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Untangling factors that drive community composition of root
associated fungal endophytes of Neotropical epiphytic orchids
Stefania Cevallos
a
,
b
, Paulo Herrera
b
, Aminael S
anchez-Rodríguez
b
,St
ephane Declerck
a
,
Juan Pablo Su
arez
b
,
*
a
Universit
e catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology, Croix du Sud, 2 box L7.05.06, B-1348 Louvain-la-Neuve,
Belgium
b
Departamento de Ciencias Biol
ogicas, Universidad T
ecnica Particular de Loja, San Cayetano Alto y Paris, C.P. 11 01 608, Loja, Ecuador
article info
Article history:
Received 23 November 2017
Received in revised form
16 April 2018
Accepted 5 May 2018
Corresponding Editor: Thorunn Helgason
Keywords:
Fungal community composition
Drivers of endophyte communities
Epiphytic orchids
Root-associated endophytes
Next generation sequencing
Fungal community analysis
abstract
In orchids, most of the root-associated fungal endophytes remain undescribed as well as the drivers that
affect their interactions with the plants. We characterized root-associated fungal endophytes of co-
existing orchids across sites in two areas of montane rainforest in the southern Ecuadorian Andes. We
amplified the nrDNA ITS2 region of 130 orchid individuals with Illumina MiSeq technology and tested
whether changes in the structure of fungal communities are associated with hosts' phylogeny or the sites
where the orchids grow. We identified 3492 OTUs corresponding to the Ascomycota, Basidiomycota,
Chytridiomycota, Glomeromycota and Zygomycota phyla. Fungal communities associated with orchids at
the lower geographic areas (between 2050 and 2800 m a.s.l.) showed that host evolution and sites are
drivers that could shape distinct fungal communities, while at the highest geographic areas (between
3000 and 3500 m a.s.l.), no distinct fungal comm unities were found neither between co-existing orchid
species nor between sites. These results suggested that among orchid species, abiotic and biotic factors
do not influence the composition of fungal communities in the same way.
©2018 Elsevier Ltd and British Mycological Society. All rights reserved.
1. Introduction
Orchid roots harbor a diverse set of fungal species with distinct
ecological attributes such as saprobes, latent pathogens or symbi-
onts (Kohout et al., 2013;Ma et al., 2015). Fungi located inside
orchid roots in the velamen or in the cortical tissue are collectively
termed root-associated endophytes (Brundrett, 2002). However,
studies focusing on root-associated endophytes frequently ignore
the species present in the velamen because this thick corky
epidermis is generally recognized as an adaptive structure for water
and nutrient conservation (Zotz and Winkler, 2013). Fungi present
in the velamen are thus most often considered as surface con-
taminants or opportunists associated with roots (e.g. Yuan et al.,
2010). Thus, the root cortical tissue is the main target of multiple
studies focusing on orchid fungal endophytes because it often
harbors fungi that may interact with the plant as symbionts
(Brundrett, 2002;Smith and Read, 2008).
Fungal endophytes in root cortical tissues comprise a poly-
phyletic group (Smith and Read, 2008) that includes mycorrhizal
and non-mycorrhizal fungi. While mycorrhizal fungi have been
widely investigated across multiple orchid species and with
recognized ecological roles in their life cycle (Dearnaley et al.,
2012), non-mycorrhizal fungi have been less studied (Ma et al.,
2015).
Most studies focusing on community composition of orchid
root-associated endophytes (both mycorrhizal and non-
mycorrhizal fungi) have been based on culture-dependent
methods (Herrera et al., 2010;Novotn
a et al., 2018). However, the
great majority of fungi cannot be grown in artificial conditions and
thus culture-independent methods (sequencing and cloning) have
been developed to increase the knowledge of fungal diversity
(Kristiansen et al., 2001). With the development of powerful mo-
lecular methods in recent years (e.g. next generation sequencing),
the range of fungi identified to the species level has increased
*Corresponding author. Universidad T
ecnica Particular de Loja, 11 01 608 Loja, Ecuador.
E-mail address: jpsuarez@utpl.edu.ec (J.P. Su
arez).
Contents lists available at ScienceDirect
Fungal Ecology
journal homepage: www.elsevier.com/locate/funeco
https://doi.org/10.1016/j.funeco.2018.05.002
1754-5048/©2018 Elsevier Ltd and British Mycological Society. All rights reserved.
Fungal Ecology 34 (2018) 67e75
markedly, making more accurate ecological inferences now
possible (Smith and Peay, 2014).
Studies conducted on the orchid root-associated fungal endo-
phytes, using culture-independent methods, mostly described
fungal diversity and abundance (Kohout et al., 2013). However, how
root-associated fungal endophyte communities are assembled in
orchids remains poorly investigated (for a review, see Dearnaley
et al., 2012) and the information about the abiotic and biotic fac-
tors that drive such fungal community composition is even more
limited.
Abiotic factors such as temperature and humidity are generally
variable across sites but also at the same site, altitudinal gradient
could result in peculiar microclimates that shape plant-fungi
interactions as was observed in orchid mycorrhizal fungi (Garnica
et al., 2013). Similarly, biotic factors such as the host phylogeny or
the interactions between co-existing species were suggested to
affect fungal distribution patterns (see Waterman et al., 2011). For
instance, conforming to the co-existence theory, species are able to
cohabit because they use different niches (G€
otzenberger et al.,
2012) determined by the host (Oliveira et al., 2014).
Nowadays, the information about the specific factors that drive
the occurrence and variation of orchid root-associated fungal en-
dophytes in natural environments is scarce (e.g. Sudheep and
Sridhar, 2012;Bunch et al., 2013) probably because of the limited
investigations of orchid species, populations or sites (Ma et al.,
2015). Studies on orchid root-associated fungal endophytes were
mostly focused on terrestrial orchids in temperate ecosystems (e.g.
Jacquemyn et al., 2015;T
e
sitelov
a et al., 2015), whereas only a few
concerned epiphytic orchids in tropical areas (see Bayman and
Otero, 2006;Herrera et al., 2010;Oliveira et al., 2014;Novotn
a
et al., 2018). This fact contrasts with the higher orchid diversity
concentrated in Neotropical ecosystems (Dressler, 1990;Pridgeon,
1995). In the Ecuadorian Andes, one of the world's hotspots of
biodiversity (Beck et al., 2008), an important variety of basidio-
mycetes and ascomycetes has been identified associated with
epiphytic orchid roots (e.g. Su
arez et al., 2006,2008,2016;Herrera
et al., 2010,2018;Riofrío et al., 2013;Novotn
a et al., 2018). However,
it is likely that a large number of fungal endophytes remain
undescribed due to methodological biases (Tedersoo et al., 2010;
Kohout et al., 2013) and also because the target of each study was
either mycorrhizal or non-mycorrhizal fungi but not both at the
same time.
To fill this gap, the first critical step is to elucidate the diversity
and community assemblage of orchid root-associated endophytes
of cortical tissues including mycorrhizal and non-mycorrhizal
fungi. In the present study, we evaluated root-associated fungal
endophytes colonizing the cortical tissues of native epiphytic
orchid species: Cyrtochilum flexuosum, Cyrtochilum myanthum and
Maxillaria calantha co-occurring in the Podocarpus National Park
(PNP) and Epidendrum marsupiale and Cyrtochilum pardinum co-
occurring in the Cajas National Park (CNP). We used a meta-
genomic approach based on the analysis of the internal transcribed
spacer 2 sequences via the Illumina MiSeq technology. Our objec-
tives were to: (i) characterize root fungal endophytes associated
with the aforementioned epiphytic orchids; (ii) compare endo-
phyte communities between co-existing orchid species; (iii) eval-
uate the root-associated fungal endophyte communities between
orchid sites/populations; and (iv) compare global orchid root-
associated fungal endophytes between sites at PNP and CNP. We
expected distinct fungal endophyte communities between co-
existing orchid species if the orchid phylogeny displays an effect on
the fungal community composition. We also expected distinct root-
associated fungal endophyte communities across sites/populations
due to effects of altitude or site over the community considering a
habitat dependent community composition hypothesis.
