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Saprotrophic fungal symbionts in tropical achlorophyllous orchids: Finding treasures among the ‘molecular scraps’?


Abstract and Figures

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific 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 mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as 'molecular scraps', since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.
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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
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) specic 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-specic, and that some
mycoheterotrophic orchids associate with saprophytic fungi. A
re-examination of earlier data suggests that lower specicity
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
tropical latitudes.
Specicity 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 unspecically 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). 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
the fungus.17
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). Plant Signaling & Behavior 5
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... Evidence that supports this scenario comes from an increasing trend to report the whole fungal communities detected in roots, in part thanks to nextgeneration sequencing. Cessation of screening data and of reporting of only the taxa known, or expected, to be mycorrhizal (Selosse et al., 2010) as well as interest in describing microbiota diversity more comprehensively have enhanced knowledge of endophytic fungi and the development of new working hypotheses. ...
... Ectomycorrhizal fungi are found in a few samples and/or in a minor amount among clones (in PCR cloning) or among next-generation sequencings reads so that the orchids are considered as rhizoctonia-associated in these studies (see references in Table 1). In the last decade, this possibility has triggered the call for reporting of all fungal species found, even if unexpected (Selosse et al., 2010). Of course, next-generation sequencing has prompted larger reports of the diversity encountered, but well before these methods arose, evidence had already been obtained from cloning the barcoding PCR products, or even from direct Sanger sequencing (Table 1). ...
... non-rhizoctonia Basidiomycotas, in a secondary evolution that also implied the transition to full mycoheterotrophy Martos et al., 2009;Merckx, 2013;Hatté et al., 2020;Suetsugu et al., 2020;Wang et al., 2021). For example, the fungal genera Psathyrella, Mycena, Coprinus and Gymnopus were repeatedly involved in such transitions within the orchid lineages (Selosse et al., 2010;Lee et al., 2015). Recently, it was demonstrated that some mixotrophic orchids rely on such non-rhizoctonia fungi Yagame et al., 2021). ...
Full-text available
Background As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g., soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations. Scope Our view is based on the ‘Waiting Room Hypothesis’, an evolutionary scenario stating that mycorrhizal fungi of the land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant’s nutrition. The association with rhizoctonias is likely the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, likely as endophytes, in extant rhizoctonia-associated orchids. Conclusion The orchid family offers one of the best documented examples of the ‘Waiting Room Hypothesis’: their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchids ancestors.
... Th ere are many neotropical terrestrial orchids, but few of their mycorrhizal symbionts are known . Th e role of the fungi reported here needs to be confi rmed, but they qualify as "valuable molecular scraps" as described by Selosse et al. (2010) , in the sense that they may be important even though their association with orchids is not obvious. ...
... Th is pattern may seem unusual, but there is an obvious biological explanation: seeds are obligately mycorrhizal, whereas adult plants are facultative. Obligate relationships (in which the plant requires mycorrhizae for survival) tend to be more specifi c than facultative ones (in which the plant can survive without mycorrhizae), and this pattern generally applies to orchid mycorrhizal fungi, although exceptions are known ( Allen, 1991 ;Taylor et al., 2002 ;Hynson and Bruns, 2010 ;Selosse et al., 2010 ). ...
... Th ird, it associates with a combination of Rhizoctonia -like fungi and saprotrophs; a combination of Rhizoctonia -like fungi and ectomycorrhizal fungi was proposed as "…an intermediate step in the evolution of full mycoheterotrophy, deriving from autotrophic orchid ancestors associated with 'rhizoctonias'" ( Dearnaley et al., 2012 , p. 216). Associations with a range of saprotrophic fungi have been reported for mycoheterotrophic orchids ( Selosse et al., 2010 ) but never before for photosynthetic orchids. Th ese associations are important not only for understanding the biology of the plants involved, but also the fl ow of carbon in plant communities ( Bidartondo et al., 2004, Selosse et al., 2006. ...
