mycological research 110 (2006) 12571270

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/mycres

Diverse tulasnelloid fungi form mycorrhizas with epiphytic

orchids in an Andean cloud forest
Juan Pablo SUAREZa,b,*, Michael WEIb, Andrea ABELEb, Sigisfredo GARNICAb,
Franz OBERWINKLERb, Ingrid KOTTKEb
a

Centro de Biologa Celular y Molecular, Universidad Tecnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja, Ecuador
Eberhard-Karls-Universitat Tubingen, Botanisches Institut, Spezielle Botanik und Mykologie, Auf der Morgenstelle 1,
D-72076 Tubingen, Germany

article info

abstract

Article history:

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the

Received 3 May 2006

state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high

Received in revised form

number of species on stems of trees in the Andean cloud forest, were unknown. Root sam-

7 August 2006

ples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and

Accepted 12 August 2006

Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ul-

Published online 31 October 2006

trastructural evidence of symbiotic interaction was combined with molecular sequencing

Corresponding Editor:

of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analy-

John W. G. Cairney

ses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome
and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates

Keywords:

were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from cod-

Heterobasidiomycetes

ing regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of

Molecular phylogeny

the internal transcribed spacers, obtained directly from the roots and from the fungal iso-

Pleurothallidinae

lates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were

Southern Ecuador

restricted to two Tulasnella sequence types while the other orchids were associated with up

Tropical mountain rain forest

to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct

Ultrastructure

from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid
growth and maintenance in the Andean cloud forest.
2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Although most land plants are associated with symbiotic
fungi forming mycorrhizas or mycorrhiza-like associations,
many epiphytes live without such associations, e.g. mosses,
many liverworts, bromeliads, and ferns (Kottke 2002). Findings on the mycorrhizal state of epiphytic orchids were controversial. Only sporadic fungal colonization was found
in a number of epiphytic Malaysian orchids (Hadley &

Williamson 1972), but a high infection rate was reported

from canopy-dwelling orchid species in Florida (Benzing
1982). Different degrees of infection including non-infected
roots were observed in epiphytic orchids in Ecuador (Bermudes & Benzing 1989). Goh et al. (1992) found high fungal colonization in the epiphytic orchid Dendrobium crumenatum from
a natural stand in Singapore, but only low or no mycorrhization in orchids from nurseries. Rivas et al. (1998) and Pereira
et al. (2005) reported intense colonization of epiphytic orchids

* Corresponding author.
E-mail address: jpsuarez@utpl.edu.ec
0953-7562/$ see front matter 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.08.004

J. P. Suarez et al.

1258

in Costa Rica and Brazil, respectively. All investigators stated

that fungal colonization was restricted to roots attaching to
the substrate; aerial roots were not colonized.
Identification of root-associated fungi was mostly achieved
by isolation on sterile media (Rasmussen 2002). However, the
distinction between endophytic fungi inhabiting only the velamen or the root surface and reliably mycorrhiza-forming
fungi colonizing the cortical tissue was mostly unclear (Currah et al. 1997; Pridgeon 1987). Xylaria (Ascomycota) was frequently isolated (Bayman et al. 1997, Tremblay et al. 1998),
but was never proven experimentally or demonstrated by ultrastructure to form mycorrhizas with orchids. Fungal isolation from pelotons as a more selective approach has been
successfully attempted in terrestrial orchids (Warcup & Talbot
1967, 1971, 1980, Bougoure et al. 2005). In cases where sexual
stages could be achieved, the isolated fungi were determined
as basidiomycetes belonging to the Sebacinales, Tulasnellales
or Ceratobasidiales (Warcup 1981; Warcup & Talbot 1967,
1971, 1980). Tulasnella anamorphs (Epulorhiza) were isolated,
e.g. from epiphytic Epidendrum conopseum in Florida (Zettler
et al. 1998), epiphytic Epidendrum rigidum, Polystachia concreta
(Pereira et al. 2003), and terrestrial Oeceoclades maculata from
Brazil (Pereira et al. 2005). DNA sequencing supported the
presence of Tulasnella, Sebacinales and Ceratobasidium in
Cypripedium spp. from the temperate Northern Hemisphere
(Shefferson et al. 2005). Molecular tools were also used to identify fungal isolates obtained from pelotons (Bougoure et al.
2005) or by direct DNA isolation from pelotons (Kristiansen
et al. 2001). A taxon distantly related to Laccaria, an ectomycorrhiza-forming fungus, was found in Dactylorhiza majalis
(Kristiansen et al. 2001) in addition to Tulasnella. Ectomycorrhiza-forming mycobionts were also proven for nonphotosynthetic orchids by DNA isolations and sequencing
directly from mycorrhizas (Taylor & Bruns 1997, 1999; Taylor
et al. 2003, Bidartondo et al. 2004, Selosse et al. 2004, Julou et al.
2005), thus widening the previous knowledge on orchid
mycobionts.
Selosse et al. (2004) confirmed their molecular finding of Tuber spp. (Ascomycota) as orchid mycobionts by ultrastructural
demonstration of ascomycetous hyphae in the cortical cells of
the orchid roots. Ascomycetes can be discerned from basidiomycetes by ultrastructure of the cell wall and the septal pore,
and different groups of basidiomycetes can be distinguished
by the parenthesomes covering the dolipores (Wells & Bandoni 2001); tulasnelloid fungi display characteristic slime bodies in the cell walls (Bauer 2004). In spite of these diagnostic
possibilities transmission electron microscopy has rarely
been used in orchid studies addressing fungal identity. However, the previous work is encouraging (Currah & Sherburne
1992; Andersen 1996) and minimizes errors resulting from
contamination during isolation of fungi or DNA directly from
mycorrhiza samples. In our study of the orchid mycobionts
of four epiphytic, pleurothallid orchid species in the Andean
cloud forest of south Ecuador, we therefore combined ultrastructural studies with DNA sequencing and isolation.
Stelis concinna, S. hallii, S. superbiens, and Pleurothallis lilijae
Foldats were selected because of the abundance and frequent
flowering of these small orchids in the tropical mountain
rainforest of the study area. Thus severe violations of the
orchid populations in this highly endangered forest could be