2. Materials and methods
2.1. Sample collection
Roots of orchids were collected in 2012 and 2013, with a total of
130 individual plants sampled along six sites of evergreen upper
montane forests in the Southern Ecuadorian Andes. Two sites close
to the PNP in Zamora-Chinchipe province were chosen: the first
one called ‘Curva Misteriosa’(site 1; 3
59
0
32
00
S, 79
06
0
29
00
W) and
the second one ‘El Tiro’(site 2; 3
59
0
20
00
S, 79
08
0
38
00
W), both sites
located between 2050 and 2800 m a.s.l. Curva Misteriosa is char-
acterized by a steep slope (51%), with trees 5e8 m high (Riofrío
et al., 2007), a mean annual temperature of 20.8
C and mean
annual precipitation of 2193 mm (Bendix et al., 2008). El Tiro has
the ridge covered with forests on the slope sides and open grass,
bromeliad, or dwarf shrub formations along the crest line (Setaro
et al., 2006), the mean annual temperature is 9.8
C and mean
annual precipitation is 3000 mm (Gradstein et al., 2008). At both
sites, the characteristic vegetation includes epiphytic plants such as
orchids, ferns and bromeliads (Mandl et al., 2010); the climate is
cool and prehumid while, the soil is poor and acidic (Gradstein
et al., 2008). The other four sites were ‘High Maz
an’(site 3;
2
52
0
13
00
S, 79
7
0
26
00
W), ‘Low Maz
an’(site 4; 2
52
0
19
00
S, 79
7
0
8
00
W),
‘High Llaviucu’(site 5; 2
50
0
26
00
S, 79
10
0
29
00
W) and ‘Low Llaviucu’
(site 6; 2
50
0
36
00
S, 79
8
0
37
00
W), located in the CNP in Azuay prov-
ince between 3000 and 3500 m a.s.l. The sites at CNP harbor around
300 species of vascular plants (Montesinos, 1996) and Orchidaceae
is the second most diverse family (ETAPA, 2005). The climate
fluctuates between 2
C and 18
C(Minga et al., 2016). The annual
precipitation is 1200 mm in average with hail and snow episodes
(Sklen
ar et al., 2011).
The study sites were selected based on the presence of common
epiphytic orchid species. At site 1 and site 2, three epiphytic orchid
species were sampled, belonging to the tribe Cymbidieae: C. flex-
uosum (species 1), C. myanthum (species 2) and M. calantha (species
3). At site 3, 4, 5 and 6, the common epiphytic orchid species were E.
marsupiale (species 4) and C. pardinum (species 5), members of
Epidendreae and Cymbidieae tribes, respectively. In total, 19, 28, 21,
21, 22 and 19 orchid individuals were collected from sites 1, 2, 3, 4, 5
and 6, respectively.
2.2. Screening of root-associated endophytic fungi
Transverse sections from each root sample were cut with a razor
blade. The sections were stained with methyl blue 0.05% solution
(C. I. 42,780, Merck) in sterile water for 3 min and observed under a
Axiostar plus microscope (Carl Zeiss, G€
ottingen, Germany) at
40 magnification to briefly verify the presence of fungal coils, as
evidence of colonized root sections. The selected samples (root
sections) were then surface-disinfected and since the fungi located
in the velamen (dead tissue) are assumed to be surface contami-
nants (e.g. Yuan et al., 2010), the velamen was eliminated. Only the
cortical tissue (alive tissue) was kept for DNA extraction.
2.3. Molecular analysis
For DNA extraction, two/three pieces of colonized roots (1e2cm
long) were used per plant individual. Genomic DNA was extracted
using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) as
described by the manufacturer's instructions. To amplify the fungal
internal transcribed spacer 2 (ITS2) region, the primer pair ITS86F
(Turenne et al., 1999) and ITS4 (White et al., 1990) was used ac-
cording to Jacquemyn et al. (2016). One 20
m
l polymerase chain
reaction (PCR) contained 11.6
m
l of sterile water, 4
m
l of the 5X
Phusion HF Buffer, 0.4
m
l of each primer (0.5
m
M), 0.4
m
l of the
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e7568
10 mM dNTPs, 0.2
m
l of the Phusion High-Fidelity DNA Polymerase
(Thermo Scientific, Wilmington, DE, USA) and 3
m
l of total DNA
extract. PCR conditions were as follows: initial denaturation at
98
C for 30 s followed by 30 cycles at 98
C for 10 s, 60
C for 20 s,
72
C for 30 s, and a final extension step at 72
C for 10 min. A
negative control reaction without DNA template was included in
each PCR. Afterwards, PCR amplification success was tested in 1%
agarose gel electrophoresis and amplicons within the appropriate
size range purified using Wizard
®
SV Gel and Clean-up System
(Promega, Madison, WI, USA). Concentrations and purity of
amplicons were determined using a 2000c Spectrophotometer
NanoDrop
®
(Thermo Scientific, Wilmington, USA). Finally, the
target amplicons were sequenced using Illumina MiSeq
®
technol-
ogy (IMGM Laboratories GmbH, Martinsried, Germany) that
generated 300 bp long paired-end reads (See Supplementary
information).
2.4. Bioinformatics analysis
Operational taxonomic units (OTUs) from the raw Illumina data
were reconstructed using the UPARSE software (Edgar, 2013). First,
single sequences were obtained after the assembly of the over-
lapping paired reads using the ‘fastq_mergepairs’command. Sec-
ond, a quality filter was applied using the ‘fastq_filter’command
with a maximum expected error threshold of 0.3 for single se-
quences. The truncation length of sequences was set to 240 bp to
maximize the number of retained sequences after quality filters.
Third, to remove singletons the ‘derep_fullength’command was
used. Finally, sequences with 97% homology were clustered into the
same OTU using the ‘cluster_otus’command.
Taxonomic assignment of OTUs was performed using the
BLASTN algorithm implemented in UNITE database http://unite.ut.
ee;(Abarenkov et al., 2010a) through the PlutoF (Abarenkov et al.,
2010b) web-based sequence management workbench (2017-06-
28 release). The taxonomically defined OTUs were parsed against
the FunGuild v1.0 (http://www.stbates.org/guilds/app.php) data-
base to designate putative trophic strategies.
2.5. Data analysis and statistics
Read counts of the root-endophyte-OTUs per sample were
converted into presence/absence matrix to evaluate the relation-
ships between root-associated fungal endophyte community and
the host orchid species or study site. Accumulation curves were
constructed for each site with the sample-based rarefaction
method using 100 permutations applied to binary data using Es-
timateS 9.1.1 software (Colwell, 2013). The observed root-
endophyte richness per site was evaluated using the Clench equa-
tion (Sn ¼a*n/(1 þb*n)) executed in STATISTICA (StatSoft, Tulsa,
OK, USA), where ais the rate of new species increment, nis the
sampling effort, and bis a parameter related to the shape of the
curve (Jim
enez-Valverde and Hortal, 2003). In addition, differences
in root-associated fungal endophyte communities from co-existing
orchid species were evaluated using permutational analysis of
variance (PERMANOVA) with 999 permutations using the adonis
function of the vegan package (Oksanen et al., 2016)inR(R
Development Core Team, 2014). Furthermore, to investigate
which fungal communities were more similar to each other, a
pairwise assessment using Jaccard index implemented in the sta-
tistical software SPSS 22 (IBM Corp., Somers, NY, USA) was per-
formed and implemented as in Cevallos et al. (2017).