Full-text available
... This hypothesis proposes that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors. As such, the investigated Cypripedium species may represent another fascinating example of orchid species whose mycobionts may evolve mycorrhizal habits from endophytic ancestors (Selosse and Roy 2009;Selosse et al. 2010;Jacquemyn et al. 2017). ...
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Orchids commonly rely on mycorrhizal fungi to obtain the necessary resources for seed germination and growth. Whereas most photosynthetic orchids typically associate with so-called rhizoctonia fungi to complete their life cycle, there is increasing evidence that other fungi may be involved as well and that the mycorrhizal communities associated with orchids may be more diverse. Coexisting orchid species also tend to associate with different fungi to reduce competition for similar resources and to increase long-term population viability. However, few studies have related the mycorrhizal communities in the rhizosphere to communities found in the roots of closely related coexisting orchid species. In this study, we used high-throughput sequencing to investigate the diversity and community composition of orchid mycorrhizal fungi in the roots and the rhizosphere of four Cypripedium species growing in forests in Northeast China. The results showed that the investigated Cypripedium species associated with a wide variety of fungi including members of Tulasnellaceae, Psathyrellaceae, and Herpotrichiellaceae, whereas members of Russulaceae, Cortinariaceae, Thelephoraceae, and Herpotrichiellaceae showed high abundance in rhizosphere soils. The diversity of fungi detected in the rhizosphere soil was much higher than that in the roots. The observed variation in fungal communities in Cypripedium roots was not related to forest site or orchid species. On the other hand, variation in mycorrhizal communities of rhizosphere soil was significantly related to sampling site. These results indicate that orchid mycorrhizal communities in the rhizosphere display considerable variation among sites and that orchids use only a subset of the locally available fungi. Future studies focusing on the fine-scale spatial distribution of orchid mycorrhizal fungi and more detailed assessments of local environmental conditions will provide novel insights into the mechanisms explaining variation of fungal communities in both orchid roots and the rhizosphere.
... From the fungal point of view: certain fungi exhibit a metabolic route change from saprotrophic to biotrophic metabolism. Some species of Mycena and Gymnopus are known as saprotrophic fungi that were recruited as mycorrhizal symbionts of certain mycoheterotrophic orchids (Selosse et al. 2010). Mycena, root-associated saprotrophic fungi, were found to occupy tree roots and to be involved in carbon and phosphorous transfer between fungi and their host in a manner similar to that of mycorrhizal species (Thoen et al. 2020, Harder et al. 2021. ...
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Mechanisms of host-microbe interactions and their direct impact on both parties have been extensively researched, however, much less is known on the effect of these interactions on the ecology of the host-community. Here we investigate tree-fungi mycorrhizal interactions, focusing on mycorrhizal-meditated resource sharing among trees, while examining the dynamics between specialist and generalist fungi and their implications on the forest ecology. Using genetic meta-barcoding, we identified the fungal community colonizing different trees in a mixed forest, and generated an extensive mapping connecting fungal sequences to their tree hosts. The mycorrhizal fungal community diverged between ectomycorrhizal and arbuscular host trees, but, unexpectedly, multiple ectomycorrhizal species colonized roots of non-ectomycorrhizal host trees. We complemented these findings by a novel computational framework, modeling competition between generalist and specialist mycorrhizal fungi, accounting for fungal-mediated resource sharing among neighboring trees. The analysis of the model revealed that generalist mycorrhizal networks may affect the entire tree community, and contribute to the maintenance of forest diversity in the long run. Furthermore, higher initial spatial mixing of trees can promote the evolution of generalist mycorrhizal species. These novel belowground interactions among trees and fungi may significantly impact forest biodiversity.
... Moreover, AM fungi cannot grow independently of their hosts 12 . In contrast, individual fungal species of other types of mycorrhizae, particularly EEM, have adaptations for aerial dispersal of spores 13,14 and can grow independently of their host through saprophytic activity 12,[15][16][17][18][19][20] (but see Lindahl et al. 21 ). ...