minimized. The genera Stelis and Pleurothallis belong to

subtribe Pleurothallidinae, the largest subtribe in the tribe
Epidendreae of Orchidaceae (Luer 1986a,b), which is widely distributed in tropical America. These two genera include 485
Pleurothallis and 465 Stelis species reported until now for Ecuador (L. Endara, pers. comm.). Many of these epiphytes are endemic species of Ecuadorian tropical forests. Only a few of
them are in culture so far. The rapid loss of habitats requires
an understanding of the symbiotic relationships in order
to support conservation efforts for these orchids. According
to Hamilton et al. (1995) approximately 90 % of the Northern
Andean forests have been already destroyed. Consequently,
the orchids and their fungi might be lost in the near future
if not taken into culture. As the mycorrhizal state and mycobionts of the epiphytic Pleurothallidinae were unknown, no
advice could be given to laboratories interested in orchid culturing or to local forest management aiming to rehabilitate
the tropical mountain forest with its epiphytic orchid diversity
(see: http://www.bergregenwald.de of which this work is a
part). We therefore started with light- and transmission electron microscopic investigation of the selected orchid species,
and continued with DNA isolation and sequencing of the
most frequently observed fungal group, the Tulasnellales. In
parallel, isolation of mycelia was carried out, yielding several
Tulasnella isolates. We were especially interested to see
whether the Tulasnellales present as mycobionts of epiphytic
orchids in the tropical mountain rain forest were distinct
from those described for other habitats of the Northern Hemisphere and Australia. This knowledge would help to decide if
local or ubiquitous fungal isolates were appropriate for cultivation of the local orchids, and would support evaluation of
loss of local fungi for rehabilitation of orchids in the tropical
mountain forest area.

Materials and methods

Study site
The study site is located on the eastern slope of the Cordillera El
Consuelo in the northern Andes of southern Ecuador. The area
of about 1000 ha belongs to the Reserva Biologica San Francisco
and borders the Podocarpus National Park in the north, half
way between Loja and Zamora, Zamora-Chinchipe Province
(3 58 S, 79 04 W). The tropical mountain rainforest covers the
steep slopes between 1850 and 2700 m a.s.l. Characteristic
and most frequent trees are Melastomataceae, Rubiaceae, Lauraceae and Euphorbiaceae reaching a height of 25 m. Crown density
as measured by a spherical densitometer is 94 % on average,
only 7.5 % were open canopy (Homeier 2004).
The richness and abundance of epiphytes is due to the
semi- to sub-humid climate with rainfall during ten months
and even more frequent fog combined with moderate temperatures (Richter 2003). Mean annual precipitation at 1950 m
a.s.l. is 2200 mm, annual mean temperature is 15,5  C (14,417,5  C). Precipitation increases with higher elevation and
reaches 4000 mm at 2600 m asl. Air humidity in two months
is 96 % on average and does not fall below 70 % during the
drier season (Noske 2004). The high air humidity is especially
important for stem epiphytes.

Diverse tulasnelloid fungi form mycorrhizas

1259

Sampling

Fungal isolation

Sampling was carried out at small paths at an altitudinal gradient between 1850 and 2100 m a.s.l. Stelis hallii was found in
the forest covering the steep slopes between 1800 and
1900 m a.s.l., while S. superbiens and Pleurothallis lilijae were
collected in the forest covering the mountain ridge between
1900 and 2100 m a.s.l. Stelis concinna was restricted to the upper part of the mountain ridge where the forest was less
dense, with only 92 % crown density, and exposition to frequent and heavy winds.
Roots were collected continuously during three years
from 2003 until 2005 from a total of 77 flowering individuals,
22 of S. hallii, 17 of S. superbiens, 25 of S. concinna, and 13 of
Pleurothallis lilijae. All selected plants were epiphytes on
trunks or branches of standing trees at 50 cm to 200 cm
above the forest floor. Distances between trees with flowering orchids varied between 50 cm and several metres (up to
20 m). Identification of trees was not taken into consideration. Roots of one flowering individual orchid per tree
stem were collected. One to four roots per plant individual
were packed in aluminum foil to prevent desiccation
and transported to the laboratory the same day. As preinvestigation had shown that mycorrhizal fungi colonized
only roots in contact with the stems, best when also covered
by mosses or a minute humus layer, later on only such roots
were selected. Root samples were processed the day of
collection as pre-investigation had revealed a fast loss of
vitality in the symbiotic fungi. Vouchers of the orchid
specimens were deposited in the Herbarium of UTPL, Loja,
Ecuador, including flowers fixed in ethanol. Vouchers of
the mycorrhizas were embedded in resin and deposited in
the Herbarium of Tubingen University (TUB).