Analyses related to root-associated fungal endophyte commu-
nity composition across study sites-orchid populations and across
both areas of montane rainforest (PNP and CNP) were performed
based on the binary data (presence/absence matrix) independently
per species. Using SPSS 22 non-metric multidimensional scaling
(NMDS) plots were generated to visualize differences in root-
associated endophytic communities between orchid populations
and between the two areas of montane rainforest. The effect of the
site-population was also tested for significance using PERMANOVA
analysis under the same conditions as aforementioned (999 per-
mutations using the adonis function of the vegan package in R).
Although, endophyte communities' comparison between both
areas of montane rainforest is biased by local environment condi-
tion and by the orchid species, it could give us some insights about
the factors that could affect the endophytes communities'
composition.
3. Results
3.1. Taxonomic coverage of root-associated fungal endophyte
communities and putative life strategy
MiSeq sequencing of the 130 orchid individuals sampled at the
six studied sites yielded a total of 76721 quality-filtered sequences
with a length of 240 bp. During the OTUs reconstruction from the
quality-filtered sequences, singletons, as well as chimeric se-
quences (5.5% of all reconstructed sequences), were discarded to
obtain 3413 OTUs (3% sequence dissimilarity cutoff) assigned to
root-associated fungal endophytes. The data were deposited in
GenBank (Bioproject PRJNA344001 and PRJNA417757). Endophyte
communities identified in association with C. flexuosum, C. myan-
thum, M. calantha, E. marsupiale and C. pardinum included members
of 103 orders (Table S1) in the phyla Ascomycota, Basidiomycota,
Chytridiomycota, Glomeromycota and Zygomycota. Summed over
the two areas of montane rainforest (e.g. PNP and CNP), the highest
number of OTUs (414) was assigned to the Agaricales, while
considering the two areas of montane rainforest separately, Hel-
otiales was the highest at PNP (156 OTUs) and Agaricales at CNP
(385 OTUs). On average, individuals from C. pardinum harbored
more OTUs (Fig. 1). The most frequent OTU was OTU1 belonging to
the Xylariales. It was identified in all the individuals sampled at PNP
and CNP.
Fig. 1. Richness average of operational taxonomic units (OTUs) identified in association
with Cyrtochilum flexuosum,Cyrtochilum myanthum,Maxillaria calantha,Epidendrum
marsupiale and Cyrtochilum pardinum. The whiskers represent the highest and lowest
number of OTUs that an orchid individual could have had.
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e75 69
Using FunGuild database, the putative life strategy was assigned
only to OTUs with taxonomic assignment at ‘species’level (1200
OTUs). Analyzed OTUs were assigned to saprotroph pathotroph-
saprotroph, saprotroph-symbiotroph, pathotroph-saprotroph-
symbiotroph, pathogen-saprotroph-symbiotroph, symbiotroph,
pathotrophs or pathotroph-symbiotroph guilds (Fig. 2). Finally,
evaluation of the endophyte communities using accumulation
curves showed that the fungal communities did not reach a plateau
in any of the study sites (Fig. 3). It was calculated that 48%, 51%, 48%,
46%, 43% and 45% of the estimated richness was observed at site 1,
2, 3, 4, 5 and 6, respectively.
3.2. Root-associated fungal endophyte communities between co-
existing orchid species
PERMANOVA analysis revealed that C. flexuosum, C. myanthum
and M. calantha, co-existing at site 1 and site 2 located at PNP,
harbored significantly different fungal endophyte communities
(P<0.0001 and 0.0003, respectively). Conversely, no significant
changes in the fungal endophyte communities were noticed for
E. marsupiale and C. pardinum that co-exist at CNP, specifically at
sites 3, 4, 5 and 6 (P¼0.2521, 0.6999, 0.4662 and 0.6936, respec-
tively). In addition, the similarity between fungal endophyte com-
munities was assessed in co-existing orchid species pairwisely
using Jaccard indexes calculated by contrasting binary vectors that
represent the presence/absence of OTUs associated with in-
dividuals of each species pair. This analysis could be conducted at
PNP. Analyzing the variance of Jaccard indexes (dependent vari-
able) as a function of the host phylogenic distance(factor) by means
of a one-way ANOVA, we found that at site 1, the similarity was
significantly higher between fungal endophyte communities asso-
ciated with C. flexuosum and C. myanthum than endophyte com-
munities associated with either species and with M. calantha.In
contrast, at site 2 the similarity between the fungal endophyte
communities associated with C. flexuosum and M. calantha were
significantly higher than the similarity between endophyte com-
munities associated with the close relatives Cyrtochilum species
(Table 1).
Fig. 2. Frequency distribution displaying the number of operational taxonomic units (OTUs) belonging to the different trophic guilds identified at Podocarpus National Park (gray
bars) and Cajas National Park (black bars).
Fig. 3. Species accumulation curves describing the identified number of operational taxonomic units (OTUs) of root-associated fungi as a function of the sampled orchids per
studied sites.
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e7570
3.3. Root-associated fungal endophytes between orchid populations
The NMDS ordinations did not show clear distinctive patterns in
fungal endophyte communities between orchid populations
(Supplementary Material Fig. S1). However, the PERMANOVA
analysis revealed that endophytes associated with C. flexuosum, C.
myanthum and M. calantha (orchids distributed at PNP) differed
significantly between orchid populations (P¼0.0031, 0.0029 and
0.0303, respectively). Meanwhile, endophyte communities associ-
ated with E. marsupiale and C. pardinum (orchid distributed at CNP)
were not significantly different between orchid populations
(P¼0.6992 and 0.4948, respectively). The subsequent pairwise
Tukey test performed to compare the means showed that the
endophyte community associated with E. marsupiale at site 3 was
less similar to the endophyte communities at sites 4, 5 and 6
(Supplementary Material Table S2), whereas, the root endophyte
community associated with C. pardinum at site 4 was the most
divergent.
Finally, we identified a set of root-associated fungal endophytes
that overlapped across populations of all evaluated orchid pop-
ulations and another set of endophytes identified only at a partic-
ular site-population combination (Supplementary Material
Table S3 and Fig. S2). Within the overlapped OTUs, members of
Helotiales were the more abundant in populations of C. flexuosum,
C. myanthum and M. calantha while Glomerales and Hypocreales
were the most abundant in the populations of E. marsupiale and
C. pardinum, respectively.
3.4. Differences in root-associated fungal endophyte communities
between the two areas of montane rainforest (PNP vs CNP)
The NMDS ordination of the root-associated fungal endophyte
communities between orchid species at PNP versus orchid spe-
cies at CNP, yielded distinct endophyte communities (Fig. 4). This
was confirmed by the PERMANOVA analysis (P<0.0001). How-
ever, 45 similar OTUs were found at both areas of montane
rainforest and identified in at least one individual per sampled
orchid population. The order with the highest number of OTUs
was Helotiales with six OTUs, followed by Cantharellales and
Sebacinales with four OTUs each; Xylariales, Thelephorales, Pol-
yporales, Pleosporales with three OTUs each; Atractiellales,
Chaetothyriales, Agaricales with two OTUs each; Sordariales,
Hymenochaetales, Capnodiales, Pezizales, Glomerellales, Leca-
norales, Hypocreales, Diaporthales, Gloeophyllales, Ostropales,
Malasseziales, Mucorales with one OTU and one undefined
Ascomycota OTU.