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Plant colonization of islands may be limited by the availability of symbionts, particularly arbuscular mycorrhizal (AM) fungi, which have limited dispersal ability compared to ectomycorrhizal and ericoid (EEM) as well as orchid mycorrhizal (ORC) fungi. We tested for such differential island colonization within contemporary angiosperm floras worldwide. We found evidence that AM plants experience a stronger mycorrhizal filter than other mycorrhizal or non-mycorrhizal (NM) plant species, with decreased proportions of native AM plant species on islands relative to mainlands. This effect intensified with island isolation, particularly for non-endemic plant species. The proportion of endemic AM plant species increased with island isolation, consistent with diversification filling niches left open by the mycorrhizal filter. We further found evidence of humans overcoming the initial mycorrhizal filter. Naturalized floras showed higher proportions of AM plant species than native floras, a pattern that increased with increasing isolation and land-use intensity. This work provides evidence that mycorrhizal fungal symbionts shape plant colonization of islands and subsequent diversification.
... (Psathyrellaceae) or Mycena spp. (Mycenaceae; Martos et al. 2009;Ogura-Tsujita et al. 2009;Selosse et al. 2010), but no MX orchid associating with non-rhizoctonia saprobic fungi, such as Psathyrellaceae, Mycenaceae or Tricholomataceae, has been reported so far. ...
Mixotrophy (MX, also called partial mycoheterotrophy) in plants is characterized by isotopic abundances that differ from those of autotrophs. Previous studies have evaluated mycoheterotrophy in MX plants associated with fungi of similar ecological characteristics, but little is known about the differences in the relative abundances of 13C and 15N in an orchid species that associates with several different mycobionts species. Since the chlorophyllous orchid Cremastra variabilis Nakai associates with various fungi with different ecologies, we hypothesized that it may change its relative abundances of 13C and 15N depending on the associated mycobionts. We investigated mycobiont diversity in the chlorophyllous orchid C. variabilis together with the relative abundance of 13C and 15N and morphological underground differentiation (presence or absence of a mycorhizome with fungal colonization). Rhizoctonias (Tulasnellaceae, Ceratobasidiaceae, Sebacinales) were detected as the main mycobionts. High differences in δ13C values (– 34.7 to – 27.4 ‰) among individuals were found, in which the individuals associated with specific Psathyrellaceae showed significantly high relative abundance of 13C. In addition, Psathyrellaceae fungi were always detected on individuals with mycorhizomes. In the present study, MX orchid association with non-rhizoctonia saprobic fungi was confirmed, and the influence of mycobionts on morphological development and on relative abundance of 13C and 15N was discovered. Cremastra variabilis may increase opportunities to gain nutrients from diverse partners, in a bet-hedging plasticity that allows colonization of various environmental conditions.
... Additionally, a few photosynthetic orchids associate with saprotrophic Auriculariales, Psathyrellaceae, Tricharina, Clavulina, Armillaria, Marasmius, and Scleroderma fungi belonging to the common ECM fungal taxa (Waterman et al., 2011;Yagame et al., 2013;Jacquemyn et al., 2016b;González-Chávez et al., 2018;Qin et al., 2019;May et al., 2020;Salazar et al., 2020). The presence of such fungi, likely in minor amounts, was greatly enhanced by the use of HTS and the reporting of all the diversity found, without a priori screening (Selosse et al., 2010). Although some MX orchids preserve their autotrophic ability, as shown by intact photosynthetic genes in plastid genomes (Lallemand et al., 2019), they possess few or no rhizoctonias in their roots, whereas ECM fungi Frontiers in Plant Science | ...
