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Untangling factors that drive community composition of root associated fungal endophytes of Neotropical epiphytic orchids

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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 communities 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.
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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
amplied 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 identied 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 inuence 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 articial 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 identied 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 specic 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 identied 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 ll this gap, the rst 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 exuosum, 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 rst
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
uctuates 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. ex-
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 magnication to briey 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 Scientic, 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 nal extension step at 72
C for 10 min. A
negative control reaction without DNA template was included in
each PCR. Afterwards, PCR amplication success was tested in 1%
agarose gel electrophoresis and amplicons within the appropriate
size range puried 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 Scientic, 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_mergepairscommand. Sec-
ond, a quality lter was applied using the fastq_ltercommand
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 lters.
Third, to remove singletons the derep_fullengthcommand was
used. Finally, sequences with 97% homology were clustered into the
same OTU using the cluster_otuscommand.
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 dened 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 signicance 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-ltered sequences
with a length of 240 bp. During the OTUs reconstruction from the
quality-ltered 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 identied in association with C. exuosum, 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 identied in all the individuals sampled at PNP
and CNP.
Fig. 1. Richness average of operational taxonomic units (OTUs) identied in association
with Cyrtochilum exuosum,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 specieslevel (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. exuosum, C. myanthum
and M. calantha, co-existing at site 1 and site 2 located at PNP,
harbored signicantly different fungal endophyte communities
(P<0.0001 and 0.0003, respectively). Conversely, no signicant
changes in the fungal endophyte communities were noticed for
E. marsupiale and C. pardinum that co-exist at CNP, specically 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
signicantly higher between fungal endophyte communities asso-
ciated with C. exuosum 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. exuosum and M. calantha were
signicantly 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 identied at Podocarpus National Park (gray
bars) and Cajas National Park (black bars).
Fig. 3. Species accumulation curves describing the identied 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. exuosum, C.
myanthum and M. calantha (orchids distributed at PNP) differed
signicantly 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 signicantly 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 identied a set of root-associated fungal endophytes
that overlapped across populations of all evaluated orchid pop-
ulations and another set of endophytes identied 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. exuosum,
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 conrmed by the PERMANOVA analysis (P<0.0001). How-
ever, 45 similar OTUs were found at both areas of montane
rainforest and identied 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 undened
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 ve epiphytic orchid spe-
cies (i.e. C. exuosum, 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 identied (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 identication 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) identied 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) identied 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 (identied in
Table 1
Analysis of variance of the similarity between root-associated fungal endophytic communities associated with Cyrtochilum exuosum, 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. exuosum-C. myanthum vs. C. myanthum-M. calantha 0.04279* ±0.02314 0.001 0.00428 ±0.01602 0.815
C. exuosum-C. myanthum vs. C. exuosum-M. calantha 0.03200* ±0.01981 0.001 0.03839* ±0.01754 0.001
C. myanthum-M. calantha vs. C. exuosum-C. myanthum 0.04279* ±0.02314 0.001 0.00428 ±0.01654 0.815
C. myanthum-M. calantha vs. C. exuosum-M. calantha 0.01079 ±0.02069 0.434 0.03411* ±0.01939 0.001
C. exuosum-M. calantha vs. C. exuosum-C. myanthum 0.03200* ±0.01982 0.001 0.03839* ±0.01754 0.001
C. exuosum-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 signicant (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 identied 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
ow (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 exuosum (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 gures) and
Cajas National Park (white gures). 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 identication (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 inuence 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 benets 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 benets (or at least a
transitory backow), 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
identied 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 ndings 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 inuenced 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 identied 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 specic across ecosystems
(Barnes et al., 2016). Here we showed that root endophyte com-
munities associated with C. exuosum, 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 specic 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 classied 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-
tied fungi were not specically 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 specic environ-
mental conditions that resulted in a particular ecosystem structure
(Baquero et al., 2004) in addition to the specic 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 congure 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 identied 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 ll 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
dEnseignement 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.
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... Finally, our results match the pattern of detecting a few highly abundant fungal taxa accompanied by a large number of OTUs with low abundances or representing rare taxa in orchid-fungal studies (Jacquemyn et al. 2011Okayama et al. 2012;Martos et al. 2012;Sakamoto et al. 2015;Waud et al. 2016;Cevallos et al. 2017Cevallos et al. , 2018aCevallos et al. , 2018bXing et al. 2017;Qin et al. 2019). Highly-abundant mycorrhizal fungi are expected to play a major part in nutrient and water acquisition and increase a plant's adaptability and resilience (Guimarães et al. 2011;Cevallos et al. 2017), while the role of sequence-scarce and rare OTUs is less clear. ...
