Content uploaded by Thierry Pailler
All content in this area was uploaded by Thierry Pailler
Content may be subject to copyright.
www.landesbioscience.com Plant Signaling & Behavior 1
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Plant Signaling & Behavior 5:4, 1-5; April 2010; © 2010 Landes Bioscience
Considerable advances were recently made in the ecology of
achlorophyllous, heterotrophic plants that obtain carbon from
their mycorrhizal fungi (Fig. 1). Most plants have contact with
soil through mycorrhizal symbioses, in which roots associate with
a suitable fungal partner. Fungi utilize soil mineral nutrients, and
while sharing them with host plants, they generally receive carbon
as a reward. In contrast, some achlorophyllous plants living in the
shaded forest understorey have reversed the process. They receive
carbon from their mycorrhizal fungi exclusively, hence the desig-
nation ‘mycoheterotrophic’ (MH) plants.1 Mycoheterotrophy has
appeared several times during the evolution of land plants, and
more than 20 times among orchids that encompass half of all
MH species.2 In the last decade, the development of molecular
tools has enabled researchers to identify many fungal symbionts,
which are often uncultivable. The fungi occurring in the densely
colonized roots of MH species often produce a stronger PCR
*Correspondence to: Marc-André Selosse; Email: ma .firstname.lastname@example.org
Submitted: 11/25/09; Accepted: 11/25/09
Previously published online:
Mycoheterotrophic plants are achlorophyllous plants that
obtain carbon from their mycorrhizal fungi. They are usually
considered to associate with fungi that are (1) specic of each
mycoheterotrophic species and (2) mycorrhizal on surrounding
green plants, which are the ultimate carbon source of the entire
system. Here we review recent works revealing that some my-
coheterotrophic plants are not fungal-specic, and that some
mycoheterotrophic orchids associate with saprophytic fungi. A
re-examination of earlier data suggests that lower specicity
may be less rare than supposed in mycoheterotrophic plants.
Association between mycoheterotrophic orchids and sapro-
phytic fungi arose several times in the evolution of the two
partners. We speculate that this indirectly illustrates why tran-
sition from saprotrophy to mycorrhizal status is common in
fungal evolution. Moreover, some unexpected fungi occasional-
ly encountered in plant roots should not be discounted as ‘mo-
lecular scraps’, since these facultatively biotrophic encounters
may evolve into mycorrhizal symbionts in some other plants.
signal than any fungal contaminant, making molecular tools
very effective for this ﬁeld of study.
Unexpectedly Diverse Fungal Partners
Although most MH species occur in tropical region, most
MH plants currently investigated are from northern latitudes.
In temperate MH plants, two main features were consistently
found: high speciﬁcity and indirect below ground connection
with nearby autotrophic plants.1,3 Regarding speciﬁcity, each of
these temperate MH species only associate with a narrow range
of closely related fungal taxa, and these taxa differ among MH
species. High speciﬁcity is often assumed as an outcome of an
arms race between coevolving fungi and MH plants; each MH
species successfully exploits only one fungus, similar to special-
ized parasites (epiparasites4). Fungal partners of temperate MH
species also form mycorrhizae with nearby autotrophic trees that
represent the ultimate carbon source of the entire system.5 Direct
evidence of a below-ground connection between a MH plant and
surrounding trees was obtained by labelling photosynthates.6
In most studies, indirect evidence was provided by using stable
isotopes;7 using 13C, natural abundances in MH plants were
found to be similar to that of their mycorrhizal fungi but higher
than that of nearby autotrophic plants and using 15 N, natural
abundances were found to increase from fungi to MH plants, as
is expected along trophic chains.
There are older as well as more recent reports on associations
between Asiatic tropical MH orchids and saprotrophic fungi,
which were mainly identiﬁed by using in vitro isolation,8,9 a tech-
nique that often selects saprotrophic contaminants. Some MH
orchids living in wet forests in Asia and Australia have been pre-
sumed to be associated with litter decaying fungi10, 11 (reviewed
in ref. 3). More recently, saprotrophic fungi were identiﬁed in
situ using molecular tools on colonized roots or rhizomes. In
the Asiatic Epipogium roseum, association with saprotrophic
Psathyrellaceae12 (=Coprinaceae; Fig. 2) was further supported
by in vitro reconstruction of the symbiosis that enabled success-
ful development of the orchid.13 Psathyrellaceae were molecu-
larly identiﬁed in the Asiatic MH Eulophia zollingeri14 (Fig. 2),
Gymnopus-related fungi in the Australian MH Erythrorchis
cassythoides,15 Mycenaceae in the Japanese Gastrodia confusa16
Saprotrophic fungal mycorrhizal symbionts
in achlorophyllous orchids
Finding treasures among the ‘molecular scraps’?