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Orchids form mycorrhizal symbioses with fungi in natural habitats that affect their seed germination, protocorm growth, and adult nutrition. An increasing number of studies indicates how orchids gain mineral nutrients and sometime even organic compounds from interactions with orchid mycorrhizal fungi (OMF). Thus, OMF exhibit a high diversity and play a key role in the life cycle of orchids. In recent years, the high-throughput molecular identification of fungi has broadly extended our understanding of OMF diversity, revealing it to be a dynamic outcome co-regulated by environmental filtering, dispersal restrictions, spatiotemporal scales, biogeographic history, as well as the distribution, selection, and phylogenetic spectrum width of host orchids. Most of the results show congruent emerging patterns. Although it is still difficult to extend them to all orchid species or geographical areas, to a certain extent they follow the “everything is everywhere, but the environment selects” rule. This review provides an extensive understanding of the diversity and ecological dynamics of orchid-fungal association. Moreover, it promotes the conservation of resources and the regeneration of rare or endangered orchids. We provide a comprehensive overview, systematically describing six fields of research on orchid-fungal diversity: the research methods of orchid-fungal interactions, the primer selection in high-throughput sequencing, the fungal diversity and specificity in orchids, the difference and adaptability of OMF in different habitats, the comparison of OMF in orchid roots and soil, and the spatiotemporal variation patterns of OMF. Further, we highlight certain shortcomings of current research methodologies and propose perspectives for future studies. This review emphasizes the need for more information on the four main ecological processes: dispersal, selection, ecological drift, and diversification, as well as their interactions, in the study of orchid-fungal interactions and OMF community structure.
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Many vascular plants and aquatic organisms are not strictly autotrophic or heterotrophic but rather mixotrophic. Mixotrophy is a widespread nutritional strategy, merging autotrophy and heterotrophy to acquire organic carbon (C). Because all orchids are mycoheterotrophic during the early stages, many species are also predisposed to mycoheterotrophic nutrition during the mature stage. Consequently, many green orchids adopt partially mycoheterotrophy, a kind of mixotrophic strategy, obtaining carbon through autotrophy and mycoheterotrophy. However, the proportions of fungal‐derived carbon reported for these mixotrophic orchids show considerable variation, even within species under similar light conditions and at similar growth stages. The factors promoting such variation remain to be fully elucidated. We investigated the nutritional mode of a green orchid Calypso bulbosa to determine whether coralloid rhizomes, which are often found in fully mycoheterotrophic orchids, affect the degree of mycoheterotrophy. To this end, we performed molecular barcoding and 13C and 15N analyses to identify the mycorrhizal partners and fungal dependency of C. bulbosa specimens with and without coralloid rhizomes. We found that C. bulbosa individuals were consistently colonized by an OTU of the wood‐decaying fungal genus Protomerulius (Auriculariales), regardless of the presence of rhizomes. Furthermore, although both types of C. bulbosa specimens are partially mycoheterotrophic, as indicated by their higher 13C and 15N abundances, those with coralloid rhizomes exhibited a higher degree of mycoheterotrophy. Our results indicate that partial mycoheterotrophy in C. bulbosa is a flexible mechanism driven by subterranean morphology. Although mixotrophy has evolved repeatedly in vascular plants and aquatic organisms, their trophic plasticity remains largely unexplored. Here we propose a novel mechanism modulating the degree of mycoheterotrophy in green orchids, providing a new perspective on the ecological plasticity of nutritional mode in mixotrophic organisms.
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• Mycorrhizal fungi are central to the biology of land plants. However, to what extent mycorrhizal shifts - broad evolutionary transitions in root-associated fungal symbionts - are related to changes in plant trophic modes remains poorly understood. • We built a comprehensive DNA dataset of Orchidaceae fungal symbionts and a dated plant molecular phylogeny to test the hypothesis that shifts in orchid trophic modes follow a stepwise pattern, from autotrophy over partial mycoheterotrophy (mixotrophy) to full mycoheterotrophy, and that these shifts are accompanied by switches in fungal symbionts. • We estimate that at least 17 independent shifts from autotrophy towards full mycoheterotrophy occurred in orchids, mostly through an intermediate state of partial mycoheterotrophy. A wide range of fungal partners was inferred to occur in the roots of the common ancestor of this family, including 'rhizoctonias', ectomycorrhizal, and wood- or litter-decaying saprotrophic fungi. Phylogenetic hypothesis tests further show that associations with ectomycorrhizal or saprotrophic fungi were most likely a prerequisite for evolutionary shifts towards full mycoheterotrophy. • We show that shifts in trophic mode often coincided with switches in fungal symbionts, suggesting that the loss of photosynthesis selects for different fungal communities in orchids. We conclude that changes in symbiotic associations and ecophysiological traits are tightly correlated throughout the diversification of orchids.