... In particular, OTU23 was highly abundant in both species but mostly absent from E. odontochilum and O. klotzschianum (Supplementary Table 2). Although there is evidence that supports orchid niche partitioning through specific mycorrhizal associations (Jacquemyn et al. 2012Pellegrino et al. 2014;Cevallos et al. 2017), a few studies carried out in tropical epiphytic orchids have also encountered important OMF overlaps among coexisting orchid species (Cevallos et al. 2018b;Herrera et al. , 2019. For instance, sympatric individuals of Cyrtochilum pardinum and Epidendrum marsupiale associated with a similar OMF community in at least four different sites in the Andes forests (Cevallos et al. 2018b), which was later supported by Herrera et al. (2019). ...
... Although there is evidence that supports orchid niche partitioning through specific mycorrhizal associations (Jacquemyn et al. 2012Pellegrino et al. 2014;Cevallos et al. 2017), a few studies carried out in tropical epiphytic orchids have also encountered important OMF overlaps among coexisting orchid species (Cevallos et al. 2018b;Herrera et al. , 2019. For instance, sympatric individuals of Cyrtochilum pardinum and Epidendrum marsupiale associated with a similar OMF community in at least four different sites in the Andes forests (Cevallos et al. 2018b), which was later supported by Herrera et al. (2019). Tentatively, co-existing species are not capable of thriving if they use similar resources (Tilman 1982) unless characteristics of the microsite allow for habitat heterogeneity and the segregation of the niche; for example, separation by abiotic conditions of the canopy (Rasmussen and Rasmussen 2018). ...
Article
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Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization (i.e., sets of unique and dominant mycorrhizal fungi associated with a particular host species). However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and composition of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously recognized OMF taxa, and that the four coexisting orchid species have both a set of shared mycorrhizal fungi and a group of fungi unique to an orchid species. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time. This study provides evidence for the utilization of specific OMF that may be involved in niche segregation, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others.
... In particular, OTU23 was highly abundant in both species but mostly absent from E. odontochilum and O. klotzschianum (Supplemental Table 2). Although there is a large body of work that supports orchid niche partitioning trough speci c mycorrhizal associations, a few studies carried out in tropical epiphytic orchids have also encountered important OMF overlaps among coexisting orchid species (Cevallos et al. 2018b;Herrera et al. 2018Herrera et al. , 2019. For instance, sympatric individuals of Cyrtochilum pardinum and Epidendrum marsupiale associated with a similar OMF community in at least four different sites in the Andes forests (Cevallos et al. 2018b), which was later supported by Herrera et al. (2019). ...
... Although there is a large body of work that supports orchid niche partitioning trough speci c mycorrhizal associations, a few studies carried out in tropical epiphytic orchids have also encountered important OMF overlaps among coexisting orchid species (Cevallos et al. 2018b;Herrera et al. 2018Herrera et al. , 2019. For instance, sympatric individuals of Cyrtochilum pardinum and Epidendrum marsupiale associated with a similar OMF community in at least four different sites in the Andes forests (Cevallos et al. 2018b), which was later supported by Herrera et al. (2019). Tentatively, co-existing species are not capable of thriving if they use similar resources (Tilman, 1982) unless characteristics of the microsite allow for habitat heterogeneity and the segregation of the niche; for example, separation by abiotic conditions of the canopy (Rasmussen and Rasmussen 2018). ...
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Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization. However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and structure of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously-recognized OMF taxa, and that the coexisting orchid species display distinct (OMF) communities while keeping a base of shared fungi. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time, a strategy that may serve as a complementary mechanism to fulfill the nutritional needs associated with reproduction. This study provides evidence for niche partitioning in coexisting tropical epiphytic orchids through the utilization of specific OMF, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others.
... Interestingly, in contrast to the root mycobiome as a whole, no differences in putative mycorrhizal (ITS86-F/ ITS4) community composition were found between root forming mutualistic relationships with plants, including dark septate endophytic associations (e.g., Mandyam and Jumpponen 2005;Oliveira et al. 2014;Cevallos et al. 2018;Selosse et al. 2022). ...
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The mycorrhizal fungi of cultivated Vanilla spp. have mainly been studied in America, while a recent study has investigated them on Réunion Island (Indian Ocean). However, there are many different types of cultivation on Réunion, from shade-house crops to forest farms of endemic or exotic trees. Here we fill a gap in the study of the root mycobiome of Vanilla by sampling vines in forest plantations on recent lava flows in the southeast of Réunion. Specifically, we aimed to characterize the fungal communities between terrestrial and epiphytic roots, between forest farms that differ mainly in the species of trees, and between Vanilla roots and ECM-like roots of nearby trees. By sequencing fungal ITS2, we showed that the Vanilla root mycobiome is diverse and differed between the root types and forest farms. Epiphytic and terrestrial roots host endophytic fungi, while a putative rust with visible urediniospores was abundant in terrestrial roots mainly. Other pathogens were detected in epiphytic roots (Colletotrichum) with no sign of disease. Following sequencing and electron microscopy, Tulasnellaceae, characterized by imperforate parenthesomes and cell wall expansion with an amorphous matrix, were shown to be the main mycorrhizal fungi in both vanilla root types. Interestingly, the dominant Tulasnellaceae OTU was found in ECM-type roots of trees belonging to the ectomycorrhizal family Sapotaceae. Further observations are needed to confirm the ectomycorrhizal association of endemic trees with Tulasnella. Moreover, labeling experiments will be instrumental in investigating the transfer of nutrients between the trees and the Vanilla through the network of mycorrhizal associations in the soil.