Isolation of fungi was initiated the day of sampling. Colonized

root pieces were surface-sterilized. Roots were rinsed in
distilled water with some drops of liquid soap, immersed in
ethanol (70 %) for 30 s, immersed in Ajax chloro 20 % (household bleach, sodium hypochlorite 5.25 %) for 10 min and
finally rinsed in sterile distilled water. The velamen was
then removed using a stereo microscope, a thin blade and forceps. Five square sections of 1-3 mm thickness were cut by
hand from the middle part of the root and transferred to
a plate with MYP media (malt extract 7 g, peptone 1 g, and
agar agar 15 g l1) or MMNC media (modified Melin-Norkrans;
Kottke et al. 1987; NaCl 0,025 g, KH2PO4 0,5 g, (NH4)2HPO4 0,25 g,
CaCl2 0,05 g, MgSO4  7H2O 0,15 g, FeCl3 (1 %) 1 ml, thiamin
1 ml, malt extract 5 g, glucose 10 g, caseinhydrolysate 1 g,
agar 20 g, riboflavin 1 ml of 0.01 % solution, trace elements
10 ml according to Fortin and Piche 1979). No antibiotics
were added.

Light and transmission electron microscopy

Light microscopy was used to select material with fungal coils.
Transversal sections were cut from the middle part of each
root sample by hand using a razor blade. Sections were
stained by Methyl blue 0.05 % solution (C. I. 42780, Merck) in
lactic acid for 10 min on microscopic slides. The samples
were examined in fresh lactic acid at 100- to 1000-fold magnification (Leitz SM-LUX or Zeiss Axioskop 2).
Root pieces of 1 cm length of all the samples displaying
high frequency of vital looking hyphal coils, 56 in total and
at least ten of each species, were fixed in 2.5 % glutaraldehyde-formaldehyde in Srensen buffer (Karnovsky 1965),
post-fixed in 1 % osmium tetroxide for 1 h, dehydrated in an
acetone series and flat embedded in Spurrs resin low viscosity, longer pot-life formulation (Spurr 1969). Semithin sections
were cut from the embedded samples, stained with 0.6 % neofuchsin crystal-violet, mounted in Entellan, and observed in
the light microscope. 20 samples with apparently vital
hyphae, originating from different plant individuals, were
selected for ultrathin cutting. Sections were mounted on Formvar-coated copper grids and stained with 1 % uranyl acetate
(40 min) and lead citrate (12 min). Sections were examined using transmission electron microscopes Zeiss TEM 902 or Zeiss
TEM109.

DNA extraction, PCR and sequencing

Portions of 1-2 cm length of well colonized roots of which the
velamen was removed were collected in cups the same day or
dried and kept on silica gel for later DNA isolations. DNA was
extracted from the fresh or dried mycorrhizal tissue and from
fungal mycelium of our own isolates using a Plant Mini Kit
(Qiagen, Hilden, Germany). A first attempt to PCR amplify genomic DNA was carried out from mycorrhizal tissue using
universal fungal primer combinations ITS1F/ITS4, ITS1F/NL4,
NLMW1/LR5, NLMW1/TW14 and ITS1F/TW14 (details concerning the primers used are given in the Electronic Appendix
A). Several PCR products were obtained and sequenced. DNA
isolated from fungal cultures was amplified using the primer
combination ITS1F/NL4 or ITS1/NL4. Nested PCR was conducted to specifically amplify DNA from tulasnelloid fungi,
as the ultrastructural analysis had revealed these fungi frequently in the cortical tissue of all the orchid species under investigation. The first amplification was carried out with the
primer combination ITS1F/TW14 or ITS1/TW14 and the second, using template obtained in the first PCR in dilutions of
101, 102 and 103, with the primer combinations ITS1/
ITS4-Tul for the internal transcribed spacers (ITS1, 5.8S nuclear ribosomal gene and ITS2) and NLMW1/LR5, ITS4-TulR/
LR5 and 5.8S-Tul/NL4 for the 5 part of the nuclear large subunit ribosomal DNA (nucLSU). Primers ITS4-Tul and ITS4TulR target a Tulasnella-specific sequence at the 3 end of
ITS2. The Tulasnella-specific primer 5.8S-Tul (5-TCATTCGAT
GAAGACCGTTGC-3) designed for this study targets a specific
sequence at the 5 end of the 5.8S rDNA.
PCR conditions were as follows: initial denaturation at
94  C for 3 min; 35 cycles, each cycle consisting of one step
of denaturation at 94  C for 30 s; annealing depending of the
primer combinations for 45 s and extension at 72  C for
1 min; a final extension at 72  C for 7 min was performed to
finish the PCR. The PCR reaction volume was 50 ml, with concentrations of 1.5 mM MgCl2, 200 mM of each dNTP (Life Technologies, Eggenstein, Germany), 0.5 mM of each of the primers
(MWG-Biotech, Ebersberg, Germany), 1U Taq polymerase (Life