4. Discussion
4.1. Diversity of root-associated fungal endophytes
Root fungal endophytes are generally considered as favorable
inhabitants of plants that may contribute to their productivity and
eventually to the maintenance of ecosystem functions (Jumpponen
et al., 2017). In orchids, although the ecological implications of
these fungal communities are not yet clear, a polyphyletic fungal
group has been reported (Kohout et al., 2013). In the present study,
we applied Illumina MiSeq sequencing to characterize the diversity
of root-associated fungal endophytes of five epiphytic orchid spe-
cies (i.e. C. flexuosum, C. myanthum, M. calantha, E. marsupiale and
C. pardinum) distributed in montane forests of Southern Ecuador.
Out of the 130 collected individuals, 3413 OTUs of endophytic fungi
were detected and up to 1718 different OTUs per orchid species.
Although an important number of OTUs were identified (3413
OTUs), contrasting with earlier studies (e.g. Herrera et al., 2010;
Kottke et al., 2013), the inventories at all sites were still incomplete.
However, the use of Illumina MiSeq technology represents a more
powerful platform for the identification of the microbial commu-
nities, including rare species (Tedersoo et al., 2010) than traditional
sequencing methods (e.g. Sanger sequencing).
The detected OTUs belonged to the Ascomycota, Basidiomycota,
Chytridiomycota, Glomeromycota and Zygomycota phyla. Based on
culture-dependent studies conducted in the same area, Herrera
et al. (2010) and Novotn
a et al. (2018) identified far fewer endo-
phytes. Indeed, Novotn
a et al. (2018) isolated 49 OTUs (13 orders)
from three orchid species, including C. myanthum, while Herrera
et al. (2010) identified 58 isolates (12 orders) from four orchid
species belonging to Pleurothallidinae subtribe. Although, culture-
dependent methods are necessary to preserve the fungal diversity
in culture collections and to perform in vitro studies focused on
orchid-fungi interactions (Novotn
a et al., 2018), these methods
largely underestimate the diversity (Waterman et al., 2011) mainly
because not all fungi are able to grow under in vitro conditions
(Kageyama et al., 2008). The culture-independent method used in
the present study (e.g. NGS) yielded a much higher diversity, likely
to increase our comprehension of fungal endophyte communities
associated with orchids. However, this method is destructive and
no fungi could be preserved in collection. Both approaches should
thus be considered as complementary and useful.
Our results revealed that the Agaricales was the most OTU-rich
order (414 OTUs) summed over the six sampling sites. Members of
Agaricales have been described as mycobionts of achlorophyllous
and green orchids (Bidartondo et al., 2004;Martos et al., 2009) but
also as mycorrhizal symbionts of epiphytic orchids (Kartzinel et al.,
2013). However, the most frequent OTU was OTU1 (identified in
Table 1
Analysis of variance of the similarity between root-associated fungal endophytic communities associated with Cyrtochilum flexuosum, Cyrtochilum myanthum, and Maxillaria
calantha. Similarity between fungal communities is expressed as a Jaccard index.
Comparison Site 1 Site 2
Jaccard indexes mean difference ±std. error Pvalue Jaccard indexes mean difference ±std. error Pvalue
C. flexuosum-C. myanthum vs. C. myanthum-M. calantha 0.04279* ±0.02314 0.001 0.00428 ±0.01602 0.815
C. flexuosum-C. myanthum vs. C. flexuosum-M. calantha 0.03200* ±0.01981 0.001 0.03839* ±0.01754 0.001
C. myanthum-M. calantha vs. C. flexuosum-C. myanthum 0.04279* ±0.02314 0.001 0.00428 ±0.01654 0.815
C. myanthum-M. calantha vs. C. flexuosum-M. calantha 0.01079 ±0.02069 0.434 0.03411* ±0.01939 0.001
C. flexuosum-M. calantha vs. C. flexuosum-C. myanthum 0.03200* ±0.01982 0.001 0.03839* ±0.01754 0.001
C. flexuosum-M. calantha vs. C. myanthum-M. calantha 0.01079 ±0.0207 0.434 0.03411* ±0.01939 0.001
*Difference between Jaccard indexes considered statistically significant (Pvalue 0.05).
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e75 71
130 individuals), taxonomically assigned to Hypoxylon griseo-
brunneum, a member of Xylariales. Similar results were obtained in
previous studies conducted in Southern Ecuador where Su
arez
et al. (2006) and Novotn
a et al. (2018) found that most of the iso-
lates from the epiphytic orchids Stelis spp. belonged to the genus
Hypoxylon. Moreover, in tropical latitudes, members of Xylariales
have been reported as common, diverse and occasionally dominant
root-associated endophytes (Yuan et al., 2009), present with
different host plants, denoting preference and selectivity to some
extent (Chen et al., 2013). In other tropical regions from Central
America and La R
eunion, Cantharellales was the order with the
highest species richness (Martos et al., 2012;Kartzinel et al., 2013).
Fungal endophytes are probably part of a trade-off association
that is still not fully understood (Kia et al., 2016). Undoubtedly,
different fungal taxa impact differently orchid-fungi interactions
through their contrasting set of traits (Kia et al., 2016). Conse-
quently they may have an effect on plant productivity (Bever et al.,
2012) and resistance to pathogen damages (e.g. Chen et al., 2013).
According to their trophic mode, root-associated endophytes that
inhabit plant roots could be symbiotroph, saprotroph, pathotroph-
saprotroph, saprotroph-symbiotroph, pathotroph-symbiotroph or
pathotroph (Ma et al., 2015;Kia et al., 2016). This was also clearly
illustrated in the present study. Indeed, from the 1200 OTUs
assigned to a putative life strategy, most belonged to saprotrophs
(544 OTUs), followed by symbiotrophs (297 OTUs) and to a lesser
extend to pathotrophs (152 OTUs). There were also 79, 50, 47, 24
and 7 fungal endophyte OTUs assigned as pathotroph-saprotroph,
saprotroph-symbiotroph, pathotroph-saprotroph-symbiotroph,
pathotroph-symbiotroph and pathotroph-saprotroph-
symbiotroph, respectively. The ecological roles of endophytic
fungi, assigned putatively in this study, and their effect on orchids,
needs to be further evaluated in order to comprehend the root-
fungal Orchidobiome, the diversity and community structure of
fungi of the roots of orchids. Oliveira et al. (2014) suggested the
necessity to clarify the effects of endophytic fungi on endangered
orchids in Brazil, mainly because fungal communities potentially
contribute to orchid adaptation to changing environmental condi-
tions. Currently, endophytic fungi in orchids remain poorly char-
acterized because the proportion of OTUs identified may be not
assigned to a species in a public database (Oliveira et al., 2014;Ma
et al., 2015). Thus, for fungal taxonomic work, it is of the highest
priority to increase the DNA databases (Abarenkov et al., 2010a)
that will allow a more comprehensive characterization of fungal
communities associated with orchids but also with other plants and
ecosystems. In addition, the use of transmission electron micro-
scopy (e.g. Su
arez et al., 2006,2008) or the evaluation of nutrient
flow (e.g. Cameron et al., 2006) could provide new evidence about
functional roles of root-associated fungal endophytes.
Fig. 4. Fungal endophyte communities detected in Cyrtochilum flexuosum (species 1, black dots), Cyrtochilum myanthum (species 2, black squares), Maxillaria calantha (species 3,
black triangles), Epidendrum marsupiale (species 4, white dots) and Cyrtochilum pardinum (species 5, white squares), distributed in Podocarpus National Park (black figures) and
Cajas National Park (white figures). Stress value 0.109211.
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e7572
4.2. Communities of fungal endophytes associated with co-existing
orchid species
The few studies on root-associated fungal endophytes con-
ducted so far were mostly focused on species identification (e.g.
Bayman and Otero, 2006;Boddington and Dearnaley, 2008).