Marc-André Selosse,1,* Florent Martos,1,2 Brian A. Perry,3 Mahajabeen Padamsee,4 Mélanie Roy 1 and Thierry Pailler 2
1Centre d’Ec ologie Fo nctionnelle et Evolutive (CNRS, UMR 5175); Equipe Intera ctions Biotiques; Montpell ier, France; 2UMR C53 Peuplements végétaux et
Bioagresseurs en milieu tropical; Université de L a Réunion; Saint-Denis, France; 3Depar tment of Biology; Universit y of Hawai‘i at Hilo; Hilo, HI USA; 4Department
of Plant Pathology; Louisiana State U niversity Agricultural Center; Baton Rouge, LA USA
Key words: endophytic fungi, evolution of mycorrhizae, mycoheterophy, mycorrhizae, saprophytic fungi, specificity
2 Plant Signaling & Behavior Volume 5 Issue 4
reported. As shown in the analysis of the papers cited
above, it is valuable to consider their putative mycor-
rhizal abilities. In the light of recent works, we could
indeed ﬁnd treasures among molecular scraps.
Nevertheless, we still ignore whether all fungal
partners actually provide carbon to the unspecial-
ized MH orchids. To date, there is even no direct
evidence or experimental design (e.g., by labelling
experiments) showing that MH orchids associated
with saprotrophs indirectly exploit carbon from
dead organic matter. This is only supported by rhi-
zomorphs (elongated bundles of fungal hyphae) that
occasionally link dead leaves to mycorrhizal roots10,17
and 13C and 15 N natural contents, consistent with
the use of saprotrophic fungi as a food source.16, 17
Wullschlaegelia aphylla is linked to rhizomorphs of
Mycenaceae and Gymnopus-related fungi,17 but this
does not entail that they all provide carbon; more-
over, isotopic abundances only distinguish the ecol-
ogy of the fungal species furnishing carbon, not its
(or their) species.
Mycorrhizal Evolution: Promising Molecular Scraps?
The observation of simultaneous morphogenetic processes
between plants and saprotrophic fungi (Fig. 1B), which usually
do not colonize living material, is fascinating. This supports a lead
role for MH orchids in the mycorrhizal morphogenetic process,
since saprotrophic fungi were never selected for such a trait, and
raises the question of how the orchids manipulate these fungi.
Moreover, although investigating only a few models, current stud-
ies demonstrate that MH orchids recruited several independent
saprotrophic lineages in the Hymenochaetales, Psathyrellaceae,
Mycenaceae and Marasmiaceae (Fig. 2 and 3). Although some
clades were targeted twice, such Psathyrellaceae in the unrelated
orchids Epipogium roseum and Eulophia zollingeri (Fig. 2), no
speciﬁc fungal clade is predisposed to associate with MH orchids.
One can thus speculate that fungi do not need speciﬁc or rare
predispositions to be driven to living roots by MH orchids. In
other words, the transition to growing in living material might
arise fairly easily in fungal evolution, possibly even without any
speciﬁc genetic modiﬁcation. Although the genomes of myc-
orrhizal fungi contain numerous genes speciﬁcally involved in
symbiosis,22 it may be that the latter are only secondarily arisen as
an adaptation to exploiting the biotrophic niche (indeed, sapro-
trophs from MH orchids grow in roots, but do not receive carbon
from them). This may even explain why the transition to mycor-
rhizal status occurred so often in the evolution of fungi.23
In green orchids, in addition to the usual mycorrhizal
symbionts (the rhizoctonias, i.e., members of Sebacinales,
Ceratobasidiales and Tulasnellales, often considered as saprotro-
phs), other saprotrophs sometimes occur, e.g., Mycena species in
Anoectochilus roxburghii24 or Cymbidium sinense.25 In these cases,
they may be contaminants, or facultatively biotrophic encoun-
ters that either form mycorrhizal structures on very small root
portions, or colonize the tissues as endophytes (i.e., grow within
and the Caribbean Wullschlaegelia aphylla17 (Figs. 1A and 3),
Marasmiaceae in the Australian Gastrodia sesamoides,18 and a
Resinicium species (Hymenochaetales) in Gastrodia similis from
La Réunion17 (Indian Ocean). Therefore, there are now multiple
lines of evidence that several saprotrophic fungal lineages sup-
port some MH orchids, and that such symbioses are common in
Specicity of Association: Less than Expected
Additionally, the MH orchid Wullschlaegelia aphylla associates
with both Mycenaceae and Gymnopus-related fungi, and possibly
with a species close to Psathyrellaceae.17 The former fungi were
also identiﬁed by PCR from mycorrhizal pelotons—i.e., the fun-
gal coils formed in mycorrhizal root cells (Fig. 1B)—supporting
these taxa’s ability to form orchid mycorrhizae. Some MH plants
have thus non-speciﬁc associations with several fungal lineages.