... Additional sampling in more sites would be necessary to unequivocally confirm our results. For instance, previous studies have indicated that at lower altitudes, both species host and site significantly influence the fungal community composition, whereas at higher altitudes, these effects were not as pronounced [70]. ...
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Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.
... No obstante, los estudios de inventariado y registro de diversidad de hongos en Ecuador aún son pocos [4][5][6][7] y algunos están dirigidos a fines ecológicos como diversidad de micorrizas, aplicando análisis moleculares [8][9][10][11]. Sin embargo, recientemente se ha detectado algunos avances en reporte de diversidad de hongos basados en morfología y otros estudios integrando datos moleculares [6,[12][13][14]. ...
Article
Full-text available
Ecuador es un país Megadiverso en Flora, Fauna y Funga, a más de ecosistemas únicos como el Bosque Petrificado de Puyango. Este bosque no ha sido investigado en cuanto a su diversidad fúngica. En el presente trabajo se registra preliminarmente por primera vez una lista y guía visual rápida de macrohongos principalmente descomponedores de madera para el Bosque Petrificado de Puyango. Los resultados sugieren una gran diversidad de hongos para este bosque, donde 28 observaciones corresponden a 28 morfoespecies. Esta información marca la línea base para futuros proyectos investigativos que permitan mostrar y valorizar la diversidad de hongos neotropicales en esta zona protegida declarada Patrimonio Nacional de Ecuador.
... The root is composed of a cortex covered by the velamen which serves to actively absorb water mixed with residues of organic matter. It has been shown that the velamen secretes metabolites that facilitate the recruitment of microorganisms tolerant to these conditions [32,39], which would obviously involve recruitment from the bark substrate to which the roots are attached. The thick, flat, elliptical leaves, covered with a layer of cuticular wax and stomata, receive a greater amount of light and eventually fall off to be recycled into the canopy or ground soil. ...
Article
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Orchids coexist with a diversity of endophytic fungi within their roots and other parts of the plant. These are presumed to contribute to nutrition, and may protect the plants against pathogens and herbivores; however, some may be latent pathogens and/or bring no benefit to the plant. Guarianthe skinneri is an epiphytic Central American threatened orchid used as an ornamental plant and in the rituals and celebrations of many communities. However, in the Soconusco region (Chiapas, Mexico), the pseudobulbs of mature plants are affected by the Lasiodiplodia theobromae fungus, causing the disease “black blotch”. We evaluated and compared the diversity of the endophytic fungal community within the leaves, pseudobulbs and roots of mature plants in two conditions, asymptomatic and symptomatic. Thirty samples from each condition and tissue were amplified with ITS and sequenced by Illumina MiSeq. Sequences were obtained and analyzed to determine taxonomic assignment and functionality with FUNGuild, obtaining 1857 amplicon sequence variants (ASVs). Alpha diversity was similar between plant conditions. In symptomatic plants, significant differences were found between the three types of tissue. According to the FUNGuild functionality analysis, 368 ASVs were determined to be endophytic fungi. The tissues of G. skinneri plants are reservoirs of fungal endophytes that should be considered for further exploration for research and conservation purposes.
... The ITS2 amplicons were prepared for Illumina sequencing using a multiplex strategy in two rounds PCR(Cevallos et al. 2018). The rst round of PCR was performed using forward (fITS7 -GTGARTCATCGAATCTTTG) and reverse (ITS4 -TCCTCCGCTTATTGATATGC) primers containing the proper overhang sequence for Nextera XT DNA Library Preparation Kit. ...