However, the potential abiotic and biotic drivers determining
fungal endophyte community assembly have usually not been
considered. In the present study, the influence of the orchid species
co-existence in root-associated fungal endophytes was evaluated as
a potential driver for endophyte communities. In natural ecosys-
tems co-existent species, in theory, do not compete for the same
resources (Tilman, 1982) unless small-scale habitat heterogeneity is
present and consequently a segregation of niches could be expected
(Selosse, 2014). Studies on sympatric orchids reported distinctive
mycorrhizal communities that could represent niche partitioning
which may contribute to orchid co-existence (Jacquemyn et al.,
2014;Cevallos et al., 2017). Our results at PNP corroborate these
observations. Indeed, distinct endophyte communities were found
between co-existing orchid species at both study sites. For instance,
we found mycorrhizal fungi that promote nutrient uptake
(Rasmussen, 2002) but also non-mycorrhizal fungi that are thought
to be sources of bioactive compounds (Xing et al., 2015). Such di-
versity in fungal functional attributes could help orchids to adapt to
the changing environmental conditions (Oliveira et al., 2014).
Considering the mutualism-parasitism continuum hypothesis
(Johnson et al., 1997), changes in ecological context could cause a
transition from mutualism to parasitism or vice versa. In orchid
communities, species form complex networks of orchid-fungi in-
teractions (Martos et al., 2012) although such associations do not
always have benefits for both partners. For instance, at the early
developmental stages of the orchid, the associated fungi do not
receive any reward from the orchid seeds (Smith and Read, 2008),
and the relationship has been considered parasitism to some extent
(Dearnaley, 2007). Meanwhile, the interaction between fungi and
adult orchids, where both partners have benefits (or at least a
transitory backflow), is recognized as mutualism (Cameron et al.,
2006;Fochi et al., 2017). In some interactions, fungi that facilitate
seed germination could also associate with orchids at the adult
stage (Cameron et al., 2008). In this case, there is a transition in the
relationship, from parasitism to mutualism (van der Heijden et al.,
2015).
In addition, host phylogeny seems to drive fungal community
assemblage. This was evidenced by Jacquemyn et al. (2011) who
identified similar mycorrhizal fungi associated with different Orchis
species. Likewise, in the Angraecoid orchid subtribe the reported
mycorrhizal fungi were closely related species (Martos et al., 2012).
Interestingly, in the present study we found (in site 1) that closely
related Cyrtochilum species shared more similar endophytic fungal
communities among each other than with M. calantha, in accor-
dance with the results of Cevallos et al. (2017). This observation
was, however, not repeated at site 2. Therefore, increasing the
number of individuals, at both sites, should help to corroborate
whether the host phylogeny is a driver of endophyte community
composition.
In contrast to the findings at sites 1 and 2 (PNP), co-existing
orchids E. marsupiale and C. pardinum, at CNP (sites 3, 4, 5 and 6),
had similar fungal endophyte communities although both orchid
species belong to distinctive tribes (Epidendreae and Cymbidieae,
respectively). In this case host phylogeny is not a determining
driver for endophyte community structure. Deep phylogenetic
studies evaluating the evolutionary histories between orchids
could contribute to deduce more precise ecological premises (e.g.
Freudenstein and Chase, 2015).
Apart from host phylogeny, fungal endophyte community
composition could be influenced by many abiotic and biotic factors
(Barnes et al., 2016). For instance, altitude is widely recognized as
an important factor that determines the fungal communities in soil,
especially in the Andes (Geml et al., 2014). Although microorgan-
isms are considered globally cosmopolitan, abiotic factors probably
impact the fungal diversity, especially in habitats with harsh
environmental conditions (Jumpponen et al., 2017). However, the
45 OTUs identified at both PNP and CNP, seem not to be restricted
by abiotic factors. It is likely that the fungi that overlapped between
all studied orchids, at both areas of montane rainforest, have
exceptional characteristics and are prone to develop under a wider
gradient of abiotic conditions (Cray et al., 2013).
4.3. Root-associated fungal endophytes across orchid populations
Factors that could drive fungal endophyte community compo-
sition across orchid populations are still not fully described or
assessed. Environment and geography affect the diversity and
composition of some root-associated fungal communities but the
effect of each factor could be variable or specific across ecosystems
(Barnes et al., 2016). Here we showed that root endophyte com-
munities associated with C. flexuosum, C. myanthum and
M. calantha, evaluated independently per orchid species, varied
substantially across orchid population, suggesting that host phy-
logeny is not a determining factor for endophyte community
composition. Our results corroborate previous observations of
Jacquemyn et al. (2016) on mycorrhizal fungi, revealing that fungi
can be characteristic of a specific site as a result of local conditions.
This supported the theory that orchid endophytic fungi are sub-
jected to local selection because of particular environmental con-
ditions (Ma et al., 2015) at each sample site. At PNP, the study sites
have similar forest structure and are classified as evergreen upper
montane forests (Beck et al., 2008), but each site has particular
temperature and rain conditions. Oliveira et al. (2014) reported
similar results in an early study in a Neotropical region in Brazil,
where Hadrolaelia jongheana, Hoffmannseggella caulescens, and
Hoffmannseggella cinnabarina displayed different endophyte com-
munity composition with few overlapping fungal endophytes as a
consequence of local factors (soil conditions, vegetation and
climate).
Our results at CNP showed that populations of E. marsupiale and
C. pardinum, evaluated independently per orchid species, had
similar endophyte communities. This was probably because the
sites at CNP are located in the same mountain foothill under similar
temperature and rainfall conditions (ETAPA, 2005), corroborating
the effect of the site on fungal endophyte communities. Moreover,
it is likely that proximity between sites could facilitate fungal
dispersal (Jumpponen et al., 2017). As a consequence, overlapping
endophytic fungi are more probable. Notwithstanding that very
little is known about the restrictions of fungal endophyte distri-
bution (Queloz et al., 2011), the possibility that our results were
somewhat the consequence of geographic proximity between sites
or an effect of contrasting environmental conditions due to high
altitudinal levels at mountain areas, cannot be ruled out. Moreover,
it is not excluded that orchid species in CNP present an ancestral
ecological conservatism in endophyte preferences. Because iden-
tified fungi were not specifically associated with a single orchid
species, it is probable that orchids had preferences for several
widely distributed fungal groups (Otero et al., 2007).
In conclusion, the fungal endophyte communities assessed on
epiphytic orchids seem to follow different strategies of assembly.
Fungal endophytes at PNP appear to be impacted by host phylogeny
and sites, while the results at CNP suggest that neither host phy-
logeny nor the sites had an effect on fungal endophyte commu-
nities. Either way, the assessment of additional orchid species and
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e75 73
sites could help elucidate which factors determine root-associated
fungal endophytes.
4.4. Comparison of endophyte communities between the two areas
of montane rainforest (PNP vs CNP)
Sampling approaches at both areas of montane rainforest were
not totally comparable because each one has specific environ-
mental conditions that resulted in a particular ecosystem structure
(Baquero et al., 2004) in addition to the specific orchid species
sampled per area. Thus, a thorough analysis combining data from
both areas of montane rainforest to make inferences about the ef-
fect of biotic or abiotic factors on OMF communities was beyond the
scope of this study. However, the contrast of all the fungal endo-
phytes associated with epiphytic orchids (independently of orchid
species) between the two areas of montane rainforest could give
some insights about the fungal community structure at a larger
scale. We showed that orchid root-associated fungal endophyte
communities were highly different between PNP and CNP. Our re-
sults corroborate the hypothesis of Baas Becking (1934) that local
environmental conditions could configure fungal endophyte com-
munities, assuming the hypothesis that microorganisms are widely
distributed and the environment is shaping which ones are able to
grow. In addition to the distinct fungal endophyte community
composition at PNP and CNP, we also identified a set of endophytic
fungi that were present in both areas of montane rainforest.