The recent ﬁnding of multiple fungal taxa within a plant or even
within a root in two other MH species supports that speciﬁcity is
not the rule: several fungi, all mycorrhizal on surrounding trees,
occur in the orchid Aphyllorchis spp. from Thailand19 and in the
North-American ericaceous Pyrola picta.20
Previous reports of specialized MH plants may need to be re-
investigated to determine whether they are truly non-speciﬁc. The
African MH Afrothismia hydra (Thismiaceae) revealed mostly
Glomus species and also an Acaulospora, which was perceived
as a facultative partner with a reduced role in plant nutrition.21
In the MH orchid Gastrodia confusa, additional saprotrophic
Marasmiaceae (Marasmiellus, Clitocybula) were occasionally
recovered in individuals lacking the frequently ampliﬁed Mycena
spp.;16 the authors hypothesized that some plant populations may
differ in their mycorrhizal fungus, but Marasmiellus and Mycena
fungi also co-existed in some plants. Therefore, fungi identiﬁed
by molecular tools that have been discounted from relevant sym-
bionts in the initial study (the “molecular scraps”, i.e., the fungal
sequences that are not granted any relevant role) deserve to be
Figure 1. Wullschlaegelia aphylla, a mycoheterotrophic orchid unspecically associated
with saprotrophic Mycena and Gymnopus species. (A) Whole plant at owering time,
with reduced, tuberoid root system at that period. (B) Section of mycorrhizal root
showing intracellular hyphal pelotons at early st age (p), or late stage (undergoing lysis,
lp); among orchids, the coloniz ation of dead cortical cell (cc) is a unique feature to
some saprotrophic fungi (picture by A . Faccio, University of Torino).
www.landesbioscience.com Plant Signaling & Behavior 3
for our understanding of the evolution of the symbiosis! In this
respect, a particular issue is that of the fungal shifts that occur in
the evolution of some speciﬁc MH lineages, such as in Ericaceae,
where closely related MH species speciﬁcally target different fun-
gal taxa.28 How did the common ancestor shift to a new symbi-
ont? Indeed, one of these facultatively biotrophic encounters may
evolve into an exclusive symbiont.26
On the orchids’ side, the ability to exploit fungal carbon arose
several times2 using fungi mycorrhizal on other nearby autotro-
phs, or saprotrophic fungi. Even though some MH taxa only
use one kind of fungus, such as tree mycorrhizal fungi in the
Neottieae tribe or saprotrophs in the genus Gastrodia, there is
evidence that associations with saprotrophic versus mycorrhizal
living plant tissues, causing an unapparent infection, but do not
form true mycorrhizae, nor cause any disease symptoms). The
latter cases may be on the pathway from which closer mycorrhizal
relationships evolved. Indeed, evolution from saprotrophism to
endophytism and then to mycorrhizal association is a possible
pathway for the repetitive evolution of mycorrhizal associations
seen in fungi.26 The same applies for the ﬁnding of tree mycor-
rhizal fungi in some rhizoctonia-associated green orchids, e.g.,
overlooked Russula species in Cypripedium.27 This may be the
starting point for evolution to MH orchids connected by myc-
orrhizal fungi with surrounding autotrophs. Thus, when inves-
tigating green orchids, the report of minor ‘contaminant’ taxa,
usually disregarded as molecular scraps, is also extremely relevant
Figure 2 . A phylogeny of Psathyrellaceae (maximum likelihood) shows that several lineages were recruited as mycorrhizal symbionts of the myco-
heterotrophic orchids Eulophia zollingeri and Epipogium roseum. Mycorrhizal taxa fall within a clade consisting of Psathyrella candolleana represent a-
tives, while others fall within the Coprinellus clade (sequences from references 12 and 14; the support values on branches correspond to maximum
likelihood bootstrap probabilities, and only values above 70% are shown).