J. P. Suarez et al.

1260

Technologies, Eggenstein, Germany), with an amplification

buffer (Life Technologies, Eggenstein, Germany).
In every PCR a control including PCR mix without DNA
template was included. Success of the PCR amplifications
was tested in 0.7 % agarose, stained in a solution of ethidium
bromide 0.5 mg ml1. PCR products were purified using the
QIAquick protocol (Qiagen). Cycle sequencing was conducted
using BigDye version 3.1 chemistry, and sequencing was
done on an ABI 3100 Genetic Analyzer (Applied Biosystems,
Foster City, CA). Both strands of DNA were sequenced.
Sequence editing was performed using Sequencher version
4.5 (Gene Codes, Ann Arbor, MI). The sequences obtained
in this study are available from GenBank under accession
numbers DQ178029-DQ178118 (Table 1).
We also included in this study sequence data from Tulasnella reference strains kindly provided by the National Institute of Agrobiological Sciences (NIAS), Japan, which were
previously isolated from Australian orchids and determined
by J. H. Warcup (Warcup & Talbot 1967, 1971).

MrModeltest, version 2.2 (Nylander 2004) involving four

incrementally heated Markov chains over four million generations and using random starting trees. Trees were sampled
every 100 generations resulting in a total of 40000 trees from
which the last 24000 were used to compute a 50 % majority
rule consensus tree. Each analysis was repeated to check
the reproducibility of the results (Huelsenbeck et al. 2002).
An accumulation curve of clades vs number of collected individuals from the four orchid species was computed with EstimateS (Version 7.5, R. K. Colwell, unpubl.).
We determined the proportional differences between sequences within each clade of the nucLSU D1/D2 in order to define sequence types. We compared the number of Tulasnella
sequence types within single and between different orchid
species. The proportional differences between sequences
were pooled into five tables (Electronic Appendix B).

Phylogenetic analyses

Microscopical and ultrastructural features of the mycorrhizas

We used BLAST (Altschul et al. 1997) against the NCBI nucleotide database (GenBank; http://www.ncbi.nlm.nih.gov/) to detect published sequences with a high similarity to the nucLSU
sequences obtained from the Ecuadorian epiphytic orchids.
For thorough phylogenetic analysis of the Tulasnella sequences we analyzed nucLSU and ITS-5.8S alignments including the closest BLAST matches together with the sequences
from the Warcup Tulasnella reference isolates (see above)
and other sequences from Tulasnellaceae and related groups
retrieved from GenBank.
Sequences were aligned using the G-INS-i or L-INS-i strategy as implemented in MAFFT v5.667 (Katoh et al. 2005). Due
to the heterogeneity of the Tulasnella sequences we had to exclude considerable portions of the nucLSU sequences for phylogenetic analysis. Even the 5.8S ribosomal region, considered
as universally conserved, exhibited a remarkable heterogeneity as was already mentioned by Bidartondo et al. (2003). As
expected, the ITS1 and ITS2 rDNA could not be aligned over
the whole data set. Therefore, we used the 5.8S region to calculate phylogenetic trees of a wider phylogenetic spectrum
and produced several other phylogenetic analyses including
subsets of related sequences, for which we used portions of
the ITS1 and ITS2 regions in addition to the 5.8S sequences.
The alignments used can be obtained from TreeBASE (http://
www.treebase.org/) under accession number S1629.
Neighbour-joining (NJ) and a Bayesian likelihood approach
were used to estimate the phylogenetic relationships. The
neighbour-joining analysis was performed in PAUP* (Swofford
2002) using the BIONJ modification of the NJ algorithm to
accomplish the observed high genetic variability in the sequences used (Gascuel 1997). DNA substitution models and individual model parameters were estimated using the Akaike
information criterion (AIC) as implemented in Modeltest, version 3.7 (Posada & Crandall 1998). For the Bayesian approach
based on Markov chain Monte Carlo (MCMC) we used
MrBayes, version 3.0b4 (Huelsenbeck & Ronquist 2001). Each
dataset was analyzed using the DNA substitution models estimated using the Akaike information criterion (AIC) in