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In 2017, a 560-ha area of hybrid poplar plantation in northern Poland showed symptoms of tree decline. The leaves appeared smaller, yellow-brown, and were shed prematurely. Twigs and smaller branches died without distinct cankers. Trunks decayed from the base. The phloem and xylem showed brown necrosis. Ten percent of the trees died 1–2 months after the first appearance of the symptoms. None of these symptoms were typical for known poplar diseases. The trees’ mycobiota were analysed using Illumina sequencing. A total of 69 467 and 70 218 operational taxonomic units (OTUs) were obtained from the soil and wood. Blastocladiomycota and Chytridiomycota occurred only in the soil, with very low frequencies (0.005% and 0.008%). Two taxa of Glomeromycota, with frequencies of 0.001%, occurred in the wood. In the soil and wood, the frequencies of Zygomycota were 3.631% and 0.006%, the frequencies of Ascomycota were 45.299% and 68.697%, and the frequencies of Basidiomycota were 4.119% and 2.076%. At least 400 taxa of fungi were present. The identifiable Zygomycota, Ascomycota, and Basidiomycota were represented by at least 18, 263 and 81 taxa, respectively. Many fungi were common to the soil and wood, but 160 taxa occurred only in soil and 73 occurred only in wood. The root pathogens included species of Oomycota. The vascular and parenchymal pathogens included species of Ascomycota and of Basidiomycota. The initial endophytic character of the fungi is emphasized. Soil, and possibly planting material, may be the sources of the pathogen inoculum, and climate warming is likely to be a predisposing factor. A water deficit may increase the trees’ susceptibility. The epidemiology of poplar vascular wilt reminds grapevine trunk diseases (GTD), including esca, black foot disease and Petri disease.
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Erythrorchis cassythoides is a common climbing orchid in Eastern Australia. The plant lacks chlorophyll and typically is rooted at the base of mature trees suggesting the orchid receives its carbon supply via root fungi from either rotting vegetation or indirectly from living tree roots. We have analysed the fungal DNA within roots of the orchid using ITS-PCR analysis, cloning and molecular sequencing to gain insight into the mode of nutrition of this orchid. Fungal ITS rDNA sequences were successfully amplified and cloned from roots of three orchid plants occurring at different localities in SE Queensland. Comparison of these sequences with ITS rDNA in GenBank revealed that the fungal community of E cassythoides roots consists of a saprotrophic homobasidiomycete and ectomycorrhizal fungal species thus suggesting that the orchid is potentially capable of both saprophytic and parasitic modes of nutrition. J.D.W. Dearnaley (2006). The fungal endophytes of Erythrorchis cassythoides—is this orchid saprophytic or parasitic? Australasian Mycologist!^ (2): 51-57.
During a study to isolate and identify endophytic fungi present in cortical tissues of the roots of tropical orchids of Xishuangbanna, Yunnan, some white isolates were obtained from Anoectochilus roxburghii. One of them, with distinctive clamp connections, was found to belong to Mycena, Sect. Sacchariferae, and is evidently distinguished from previously described species of Mycena by clavate cheilocystidia which are covered with several to numerous ellipsoid to ovoid excrescences. A new taxon, M. anoectochila, is described and illustrated to accommodate this isolate. A sequence of symbiotic germination tests between seeds of 12 orchids and M. anoectochila demonstrated M. anoectochila was able to stimulate germination of 4 orchids, but was not effective in promoting seed germination or protocorm development in other orchids. This shows that M. anoectochila has the activity to stimulate seed germination.