4 Plant Signaling & Behavior Volume 5 Issue 4
carbon that can be provided to a MH plant at little or no cost to
Certainly, MH orchids associating with saprotrophic fungi
are more tractable models for in vitro experimentation13 than
those associating with mycorrhizal fungi. These taxa offer inter-
esting models for testing the impact of MH orchids on associated
fungi, the mechanisms of recognition between the partners or the
exchanges at cell level. Beyond this, there is a great need for addi-
tional research on biological interactions in MH plants, and more
generally on plant-microorganism relationships, in the tropics.
And, please, let’s never forget reporting on the molecular scraps!
fungi are not phylogenetically constrained in MH orchids. For
example, although Epipogium roseum associates with saprotrophic
Psathyrellaceae,12,13 the related E. aphyllum associates with tree
mycorrhizal Inocybe.29 Strikingly, the saprotrophic fungi involved
(Fig. 2 and 3) belong to taxa that are abundant in temperate eco-
systems where MH plants also exist, however, to our knowledge,
these taxa never interact with MH plants outside of the tropics
(or very wet temperate forests). The ecological environment may
be more relevant than the phylogenetic position of the orchid.
A possibility is that a wetter and hotter climate may extend the
period of growth and nutrition of saprotrophs, providing surplus
Figure 3. A phylogeny of Mycenaceae (maximum likelihood) shows that several lineages were recruited as mycorrhizal symbionts of the myco-
heterotrophic orchids Gastrodia similis, G. confusa and Wullschlaegelia aphylla). Mycorrhizal taxa fall out among species of Mycena, while four addition-
al isolates fall out with Mycena pura (= Prunulus purus), or in more basal positions within, or sister to, Mycenaceae (sequences from references 16 and
17; the support values on branches correspond to parsimony bootstr ap/maximum likelihood bootstrap probabilities; only values above 70% are shown).
www.landesbioscience.com Plant Signaling & Behavior 5
20. Hynson N, Bruns T D. Evidence of a myco-het-
erotroph in the pla nt fa mily Ericaceae that lack s
mycorrhi zal specificity. Proc Royal Soc Lond B 2009;
276 :4 053-9.
21. Franke T, Beenken L, Döring M, Kocyan A, Agerer
R. Arbuscula r mycorrhiz al fungi of the Glomu s-
group A l ineage (Glomerale s; Glomeromyc ota)
detected in myco-heterotrophic pl ants from tropical
Africa. Mycol Progr 2 006 ; 5:24-31.
22. Martin F, Selosse M-A. The Laccar ia genome : a sym-
biont blueprint decoded. New Phytol 20 08; 180:296-
23. Hibbett DS, Matheny PB. Relative ages of ectomyc-
orrhiz al mushrooms and their plant hosts. BMC Biol
2009 ; 7:13.
24. Guo S-X, Fan L, Cao W-Q, Xu J-T, Xiao P-G. Mycena
anoectochila sp. Nov. isolated from mycorrhiza l roots
of Anoectochilus roxburghii f rom X ishua ngbanna,
China . Mycologia 1997; 89: 952-4.
25. Fan L, Guo S, C ao W, Xiao P, Xu J, Fan L , et al.
Isolation, culture, identiﬁcation and biologica l act iv-
ity of Mycena orchidicola sp. nov. in Cymbidium sin-
ense (Orch idaceae). Acta Mycol Sin 1996 ; 15:251-5.
26. Selosse M-A, Dubois M-P, Alvarez N. Are Sebacinales
common root endophy tes? Mycol Res 20 09;
113 :10 62-9.
27. Sheffer son RP, Taylor DL, Garnica S, McCormick
MK, Adam s S, Gray HM, et al. The evolutiona ry
histor y of mycorrhizal specif icity among lady’s slipper
orchids . Evolution 2007; 61:1380-9 0.
28. Bidartondo MI, Bruns TD. Fine-level mycorrhiz al
specif icity in t he Monotropoideae (Ericaceae ): sp eci-
ficit y for fungal species groups. Mol Ecol 2002;
29. Roy M, Yagame T, Yamato M, Iwase K, Heinz C,
Faccio A, et al. Ectomycorrhiza l Inocybe species as so-
ciate with the mycoheterotrophic orchid Epipogium
aphyllum Sw., but not with its asexual propagule s,
throughout its Eura siatic range. A nn Bot 2009b;
104 :595 -610.