Fungal pelotons were present in nearly all cross-sections of

roots sampled directly from the tree bark. No fungal pelotons
were observed in aerial roots. This observation was confirmed
by sampling roots of another 65 epiphytic Stelis and Pleurothallis orchids, indicating that the roots became colonized only
where the fungi contacted the bark or the thin humus layer.
Pelotons were distributed throughout the cortex, with no difference between cortical layers. Vital, blue staining and collapsed, slightly yellow coloured pelotons were visible in the
same cells suggesting that cells became re-infected several
times. According to the light microscopical observations,
many fungal pelotons were found collapsed after the plants
had been kept one night in the laboratory. Abundant hyphae
colonized the velamen.
TEM observations confirmed the known fungal-root interaction in orchid mycorrhizas. Hyphae of more or less equal diameter were surrounded by the plant plasma membrane, the
plant vacuole forming small compartments or a network of
small vacuoles (Fig 1). Degenerating hyphae were attached
to collapsed pelotons (Fig 2). Alive hyphae contained abundant
glycogen granules (Figs 1, 3 and 5). The hyphae formed septa,
clamps were not observed. (Fig 3). The septa showed dolipores
with imperforate, dish-shaped parenthesomes with slightly
recurved margins (Fig 6). These tulasnelloid parenthesomes
were observed in all the 20 mycorrhizas analyzed by TEM. Occasionally, the hyphal walls were split into two layers and a fibrillar or slimy mass appeared between the two layers (Figs 4
and 5, arrows). This phenomenon became very prominent in
ageing cultures and the slime was then strongly osmiophilic
(not shown). The combination of this type of parenthesomes
and the slime bodies in the cell walls was confirmed for
all the investigated mycorrhizas and in the Tulasnella isolates.
The recurved ends of the parenthesome were only detected by
serial sectioning, since the appearance of the parenthesomes
varied among the sections and may appear flattened or bowed
in a steeper angle. In three samples we additionally found flat,
imperforate parenthesomes, indicating sebacinoid fungi (Williams & Thiol 1989; not shown). In one sample a dome-shaped

Results

Diverse tulasnelloid fungi form mycorrhizas

1261

Table 1 List of sampled individuals from which tulasnelloid sequences were obtained. Letters and numbers behind the
species names correspond to species, orchid individual, and root (superscript). Superscript b marks a second sequence
obtained from the same root sample. Clades A-G correspond to the MCMC phylogenetic analysis. The two rDNA regions
from the same root listed in each line originate from a single PCR amplicon
Orchid species

Pleurothallis lilijae C2.1.1

Pleurothallis lilijae C2.1.2
Pleurothallis lilijae C2.1.3
Pleurothallis lilijae C2.15
Pleurothallis lilijae C2.17
Pleurothallis lilijae C2.21
Pleurothallis lilijae C2.1MN
Pleurothallis lilijae C2.5MN7
Pleurothallis lilijae C2.MN1
Pleurothallis lilijae C2.MN5
Pleurothallis lilijae C2MN2
Pleurothallis lilijae C2MN6
Stelis concinna 7.6
Stelis concinna 7.7
Stelis concinna 7.8
Stelis concinna 7.13.2
Stelis concinna 7.13.3
Stelis concinna 7.13.4
Stelis concinna 7.14.2
Stelis concinna 7.18.3
Stelis concinna 7.18.4
Stelis concinna 7.19.1
Stelis concinna 7.19.3
Stelis concinna 7.20.1
Stelis concinna 7.20.2
Stelis concinna 7.20.3
Stelis concinna 7.20.4
Stelis concinna 7.21.1
Stelis concinna 7.21.2
Stelis concinna 9.2
Stelis concinna 9.3 culture
Stelis concinna 9.6
Stelis concinna 9.7
Stelis concinna 9.8
Stelis concinna 9.9
Stelis hallii 1.1
Stelis hallii 1.2
Stelis hallii 1.2b
Stelis hallii 1.4
Stelis hallii 1.6
Stelis hallii 1.7
Stelis hallii 1.8
Stelis hallii 1.11 culture
Stelis hallii 1.15
Stelis hallii 1.16
Stelis hallii 1.17
Stelis hallii 1.18
Stelis hallii 1.18b
Stelis hallii 1.19
Stelis hallii 1.19b
Stelis hallii 1.21
Stelis hallii 1.21b
Stelis hallii 1.23
Stelis superbiens C3.5.2 culture
Stelis superbiens C3.5.3
Stelis superbiens C3.5.4
Stelis superbiens C3.9.2
Stelis superbiens C3 MN3
Stelis superbiens C3.MN4

nucLSU

nrDNA ITS-5.8S

clade

GenBank accession no.

clade

GenBank accession no.

A
E
A

DQ178035
DQ178067
DQ178040

DQ178099

A
F
D
F
E
B

DQ178034
DQ178047
DQ178063
DQ178049
DQ178068
DQ178045

A
E
A
E
A
F
D
F
E

DQ178100
DQ178080
DQ178102
DQ178079
DQ178098
DQ178069
DQ178116
DQ178070
DQ178081

A
A
A
A
A
A
A
A
E
A
A
A
A
A
A
E
E
A
G
A
A
A
A
B

DQ178108
DQ178106
DQ178091
DQ178109
DQ178107
DQ178110
DQ178112
DQ178095
DQ178082
DQ178094
DQ178093
DQ178096
DQ178088
DQ178090
DQ178089
DQ178075
DQ178076
DQ178111
DQ178029
DQ178084
DQ178092
DQ178097
DQ178086
DQ178113