Although the distance between both areas of montane rainforest is
approximately 125 km, 45 fungal OTUs core-species (21 orders)
were found in both situations. These fungi were mycorrhizal, sap-
robes or latent pathogens. Following the premise that endophytes
are cosmopolitan, it is likely that overlapping endophytic fungi
have a large population distribution (Fitter, 2005;Jumpponen et al.,
2017) but may not be very specialized. Overlapping endophytes
probably have ecological plasticity and are able to play different
roles in the interaction (Pecoraro et al., 2017). Based on observa-
tions on orchid mycorrhizal fungi (Cevallos et al., 2017), we hy-
pothesize that endophytic fungal core-species could represent an
advantage because being frequently available they could fill some
physiological demands (e.g. nutritional or pathogen defense) when
other fungi are not available. Clarifying the potential roles of the
fungal endophytes in orchids' life needs to be explored to better
understand the ecological dynamics of plant-fungi interactions
(Oliveira et al., 2014).
Acknowledgements
This work was supported by the Acad
emie de Recherche et
d’Enseignement Sup
erieur Wallonie-Bruxelles (ARES) within the
frame of a PRD project entitled 'Reinforcement of the fungal
expertise in Ecuador via case studies of fungal plants interactions in
selected ecosystems and the development of biotechnology-ori-
ented fungal resources' and the Secretaria de Educaci
on Superior,
Ciencia, Tecnología e Innovaci
on of Ecuador [grant number PIC-13-
ETAPA-003].
Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.funeco.2018.05.002.
References
Abarenkov, K., Henrik Nilsson, R., Larsson, K.-H., Alexander, I.J., Eberhardt, U.,
Erland, S., Høiland, K., Kjøller, R., Larsson, E., Pennanen, T., Sen, R., Taylor, A.F.S.,
Tedersoo, L., Ursing, B.M., Vrålstad, T., Liimatainen, K., Peintner, U., K~
oljalg, U.,
2010a. The UNITE database for molecular identification of fungi recent updates
and future perspectives. New Phytol. 186, 281e285.
Abarenkov, K., Tedersoo, L., Nilsson, R.H., Vellak, K., Saar, I., Veldre, V., Parmasto, E.,
Prous, M., Aan, A., Ots, M., Kurina, O., Ostonen, I., J~
ogeva, J., Halapuu, S.,
P~
oldmaa, K., Toots, M., Truu, J., Larsson, K.H., K~
oljalg, U., 2010b. Plutof-a web
based workbench for ecological and taxonomic research, with an online
implementation for fungal its sequences. Evol. Bioinforma. 6, 189e196.
Baas Becking, L.G.M., 1934. Geobiologie of inleiding tot de milieukunde. W.P. Van
Stockum &Zoon, Den Haag.
Baquero, F., Sierra, R., Ord
o~
nez, L., Tip
an, M., Espinosa, L., Rivera, MaB., Soria, P.,
2004. La vegetaci
on de los Andes del Ecuador. EcoCiencia. ESLA. EcoPar. Div.
Geogr
aficaeIGM, pp. 1e28.
Barnes, C.J., Van der Gast, C.J., Burns, C.A., McNamara, N.P., Bending, G.D., 2016.
Temporally variable geographical distance effects contribute to the assembly of
root-associated fungal communities. Front. Microbiol. 7, 195.
Bayman, P., Otero, J.T., 2006. Microbial endophytes of orchid roots. In: Schulz, B.,
Boyle, C., Sieber, T. (Eds.), Microbial Root Endophytes. Springer-Verlag, Berlin,
pp. 153e178.
Beck, E., Makeschin, F., Haubrich, F., Richter, M., Bendix, J., Valarezo, C., 2008. The
ecosystem (Reserva Biol
ogica san Francisco). In: Beck, E., Bendix, J., Kottke, I.,
Makeschin, F., Mosandl, R. (Eds.), Gradients in a Tropical Mountain Ecosystem of
Ecuador. Ecological Studies, No. 198. Springer, Heidelberg, pp. 1e14.
Bendix, J., Rollenbeck, R., Richter, M., Fabian, P., Emck, P., 2008. Climate. In: Beck, E.,
Bendix, J., Kottke, I., Makeschin, F., Mosandl, R. (Eds.), Gradients in a Tropical
Mountain Ecosystem of Ecuador. Ecological Studies, No. 198. Springer, Heidel-
berg, pp. 63e73.
Bever, J.D., Platt, T.G., Morton, E.R., 2012. Microbial population and community
dynamics on plant roots and their feedbacks in plant communities. Annu. Rev.
Microbiol. 66, 265e283.
Bidartondo, M.I., Burghardt, B., Gebauer, G., Bruns, T.D., Read, D.J., 2004. Changing
partners in the dark: isotopic and molecular evidence of ectomycorrhizal liai-
sons between forest orchids and trees. Proc. Biol. Sci. 271, 1799e1806.
Boddington, M., Dearnaley, J.D.W., 2008. Morphological and molecular identifica-
tion of fungal endophytes from roots of Dendrobium speciosum. Proc. R. Soc.
Queensl 114, 13e17.
Brundrett, M.C., 2002. Coevolution of roots and mycorrhizas of land plants. New
Phytol. 154, 275e304.
Bunch, W.D., Cowden, C.C., Wurzburger, N., Shefferson, R.P., 2013. Geography and
soil chemistry drive the distribution of fungal associations in lady's slipper
orchid, Cypripedium acaule. Botany 91, 850e856.
Cameron, D.D., Johnson, I., Read, D.J., Leake, J.R., 2008. Giving and receiving:
measuring the carbon cost of mycorrhizas in the green orchid. Goodyera
Repens. New Phytol. 180, 176e184 .
Cameron, D.D., Leake, J.R., Read, D.J., 2006. Mutualistic mycorrhiza in orchids: evi-
dence from plant-fungus carbon and nitrogen transfers in the green-leaved
terrestrial orchid Goodyera repens. New Phytol. 171, 405e416.
Cevallos, S., S
anchez-Rodríguez, A., Decock, C., Declerck, S., Su
arez, J.P., 2017. Are
there keystone mycorrhizal fungi associated to tropical epiphytic orchids .
Mycorrhiza 27, 225e232.
Chen, J., Zhang, L.C., Xing, Y.M., Wang, Y.Q., Xing, X.K., Zhang, D.W., Liang, H.Q.,
Guo, S.X., 2013. Diversity and taxonomy of endophytic Xylariaceous fungi from
medicinal plants of Dendrobium (Orchidaceae). PLoS One 8 e58268.
Colwell, R.K., 2013. EstimateS: Statistical Estimation of Species Richness and Shared
Species from Samples. User's Guide and application published at:, Version 9.
http://purl.oclc.org/estimates.
Cray, J.A., Bell, A.N.W., Bhaganna, P., Mswaka, A.Y., Timson, D.J., Hallsworth, J.E.,
2013. The biology of habitat dominance; can microbes behave as weeds?
Microb. Biotechnol 6, 453e492.
Dearnaley, J.D.W., 2007. Further advances in orchid mycorrhizal research. Mycor-
rhiza 17, 475e486.
Dearnaley, J.D.W., Martos, F., Selosse, M.A., 2012. Orchid mycorrhizas: molecular
ecology, physiology, evolution and conservation aspects. In: Hock, B. (Ed.), The
Mycota (A Comprenhensive Treatise on Fungias Experimental Systems for Basic
and Applied Research). Springer, Heidelberg, pp. 207e230. Berlin.
Dressler, R.L., 1990. The Orchids: Natural History and Classification. USA. Harvard
University Press, Cambridge MA.
Edgar, R.C., 2013. UPARSE: highly accurate OTU sequences from microbial amplicon
reads. Nat. Methods 10, 996e998.
ETAPA, 2005. Aprobado vía acuerdo ministerial el 1 de abril del Plan de manejo
integral del Parque Nacional Cajas (Cuenca, Ecuador).