10. Kusano S. Gastrod ia elata and its symbiotic associa-
tion with Armillaria mell ea. J Coll Agric Japan 1911;
11. Burgef f H. Saprophyt ismus und Symbiose. Studien
an tropischen Orchideen. Jena, Germa ny: Gustav
12. Yamato M, Yagame T, Suzuk i A, Iwase K. Isolation
and identif ication of mycorrhiza l fungi associating
with an achlorophyl lous plant, Epipogium roseum
(Orchidaceae). Mycoscience 2005; 46:73-7.
13. Yagame T, Yamato M, Mii M, Su zuki A, Iwase K.
Developmental processes of achlorophyllous orchid,
Epipogium roseum: from seed germinat ion to f lower-
ing under symbiotic cultivation with mycor rhiza l
fung us. J Pl ant Res 2007; 120:229-36.
14. Ogura-Tsujita Y, Yukawa T. High mycorrhizal spec-
ificity i n a widespread mycoheterot rophic plant,
Eulophia zollingeri (Orchidaceae). Am J Bot 2008 ;
95 : 9 3 -7.
15. Dea rna ley JDW. The funga l endoph yte s of
Erythrorchis cassythoides—is this orchid saprophytic
or para sitic? Aust Mycol 20 06; 25:51-7.
16. Ogura-Tsujita Y, Gebauer G, Hashimoto T, Umata
H, Yukawa T. Evidence for novel and specia lized
mycorrhi zal parasitism: the orchid Gastrodia confusa
gains carbon from saprotrophic Mycena. Proc Roya l
Soc Lond B 2009; 276 :761-7.
17. Martos F, Dulormne M, Pailler T, Bonfa nte P,
Faccio A, Fournel J, et al. Independent recruitment
of saprotrophic f ungi as mycorrhizal partners by
tropica l achlorophyllous orchids. New Phytol 2009;
184 : 668- 81.
18. Dearnaley JWD, Bou goure JJ. Molecular identif ica-
tion of Marasmiaceae mycobiont s in rhizomes of
Gastrodia sesamoïdes —e vidence for dir ect orchid
parasitism. Fungal Ecol 2009; In press.
19. Roy M, Whatthana S, Richard F, Vessabutr S, Selosse
M-A. Two mycoheterotrophic orchids from T hailand
tropica l dipterocarpacean forests a ssociate with a
broad diversity of ectomyc orrhiz al fungi. BMC Biol
2009a ; 7:51.
1. Leake JR. Myco-heterotroph /epiparasitic plant i nter-
actions w ith ectomycorrhizal and arbuscula r mycor-
rhiza l fungi. Cu rr Opin Plant Biol 2004 ; 7:422-8.
2. Molvray M, Kore s PJ, Cha se MW. Polyphyly of
mycoheterotrophic orchids and functiona l i nfluence
on flor al and molecular characters. In: Wilson KL,
Morrison DA, eds. Monocots : systematics and evolu-
tion. Melbourne, Australia : CSIRO 2000 ; 441-7.
3. Taylor DL, Bruns TD, Leak e JR, Read D. Mycorrhiza l
spec if icit y an d funct ion i n myc o-heterotroph ic
plants. In: Van der Heijden MGA, Sanders I, eds.
Mycorrhi zal E cology. Berlin, Ger many: Springer-
Verlag 2002; 375- 413.
4. Merckx V, Bida rtondo MI, Hynson NA. Myco-
heterotrophy: when fungi host pla nts. Ann Bot 2009 ;
104 :1255 - 61.
5. Selosse M-A, Roy M. Green plants that feed on fungi:
facts and questions about mixotrophy. Trends Plant
Sci 20 09; 14:64 -70.
6. McKendrick SL , Lea ke JR, Read DJ. Symbiotic
germination a nd development of myc o-heterot rophic
plants in nature : transfer of carb on from ectomycor-
rhiza l Salix repens and Betul a pendul a to the orchid
Corallorhiza trif ida throug h shared hyphal connec-
tions. New Phytol 2000; 145:539-48.
7. Trudell SA, Rygiewicz PT, E dmonds RL. Nitrogen
and carbon stable isotope abundances support the
myco-heterotrophic n ature a nd host-spe cificity of
certa in achlorophyllous plants. New Phytol 2003;
160 :3 91-4 01.
8. Umata H. In vitro symbiotic ass ociation of an
achlorophyllous orchid, Erythrorchis ochobiensis, w ith
orchid and non-orchid fungi. Memoirs of the Facult y
of Agriculture, Kagoshim a University 1998a; 34:97-
9. Umata H. A ne w biological fu nction of shiitake
mushroom, Lentinula edodes, in a myco-he terotroph ic
orchid, Erythror chis ochobiensis. Mycoscience 1998b;
39: 85-8 .