G
B

DQ178118
DQ178114

A
E

DQ178085
DQ178066

A
A

DQ178103
DQ178104

E
E
E
E

DQ178073
DQ178071
DQ178072
DQ178077

A
E
E
A
D
C

DQ178036
DQ178083
DQ178078
DQ178087
DQ178117
DQ178115

DQ178043

A
A
A
A
A

DQ178032
DQ178030
DQ178042
DQ178033
DQ178041

DQ178029

A
A
B
E
D

DQ178038
DQ178031
DQ178044
DQ178065
DQ178051

DQ178050

E
D
D

DQ178066
DQ178057
DQ178055

A
D
D

DQ178037
DQ178053
DQ178059

D
A

DQ178060
DQ178036

A
D
C

DQ178039
DQ178058
DQ178046

(continued on next page)

J. P. Suarez et al.

1262

Table 1 (continued)

Orchid species

Stelis
Stelis
Stelis
Stelis
Stelis
Stelis
Stelis

superbiens
superbiens
superbiens
superbiens
superbiens
superbiens
superbiens

C3.1MN
C3.2MN
C3.3MN
C3.4MN
C3.4MN4
C3.5MN5
C3.5MN5b

nucLSU

nrDNA ITS-5.8S

clade

GenBank accession no.

D
D
D
D
D
E
F

DQ178056
DQ178052
DQ178054
DQ178061
DQ178062
DQ178064
DQ178048

parenthesome was found that displayed coarse perforations

and might thus putatively be assigned to Ceratobasidium (Currah & Sherburne 1992; not shown). No simple-pored ascomycetes were found in the cortical tissue of the 20 investigated
samples, although they were present in the velamen (not
shown).

Fungal isolation and molecular identification of isolates

Fungal growth was observed in only 44 plates out of 108 used
for fungal isolation, each one containing five root pieces. Four
fungal cultures were obtained from a total of 13 plates of Stelis
hallii, 15 from 36 plates of Stelis superbiens, 22 from 55 plates of
Stelis concinna, and three from four plates of Pleurothallis lilijae
mycorrhizas. A preliminary molecular identification of the
fungal isolates was carried out by BLAST searches against
the GenBank nucleotide database retrieving the most similar
available sequences (data not shown). The isolated fungi
were mainly ascomycetes closest to Xylaria, Hypoxylon and
Cryptosporiopsis and less often basidiomycetes closest to Bjerkandera, Polyporus and Tulasnella. Three cultures were identified as Tulasnella. These Tulasnella cultures exhibited slow

Fig 1 Ultrastructure of the cortical tissue of Stelis concinna

root displaying alive hyphae (h) of equal diameter in
active host cell (c). (v) Small compartments of orchid cell
vacuoles. Bar [ 1 mm.

clade

GenBank accession no.

DQ178105

DQ178101

DQ178074

growth rates contrasting with the relative fast growth rate

observed in the fungi isolated. Ascomycetes closest to Cryptosporiopsis were the most frequently isolated fungi.

Molecular identification and phylogenetic analysis

of mycorrhiza-associated fungi
The combinations of universal fungal primers yielded PCR
products preliminarily identified by BLAST searches as closest
to Cryptosporiopsis, Fusarium, Trichoderma (Ascomycota) and
Bjerkandera, Antrodiella (Basidiomycota). Tulasnella sequences
were infrequently obtained, only the primer combination
NLMW1/LR5 yielding few PCR products. Primer combinations
including ITS1F and ITS4 failed to amplify Tulasnella DNA as
was already reported by Bidartondo et al. (2003). Sequences
of Sebacinales, basidiomycetes involved in a broad range of
mycorrhizal associations (Wei et al. 2004), were also
detected, but at lower frequence than Tulasnellales sequences.
No cultures of Sebacinales were obtained from root samples
(Kottke et al. 2007).
The total number of investigated roots was 134, considering that three or four roots were collected from each of the
77 orchid individuals. Tulasnelloid fungi were detected in 84
samples (63 %), including the PCR products obtained by the
tulasnelloid specific primer combinations without successfull

Fig 2 Degenerating hyphae adjoining collapsed hyphae

(ch) in an active cortical cell of Stelis concinna root.
Bar [ 1 mm.

Diverse tulasnelloid fungi form mycorrhizas

Fig 3 Branched hypha displaying septa (arrow) without

clamp formation in root cortical tissue of Stelis concinna.
Bar [ 1 mm.

sequencing. The nested PCR conducted in order to selectively

amplify Tulasnella DNA using the primer combination ITS1/
TW14 in the first amplification and the primer combinations
ITS1/ITS4-Tul for the ITS-5.8S region and ITS4-TulR/LR5 or
5.8S-Tul/NL4 for a part of the LSU region, respectively, in the
second PCR yielded PCR products for the majority of samples.
PCR success was higher with DNA extracted from fresh root
samples and lower with DNA from dried roots.
The phylogenetic analyses of nucLSU and ITS-5.8S sequences yielded consistent results. Seven clades, which in
the following we refer to as clades A to G, were retrieved
from the analyses of both ribosomal regions (Fig 7 and Electronic Appendix C). BIONJ (trees not shown) and MCMC
yielded similar groupings of Tulasnella clades. Only small variations were present in the clade support values. As mentioned above, the 5.8S tree (Electronic Appendix C) was less
resolved than the nucLSU tree. However, the unexpected heterogeneity displayed by the 5.8S data set made it difficult to
find a suitable outgroup sequence. Therefore, we rooted the

Fig 4 Square section of active hypha of Tulasnella, displaying mitochondria (m), glycogen rosettes, and fibrillar
slime between cell wall layers (arrowheads). Bar [ 1 mm.