Fitter, A.H., 2005. Darkness visible: reflections on underground ecology. J. Ecol. 93,
231e243.
Fochi, V., Chitarra, W., Kohler, A., Voyron, S., Singan, V.R., Lindquist, E.A., Barry, K.W.,
Girlanda, M., Grigoriev, I.V., Martin, F., Balestrini, R., Perotto, S., 2017. Fungal and
plant gene expression in the Tulasnella calospora -Serapias vomeracea symbiosis
provides clues about nitrogen pathways in orchid mycorrhizas. New Phytol. 213,
365e379. https://doi.org/10.1111/nph.14279.
Freudenstein, J.V., Chase, M.W., 2015. Phylogenetic relationships in Epidendroideae
(Orchidaceae), one of the great flowering plant radiations: progressive
specialization and diversification. Ann. Bot. 115, 665e681.
Garnica, S., Riess, K., Bauer, R., Oberwinkler, F., Weiß, M., 2013. Phylogenetic di-
versity and structure of sebacinoid fungi associated with plant communities
along an altitudinal gradient. FEMS Microbiol. Ecol. 83, 265e278.
Geml, J., Pastor, N., Fernandez, L., Pacheco, S., Semenova, T., Becerra, A.,
Wicaksono, C., Nouhra, E., 2014. Large-scale fungal diversity assessment in the
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e7574
Andean Yungas forests reveals strong community turnover among forest types
along an altitudinal gradient. Mol. Ecol. 23, 2452e2472.
G€
otzenberger, L., de Bello, F., Bråthen, K.A., Davison, J., Dubuis, A., Guisan, A., Lep
s, J.,
Lindborg, R., Moora, M., P€
artel, M., Pellissier, L., Pottier, J., Vittoz, P., Zobel, K.,
Zobel, M., 2012. Ecological assembly rules in plant communities-approaches,
patterns and prospects. Biol. Rev. 87, 111e127.
Gradstein, S.R., Homeier, J., Gansert, D., 2008. The Tropical Mountain Forest - Pat-
terns and Processes in a Biodiversity Hotspot. University Press, G€
ottingen.
Herrera, P., Kottke, I., Molina, M.C., M
endez, M., Su
arez, J.P., 2018. Generalism in the
interaction of Tulasnellaceae mycobionts with orchids characterizes a biodi-
versity hotspot in the tropical Andes of Southern Ecuador. Mycoscience 59,
38e48.
Herrera, P., Su
arez, J.P., Kottke, I., 2010. Orchids keep the ascomycetes outside: a
highly diverse group of ascomycetes colonizing the velamen of epiphytic or-
chids from a tropical mountain rainforest in Southern Ecuador. Mycology 1,
262e268.
Jacquemyn, H., Brys, R., Merckx, V.S.F.T., Waud, M., Lievens, B., Wiegand, T., 2014.
Coexisting orchid species have distinct mycorrhizal communities and display
strong spatial segregation. New Phytol. 202, 616e627.
Jacquemyn, H., Brys, R., Waud, M., Busschaert, P., Lievens, B., 2015. Mycorrhizal
networks and coexistence in species-rich orchid communities. New Phytol. 206,
112 7e113 4.
Jacquemyn, H., Merckx, V.V., Brys, R., Tyteca, D., Cammue, B.P., Honnay, O.a.,
Lievens, B., 2011. Analysis of network architecture reveals phylogenetic con-
straints on mycorrhizal specificity in the genus Orchis (Orchidaceae). New
Phytol. 192, 518e528.
Jacquemyn, H., Waud, M., Lievens, B., Brys, R., 2016. Differences in mycorrhizal
communities between Epipactis palustris, E. helleborine and its presumed sister
species E. neerlandica. Ann. Bot. 118 (1), 105e114.
Jim
enez-Valverde, A., Hortal, J., 2003. Las curvas de acumulaci
on de especies y la
necesidad de evaluar la calidad de los inventarios biol
ogicos. Rev. Ib
erica Ara-
cnol. 8, 151e161.
Johnson, N.C., Graham, J.H., Smith, F.A., 1997. Functioning of mycorrhizal associa-
tions along the mutualism-parasitism continuum. New Phytol. 135, 575e585.
Jumpponen, A., Herrera, J., Porras-Alfaro, A., Rudgers, J., 2017. Biogeography of root-
associated fungal endophytes. In: Tedersoo, L. (Ed.), Biogeography of Mycor-
rhizal Symbiosis. Springer, Cham, pp. 195e222.
Kageyama, S.A., Mandyam, K.G., Jumpponen, A., 2008. Diversity, function and po-
tential Applications of the root-associated endophytes. In: Varma, A. (Ed.),
Mycorrhiza: State of the Art, Genetics and Molecular Biology, Eco-function,
Biotechnology, Eco-physiology, Structure and Systematics. Springer Berlin
Heidelberg, Berlin, Heidelberg, pp. 29e57.
Kartzinel, T.R., Trapnell, D.W., Shefferson, R.P., 2013. Highly diverse and spatially
heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad
biogeographic or environmental features. Mol. Ecol. 22, 5949e5961.
Kia, S.H., Glynou, K., Nau, T., Thines, M., Piepenbring, M., Maci
a-vicente, J.G., 2016.
Influence of phylogenetic conservatism and trait convergence on the in-
teractions between fungal root endophytes and plants. ISME J. 1e14.
Kohout, P., T
e
sitelov
a, T., Roy, M., Vohník, M., Jers
akov
a, J., 2013. A diverse fungal
community associated with Pseudorchis albida (Orchidaceae) roots. Fungal Ecol.
6, 50e64.
Kottke, I., Setaro, S., Haug, I., Herrera, P., Cruz, D., Fries, A., Gawlik, J., Homeier, J.,
Werner, F.A., Gerique, A., Su
arez, J.P., 2013. Mycorrhiza networks promote
biodiversity and stabilize the tropical mountain rain forest ecosystem: per-
spectives for understanding complex communities. In: Bendix, J., Beck, E.,
Br€
auning, A., Makeschin, F., Mosandl, R., Scheu, S., Wilcke, W. (Eds.), Ecosystem
Services, Biodiversity and Environmental Change in a Tropical Mountain
Ecosystem of South Ecuador. Springer, Berlin Heidelberg, 187e203.
Kristiansen, K.A., Taylor, D.L., Kjøller, R., Rasmussen, H.N., Rosendahl, S., 2001.
Identification of mycorrhizal fungi from single pelotons of Dactylorhiza majalis
(Orchidaceae) using single-strand conformation polymorphism and mito-
chondrial ribosomal large subunit DNA sequences. Mol. Ecol. 10, 2089e2093.
Ma, X., Kang, J., Nontachaiyapoom, S., Wen, T., Hyde, K.D., 2015. Non-mycorrhizal
endophytic fungi from orchids. Curr. Sci. 109, 36.
Mandl, N., Lehnert, M., Kessler, M., Gradstein, S.R., 2010. A comparison of alpha and
beta diversity patterns of ferns, bryophytes and macrolichens in tropical
montane forests of southern Ecuador. Biodivers. Conserv. 19, 2359e2369.
Martos, F., Dulormne, M., Pailler, T., Bonfante, P., Faccio, A., Fournel, J., Dubois, M.-P.,
Selosse, M.-A., 2009. Independent recruitment of saprotrophic fungi as
mycorrhizal partners by tropical achlorophyllous orchids. New Phytol. 184,
668e681.
Martos, F., Munoz, F., Pailler, T., Kottke, I., Gonneau, C., Selosse, M.A., 2012. The role
of epiphytism in architecture and evolutionary constraint within mycorrhizal
networks of tropical orchids. Mol. Ecol. 21, 5098e5109.
Minga, D., Ansaloni, R., Verdugo, A., Ulloa, C., 2016. Flora del P
aramo del Cajas,
Ecuador. Universidad del Azuay, Cuenca.