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Cattleya milleri is a microendemic orchid of iron-rich rupestrian grasslands in the Brazilian savanna hotspot. It is under critical threat due to illegal collections and habitat destruction. As endophytic and mycorrhizal fungi have potential application during C. milleri propagation and conservation, we investigated its root fungal community. C. milleri roots were sampled in five natural sites and at a greenhouse. Fungal root endophytes were isolated for cultural characterization and molecular ITS (Internal Transcribed Spacer) identification. Total DNA was extracted from root endorhiza and rhizosphere to ITS amplification and sequencing. Sixteen fungal isolates were clustered in 6 Operational Taxonomic Units (OTUs), while endorhizal and rhizospheric sequences were clustered in 327 OTUs. Endorhiza richness (OTUs number) ranged from 25 to 89 OTUs, and rhizosphere presented 56 OTUs. Cluster analysis showed high divergence between natural and greenhouse environments, but a small distance among natural samples. Four phyla, 48 orders, 81 families, and 94 genera were annotated. The putative role of 134 OTUs was annotated, and 24 genera were endophytes, 2 mycorrhizas, 33 pathotrophs, 40 saprotrophs and 17 symbiotrophs. Three orders containing endophytes (Capnodiales, Hypocreales, and Pleosporales) and one containing mycorrhizae (Sebacinales) occurred in all sites. The mycorrhizae Tulasnella occurred in all natural samples. The presence of only two mycorrhizal taxa suggested the mycorrhizae may limit C. milleri distribution. However, many fungi can be recruited from the environment as non-mycorrhizal endophytes. Considering their abundance and role in orchid development, Pleosporales, Tulasnella , and Sebacinalles may be considered for C. milleri propagation and conservation.
... As reported in parasitic plants, the microbial transfer might also occur between the epiphytic plants and their host. Studies in epiphytic orchids reported that the root fungal composition of epiphytic orchids is dominated by Ascomycota and Basidiomycota (Cevallos et al. 2018;Maldonado et al. 2020;Cevallos et al. 2022), while the bacterial composition in the root is dominated by Cyanobacteria (Tsavkelova et al. 2001;Tsavkelova et al. 2003a;Tsavkelova et al. 2003b;Tsavkelova et al. 2022). However, whether the microbial community in the root of epiphytic orchids originated from the host plants is unknown since there is still no study on microbial correlation and interaction between epiphytic plants and their host currently Very few studies have investigated the microbial composition in vascular epiphytic and parasitic plants and their respective host. ...
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Differences in bacterial composition between vascular epiphyte and parasitic plants living on the same host plants. Biodiversitas 23: 5798-5805. Epiphytic and parasitic plants can grow and complete their life cycle while attached to the host. Therefore, the interactions between these plants and their host provide profound evidence for co-evolution. During these symbiotic interactions, bacteria are actively exchanged between parasitic and epiphytic plants with their hosts. Since epiphytes and parasitic plants have different ways of life, they might assemble their bacterial community differently despite living in the same host. However, direct microbiome comparisons between epiphytic and parasitic plants colonizing the same host have never been evaluated. In this study, we examined the bacterial compositions of the epiphytic Hoya sp. and parasitic Dendrophthoe sp. that grow in two host species, frangipani (Plumeria sp.) and teak (Tectona grandis). The results revealed that bacterial compositions in the root of Hoya sp. are highly similar to the peripheral tissue of the host stem. In contrast, bacterial composition in the haustoria of Dendrophthoe sp. is quite distinct from the host. These results revealed that epiphytes and parasitic plants acquire and assemble their microbiome differently, despite living in the same host species. These differences might originate from different nutrient acquisition strategies between the two plants.
... Sanger sequencing is the most widespread approach and is still used today for studies in which the strains are not required to be used in germination experiments or to be conserved in mycelium banks for later purposes. Although recent next-generation sequencing (NGS) techniques with Illumina technology offer more complex and robust information on fungi associated with orchids (Cevallos et al. 2016(Cevallos et al. , 2018, this approach is not the most used due to the costs involved, which are not necessarily within the common financial source availability of researchers from developing countries in the region. ...
Chapter
South America is undoubtedly the cradle of orchid diversity. However, few aspects of the biology of this plant group have been explored in the region. Orchids establish an important relationship with fungi that supply them with nutrients in the early stages of development to stimulate the germination of their tiny seeds. These fungi are called orchid mycorrhizal fungi (OMF), and their interaction with orchids forms a particular group of mycorrhizae: the orchid mycorrhiza (OM). In this chapter, we present the advances of the research conducted in South America, which explores some aspects of this interesting interaction. We have noticed that most studies on OM are academic documents deposited in university library repositories or published in local scientific journals. About half of the studies have focused on determining the diversity of OMF associated with a few orchid species of interest. Studies of phylogeny, morphological, and symbiotic seed germination are other of the main topics addressed. Research on ultrastructure and community ecology is hardly representative, while evolutionary implications, mutualistic networks, and metabolic aspects are the least explored topics. We encourage collaborations with the international scientific community to continue investigating complex questions that allow us to understand the role of mycorrhizas in the evolutionary success of tropical orchids. Moreover, we believe that it is important to propose attractive research that generates interest not only in the academic community but also in ordinary people who have traditionally been related to orchids (e.g., growers) in order to develop the enormous potential of the region in this field.KeywordsMycorrhizal fungi Seed germination Orchidaceae Symbiosis Rhizoctonia-like fungi
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