1263

Fig 5 Hypha in cortical root tissue of Stelis concinna

displaying fibrillar slime between cell wall layers
(arrowhead) and a doliporus with imperforate, slightly
dish-shaped parenthesomes (arrow). Bar [ 0.5 mm.

5.8S overview tree (Electronic Appendix C) in such a way

that we obtained best consistency with the rooted LSU tree
(Fig 7). Portions of ITS1 and ITS2 were added to the 5.8S alignment where phylogenetic analysis was restricted to suitable
subsets of sequences detected in the 5.8S analysis, finally
resulting in an increase of phylogenetic resolution for these
subsets (Figs 8 and 9).
Our analysis of proportional differences between sequences within each clade of the nucLSU D1/D2 yielded 13
Tulasnella sequence types. We treated sequences as belonging

Fig 6 Close-up of a median section through the doliporus.

The parenthesomes consist of two electron-dense
membranes bordering an internal electron transparent
zone and show slightly recurved borders (arrows).
Bar [ 0,3 mm.

1264

J. P. Suarez et al.

Fig 7 Phylogenetic placement of Tulasnella sequences from Stelis hallii, Stelis superbiens, Stelis concinna and Pleurothallis
lilijae inferred by MCMC analysis of nuclear rDNA coding for the 5 terminal domain of the large ribosomal subunit (nucLSU).
Numbers on branches designate neighbor-joining bootstrap values / MCMC estimates of posterior probabilities (only values
exceeding 50 % are shown). Note that genetic distances cannot be directly correlated to branch lengths in the tree, since
highly diverse alignment regions were excluded for tree construction. The tree was rooted with Multiclavula mucida
AF287875.

Fig 8 Phylogenetic placement of Tulasnella sequences, clades A-C, from Stelis hallii, Stelis superbiens, Stelis concinna and
Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S rDNA. Numbers on branches designate neighbor-joining
bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic distances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for
tree construction. The tree was rooted with Tulasnella sequences from clade D from the analysis of 5.8S rDNA (Electronic
Appendix C).

1266

J. P. Suarez et al.

Fig 9 Phylogenetic placement of Tulasnella sequences, clades E and F, from Stelis hallii, Stelis superbiens, Stelis concinna
and Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S. Numbers on branches designate neighbor-joining
bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic
distances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were
excluded for tree construction. The tree was rooted with the Tulasnella sequence from the Warcup isolate
T. violea DQ520097.

Diverse tulasnelloid fungi form mycorrhizas

to the same sequence type when proportional differences

were <1 %. Clades D and E comprised four sequence types,
while the other clades consisted only of one sequence type
each (Fig 7, Electronic Appendix B). A direct comparison between 5.8S-ITS and nucLSU phylogenies was hampered by
the fact that publicly available sequence data were restricted
to either one or the other of these two DNA regions for the
vast majority of specimens studied so far (Table 1).
Mycobionts of the same sequence type were shared among
orchid species, e.g. sequence types 2, 6, and 12. Tulasnella sequences from all four studied orchid species were present in
sequence type 1 (Fig 7). Tulasnellas belonging to different sequence types were detected in mycorrhizas from the same
plant and even from the same root piece (Table 1). Six different
Tulasnella sequence types were found in mycorrhizas of S. hallii
and S. superbiens and five in P. lilijae, while only two sequence
types were detected in mycorrhizas of S. concinna (Fig 7).

Discussion
The importance of orchid mycorrhizal fungi and their role in
orchid seed germination are known since Bernards observations (Bernard 1909). In natural conditions, the protocorm
does not develop further unless it receives an exogenous carbohydrate supply from a suitable mycorrhizal fungus (Smith
& Read 1997). Although not demonstrated in nature so far,
this dependence will also account for germination of epiphytic
orchids. Our finding of constant colonization of roots in contact with the bark supports the view of Benzing (1982) that
the role of the fungi may be crucial also for the adult epiphytic
orchids. The most important benefit for the orchid may be to
retain the fungus in order to assure further seed germination.
Saprotrophic capabilities, documented for several Tulasnella
species (Roberts 1999), could explain the growth of Tulasnella
on tree bark, including the capacity of fruiting close to colonized roots (J.P.S., pers. obs.), and promotion of seed germination. We observed decline of vitality of hyphae after only one
night of plant storage in the laboratory, independent of
whether or not the samples were cooled. The decline of vitality was first indicated by loss of stainability of the pelotons,
which appeared yellow instead of blue in the light microscope.
TEM revealed very rare occurrences of vital pelotons, but
abundant collapsed hyphae in this material. No isolates of
Tulasnellales were obtained from the stored samples and PCR
amplification success was very low, although only fully turgescent roots were processed. It is rather unlikely that the
fast decline of hyphal vitality was linked to carbon shortage
as plenty of starch grains were visible in the root tissue and
large amounts of glycogen were found in the vital hyphae. Disruption of the extraradical mycelium or increase of plant
defense reaction could be involved (Hadley 1982). So far, the
reason for this fast decline is unclear but it might erroneously
imply the impression of low hyphal colonization rate.
Our combination of ultrastructural research and DNA sequencing revealed that Tulasnella species were regularly associated with the epiphytic Stelis and Pleurothallis species. We
successfully isolated three of the associated Tulasnellas,
identity proven by DNA sequences and septal porus type.
The flat bell-shaped, imperforate parenthesomes with slightly