Montesinos, F., 1996.
Arboles y arbustos del bosque Maz
an. Tomo I. ETAPA. Empresa
Munic. Tel
efonos, Agua Potable Alcantarillado.
Novotn
a, A., Benítez,
A., Herrera, P., Cruz, D., Filipczykov
a, E., Su
arez, J.P., 2018. High
diversity of root-associated fungi isolated from three epiphytic orchids in
southern Ecuador. Mycoscience 59 (1), 24e32. http://doi.org/10.1016/j.myc.
2017.07.007.
Oksanen, A.J., Blanchet, F.G., Kindt, R., Legendre, P., Minchin, P.R., Hara, R.B.O.,
Simpson, G.L., Solymos, P., Stevens, M.H.H., Wagner, H., 2016. Package “Vegan”:
Community Ecology Package.
Oliveira, S.F., Bocayuva, M.F., Veloso, T.G.R., Bazzolli, D.M.S., da Silva, C.C.,
Pereira, O.L., Kasuya, M.C.M., 2014. Endophytic and mycorrhizal fungi associated
with roots of endangered native orchids from the Atlantic Forest, Brazil. My-
corrhiza 24, 55e64.
Otero, J.T., Flanagan, N.S., Herre, E.A., Ackerman, J.D., Bayman, P., 20 07. Widespread
mycorrhizal specificity correlates to mycorrhizal function in the neotropical,
epiphytic orchid Ionopsis utricularioides (Orchidaceae). Am. J. Bot. 94,
194 4e1950.
Pecoraro, L., Huang, L., Caruso, T., Perotto, S., Girlanda, M., Cai, L., Liu, Z.-J., 2017.
Fungal diversity and specificity in Cephalanthera damasonium and C. longifolia
(Orchidaceae) mycorrhizas. J. Syst. Evol. 9999, 1e12.
Pridgeon, A., 1995. The Illustrated Encyclopaedia of Orchids. Timber Press, Portland.
Queloz, V., Sieber, T.N., Holdenrieder, O., McDonald, B.A., Grünig, C.R., 2011. No
biogeographical pattern for a root-associated fungal species complex. Glob.
Ecol. Biogeogr. 20, 160e169.
R Development Core Team, 2014. R: a Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Austria, Vienna.
Rasmussen, H.N., 2002. Recent developments in the study of orchid mycorrhiza.
Plant Soil 245, 149e163.
Riofrío, L.R., Naranjo, C.N., Iriondo, J.M.I., Torres, E.T., 2007. Spatial structure of
Pleurothallis, Masdevallia, Lephanthes and Epidendrum epiphytic orchids in a
fragment of montane cloud forest in South Ecuador. Lankesteriana 7, 1e2.
https://doi.org/https://doi.org/10.15517/lank.v7i1-2.18447.
Riofrío, M.L., Cruz, D., Torres, E., de la Cruz, M., Iriondo, J.M., Su
arez, J.P., 2013.
Mycorrhizal preferences and fine spatial structure of the epiphytic orchid
Epidendrum rhopalostele. Am. J. Bot. 100, 1e10.
Selosse, M.A., 2014. The latest news from biological interactions in orchids: in love,
head to toe. New Phytol. 202, 337e340.
Setaro, S., Kottke, I., Oberwinkler, F., 2006. Anatomy and ultrastructure of mycor-
rhizal associations of neotropical Ericaceae. Mycol. Prog. 5 (243). https://doi.
org/10.1007/s11557-006-0516-7.
Sklen
ar, P., Duskov
a, E., Baslev, H., 2011. Tropical and temperate: evolutionary his-
tory of P
aramo Flora. Botanical Rev. 77, 71e108.
Smith, D.P., Peay, K.G., 2014. Sequence depth, not PCR replication, improves
ecological inference from next generation DNA sequencing. PLoS One 9 (2)
e90234.
Smith, S.E., Read, D., 2008. Mycorrhizal Symbiosis. Academic Press, New York, USA.
Su
arez, J.P., Eguiguren, S., Herrera, P., Jost, L., 2016. Do mycorrhizal fungi drive
speciation in Teagueia (Orchidaceae) in the upper Pastaza watershed of
Ecuador? Symbiosis 36, 135e136.
Su
arez, J.P., Weiss, M., Abele, A., Garnica, S., Oberwinkler, F., Kottke, I., 2006. Diverse
tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud
forest. Mycol. Res. 110, 1257e1270.
Su
arez, J.P., Weiß, M., Abele, A., Oberwinkler, F., Kottke, I., 2008. Members of
Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a
neotropical mountain rain forest. Mycol. Prog. 7, 75e85. https://doi.org/10.
1007/s11557-008-0554-4.
Sudheep, N.M., Sridhar, K.R., 2012. Non-mycorrhizal fungal endophytes in two or-
chids of Kaiga forest (Western Ghats), India. J. For. Res. 23, 453e460.
Tedersoo, L., Nilsson, R.H., Abarenkov, K., Jairus, T., Sadam, A., Saar, I., 2010. Methods
454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi pro-
vide similar results but reveal substantial methodological biases. New Phytol.
188, 291e301.
T
e
sitelov
a, T., Kotílinek, M., Jers
akov
a, J., Joly, F.X., Ko
snar, J., Tatarenko, I.,
Selosse, M.-A., 2015. Two widespread green Neottia species (Orchidaceae) show
mycorrhizal preference for Sebacinales in various habitats and ontogenetic
stages. Mol. Ecol. 24, 1122e1134 .
Tilman, D., 1982. Resource Competition and Community Structure. Princeton Uni-
versity Press, New Jersey.
Turenne, C., Sanche, S., Hoban, D., Karlowsky, J., Kabani, A., 1999. Rapid identifica-
tion of fungi by using the ITS2 genetic region and an automated fluorescent
capillary electrophoresis System. J. Clin. Cal. Microbiol. 37, 1846e1851.
van der Heijden, M.G.A., Martin, F.M., Selosse, M.-A., Sanders, I.R., 2015. Mycorrhizal
ecology and evolution : the past, the present, and the future. New Phytol. 205,
1406e1423.
Waterman, R.J., Bidartondo, M.I., Stofberg, J., Combs, J.K., Gebauer, G., Savolainen, V.,
Barraclough, T.G., Pauw, A., 2011. The effects of above- and belowground mu-
tualisms on orchid speciation and coexistence. Am. Nat. 177, E54eE68.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of
fungal ribosomial RNA genes for phyologenetics. In: Innis, M.A., Gelfand, D.,
Sninsky, J., White, T.J. (Eds.), PCR Protocols: a Guide to Methods and Applica-
tions. Academic Press, Inc., New York, pp. 315e322.
Xing, X., Gai, X., Liu, Q., Hart, M.M., Guo, S., 2015. Mycorrhizal fungal diversity and
community composition in a lithophytic and epiphytic orchid. Mycorrhiza 25,
289e296.
Yuan, L., Yang, Z., Li, S., Hu, H., Huang, J.L., 2010. Mycorrhizal specificity, preference,
and plasticity of six slipper orchids from South Western China. Mycorrhiza 20,
559e568.
Yuan, Z.L., Chen, Y.C., Yang, Y., 2009. Diverse non-mycorrhizal fungal endophytes
inhabiting an epiphytic, medicinal orchid (Dendrobium nobile): estimation and
characterization. World J. Microbiol. Biotechnol. 25, 295e303.
Zotz, G., Winkler, U., 2013. Aerial roots of epiphytic orchids: the velamen radicum
and its role in water and nutrient uptake. Oecologia 171, 733e741. https://doi.
org/10.1007/s00442-012-2575-6.
S. Cevallos et al. / Fungal Ecology 34 (2018) 67e75 75