During a study to isolate and identify endophytic fungi present in cortical tissues of the roots of tropical orchids of Xishuangbanna, Yunnan, some white isolates were obtained from Anoectochilus roxburghii. One of them, with distinctive clamp connections, was found to belong to Mycena, Sect. Sacchariferae, and is evidently distinguished from previously described species of Mycena by clavate cheilocystidia which are covered with several to numerous ellipsoid to ovoid excrescences. A new taxon, M. anoectochila, is described and illustrated to accommodate this isolate. A sequence of symbiotic germination tests between seeds of 12 orchids and M. anoectochila demonstrated M. anoectochila was able to stimulate germination of 4 orchids, but was not effective in promoting seed germination or protocorm development in other orchids. This shows that M. anoectochila has the activity to stimulate seed germination.
Gastrodia sesamoides, a common obligate mycoheterotrophic orchid species found in eastern Australia relies on a soil fungus to provide a source of carbon nutrition. The identity of this fungus is not known although in other studies of Gastrodia species a number of mycobionts have been suggested including Fomes and Mycena. In this study the fungal community of rhizomes of G. sesamoides has been identified via fungal ITS-DNA PCR amplification, cloning and sequencing. Although a number of fungi were identified by this approach the most common fungal ITS DNA within the orchid were saprotrophic members of the Marasmiaceae (Campanella and Marasmius spp.). Analysis of the natural carbon and nitrogen stable isotope abundances of stems of G. sesamoides showed an enrichment in 13C and low levels of 15N. These data suggest that G. sesamoides obtains its carbon parasitically from free-living saprotrophic fungi and not from an ectomycorrhizal fungal partner of a photosynthetic plant, as is common for other obligate mycoheterotrophic orchid species.
Summary • Over 400 species of achlorophyllous vascular plants are thought to obtain all C from symbiotic fungi. Consequently, they are termed 'myco-heterotrophic.' However, direct evidence of myco-heterotrophy in these plants is limited. • During an investigation of the patterns of N and C stable isotopes of various eco- system pools in two old-growth conifer forests, we sampled six species of myco- heterotrophic achlorophyllous plants to determine the ability of stable isotope ratios to provide evidence of myco-heterotrophy and host-specificity within these symbioses. • Dual-isotope signatures of the myco-heterotrophic plants differed from those of all other pools. They were most similar to the signatures of ectomycorrhizal fungi, and least like those of green plants. δ 15 N values of the myco-heterotrophic plants correlated strongly and positively with those of putative mycobionts. • Used in conjunction with other techniques, N and C stable isotope ratios can be used to demonstrate myco-heterotrophy and host-specificity in these plants when other ecosystem pools are well characterized. They also appear promising for estimating the degree of heterotrophy in photosynthetic, partially myco-heterotrophic plants.
Seedlings of the myco-heterotrophic orchid Corallorhiza trifida which had been germinated in the field in mesh bags developed hyphal links and mycorrhizas with Betula pendula and Salix repens, but not with Pinus sylvestris, when transplanted into soil microcosms. The fungus connecting the myco-heterotroph to Betula and Salix formed endomycorrhiza in the orchid with typical pelotons, but formed ectomycorrhizas with the autotrophs. The orchid plants, when linked to Betula and Salix by fungal hyphae, gained 6–14% in weight over 25–28 wk. In microcosms supporting P. sylvestris, and in control microcosms which lacked autotrophs, the Corallorhiza plants lost 13% of their weight over the same period. In the course of the 28-wk experimental period new Corallorhiza seedlings, in addition to those added as part of the experiment, appeared in the microcosms containing Salix and Betula but not in the Pinus microcosms. Shoots of Betula and Salix plants grown in association with Corallorhiza were fed with 14CO2, and the movement of the isotope was subsequently traced by a combination of digital autoradiography and tissue oxidation. Direct transfer of C from both autotrophs to the myco-heterotroph occurred in all cases where the associates had become connected by a shared fungal symbiont. Orchid seedlings lacking these hyphal connections, introduced to the microcosms as controls immediately before isotope feeding, failed to assimilate significant amounts of C. The results provide the first experimental confirmation that growth of Corallorhiza trifida can be sustained by supply of C received directly from an autotrophic partner through linked fungal mycelia.
The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi.