhizoctonias indeed predicts C abundance close 13 mycorrhizal fungi (mycoheterotrophic orchids) have a C to that of autotrophic plants. abundance identical to that of the saprotrophic or ectomy- corrhizal fungi that feed them (Figure 1), which are them- Rhizoctonias revisited 13 selves C-enriched compared with autotrophic plants There is evidence that rhizoctonias do not exclusively [3,4]. Moreover, the green orchids that are partially depend on orchid mycorrhizae for their C nutrition, but 13 heterotrophic (mixotrophic orchids) have a C content have their own, independent nutritional niche. Their saprotrophy is only indirectly suggested by the observation of in vitro growth on dead and sometimes complex organic Corresponding author: Selosse, M.-A. ([email protected]). matter [1], but there is no direct evidence under natural Keywords: root endophytic fungi; mixotrophy; mycoheterotrophy; orchid mycorrhizal 13 conditions. Strikingly, the C abundance observed in ger- symbiosis; stable isotope abundances; rhizoctonias. 13 minating seedlings [5] does not fit the high C abundance 1360-1385/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.09.005 expected for saprotrophic fungi (Figure 1) [4]. What then is the real ecological niche of rhizoctonias? Trends in Plant Science, November 2014, Vol. 19, No. 11 683 Spotlight Trends in Plant Science November 2014, Vol. 19, No. 11 (A) Green orchids Orchids associated with ectomycorrhizal fungi: Orchids associated associated with with parally heterotrophic fully heterotrophic saprotrophic fungi rhizoctonias (green, mixotrophic) (achlorophyllous) (achlorophyllous) ? δ13 C (B) C3 photosynthates Endophyc fungi, including rhizoctonias, Ectomycorrhizal Saprotrophic under the hypotheses of the present paper fungi fungi Key: 1 ‰ in δ13C TRENDS in Plant Science 13 13 Figure 1. Relative spontaneous C abundance for the main nutritional types of orchids (A) and their respective mycorrhizal fungi (B), distributed along increasing d C values. The vertical grey lines indicate trophic links, that is, exploitation of a C source: orchids may either rely on a single source or mix two sources. Fully heterotrophic 13 (mycoheterotrophic) orchids are largely enriched in C: they rely on C provided either by fungi that are ectomycorrhizal with surrounding tree roots (light brown) or by saprotrophic fungi (brown) that are even more enriched. Mixotrophic orchids (in yellow green) display intermediate enrichments: they use both C from ectomycorrhizal fungi and from their own photosynthesis. On the very left, green orchids (green) associated with rhizoctonias include the vast majority of species and are often considered 13 purely autotrophic in adulthood: they display C enrichments close to those of C3 autotrophs (blue-green range), but sometimes with a small excess or deficit. We propose 13 that the newly emerging ecological niche for rhizoctonias (blue) predicts similar C enrichments in their biomass: thus, a partial use of C from rhizoctonias (question mark) 13 13 would explain the large range of C abundances in rhizoctonia-associated orchids. Absolute values of C enrichments are not given on the diagram, because they vary according to environmental conditions, but an order of magnitude of the relative difference (expressed in %) is given in the key. Rhizoctonias belong to three independent Basidiomy- Hidden C flow from rhizoctonias to green adult orchids? cota lineages, Tulasnellaceae [1] and Ceratobasidiaceae [6] It is difficult to sample mycelia of endophytic fungi to 13 (two families of the order Cantharellales which contains assess their C abundance. However, fungi producing the chanterelle mushrooms), and Sebacinales [7]. Endo- fleshy fruitbodies, which allow analysis of isotopic abun- phytism, that is, diffuse growth within living plant tissues, dances, recently turned out to have endophytic abilities, without apparent infection symptoms or symbiotic organs such as for instance in the genus Hygrocybe [8]. Interest- 13 such as mycorrhizae, is common among fungi. Endophytic ingly, these fruitbodies reveal variable C abundances, growth in the roots of non-orchid plants now turns out to be which are rather close (equivalent, slightly lower or slight- a frequent and ancestral ability of Ceratobasidiaceae [6] ly higher) to those of autotrophic plants (Figure 1; see [8] and Sebacinales [7]. Tulasnellaceae, the most common and references therein). At first glance, this may seem orchid mycorrhizal lineage, is often missing in molecular unexpected because the ectomycorrhizal fungi, which re- 13 studies of fungal communities, which usually rely on bar- ceive C from tree photosynthesis, are enriched in C coding with the fungal ribosomal DNA (rDNA): because of (Figure 1), likely thanks to a fractionation at the root– their unusual rDNA sequences, Tulasnellaceae cannot all fungus interface. However, another mycorrhizal fungal be detected by general fungal-specific PCR primers. More group, the strictly biotrophic Glomeromycota forming environmental studies focusing on the overlooked Tulas- arbuscular mycorrhizae in many land plants [1], displays 13 nellaceae are needed, for example, using the available variable C abundances that are close to or even below Tulasnellaceae-specific rDNA primers (e.g., [4,5]), to sup- those