1267

recurved margins consist of two electron-dense membranes

bordering an internal electron transparent zone as was already shown by Andersen (1996) for Rhizoctonia repens N.Bernard Epulorhiza repens (syn.), the anamorph of Tulasnella
deliquescens (syn. T. calospora sensu Warcup & Talbot 1967 fide
Roberts 1999). The combination of this parenthesome type
and the slime bodies in the cell walls was previously described
from mycorrhiza-like associations of the liverwort Aneura pinguis housing Tulasnella (Ligrone et al. 1993), and apparently is
the best structural indication of Tulasnella (Bauer 2004). The
ascomycetes that we isolated from the roots were not found
in the cortical tissue of the orchids, but colonized only the velamen. Therefore these ascomycetes cannot be considered as
mycorrhiza-forming fungi. Without specific primers Tulasnella
is nearly undetectable (Bidartondo et al. 2003). The use of both
Tulasnella-specific primers ITS4-Tul and 5.8S-Tul that target
ITS2 and 5.8S, respectively, increased the success of Tulasnella
amplification, but a complete dataset of all tulasnelloid fungi
associated with the investigated orchids can currently not be
guaranteed. The accumulation curve of clades vs. number of
collected individuals was not saturated (Electronic Appendix D).
Tulasnella was confirmed as a genus with a range of orchid
mycorrhiza forming species (Kristiansen et al. 2004; Ma et al.
2003; McCormick et al. 2004; Shefferson et al. 2005; Warcup
1971, 1981, 1985; Warcup & Talbot 1967, 1971, 1980). Tulasnella
calospora was proposed by Hadley (1970) as an universal orchid
symbiont considering the capacity to establish in vitro symbiotic associations with a broad range of orchid species. There
are, however, taxonomic problems concerning the species
concept in Tulasnella (Roberts 1999). The T. calospora strains
(T. deliquescens fide Roberts 1999) isolated by Warcup from terrestrial Australian orchids (Warcup & Talbot 1967) belong to
two separate clades according to our nucLSU tree (Fig 7). The
strains are even more clearly distinguished in the 5.8S-ITS
tree (Fig 8), clustering with Tulasnellas detected in terrestrial
orchids from a wide range of localities, such as Spathaglottis
plicata from Singapore and Epipactis gigantea from California.
Our molecular analyses indicate that T. calospora is likely to
comprise several distinct species. Taxonomic problems account also for T. violea and T. asymmetrica, as T. violea accession
AY293216 appears close to T. pruinosa AF518662 in the nucLSU
phylogenetic tree, while the Warcup T. violea isolate from the
orchid Thelymitra sp. clustered separately; the Warcup
T. asymmetrica strains (T. pinicola fide Roberts 1999) isolated
from the orchid Thelymitra sp. fall into two groups (Fig 7). On
the other hand Tulasnellas with quite different trophic strategies are displayed as closely related in the nucLSU tree, e.g.
the mycobiont of the myco-heterotrophic liverwort Cryptothallus mirabilis (AY192482) that also forms ectomycorrhizas with
Pinus and Betula (Bidartondo et al. 2003), and T. irregularis
(AY243519), a Warcup isolate from Dendrobium dicuphum, an
Australian orchid. Attempts are currently undertaken to collect and describe Tulasnellas occurring on the bark of the
sampled trees in our research area and to induce basidia formation in the isolate cultures. Morphological description
and determination combined with sequence typing appeared
as promising tools to further clarify relationships between
these poorly studied mycobionts.
Irrespective of the taxonomic problems, available sequence data from nucLSU and ITS-5.8S made it possible to

J. P. Suarez et al.

1268

compare the seven Tulasnella clades of symbionts of epiphytic

orchids studied here with named Tulasnella species and mycorrhizal Tulasnellas mostly from terrestrial orchids. Tulasnellas of the studied epiphytic orchid species were distinct
from so far known Tulasnellas associated with terrestrial orchids. Tulasnelloid fungi associated with the terrestrial temperate orchids Cypripedium spp. (subfamily Cypripedioideae)
and Dactylorhiza majalis (AY634130) (subfamily Orchidoideae)
were displayed in a basal position with respect to the Tulasnella sequence types from Stelis and Pleurothallis in the nucLSU
phylogeny (Fig 7). In the 5.8S nrDNA phylogeny, the tulasnelloid fungi associated with Dactylorhiza majalis (AY634130),
Orchis purpurea (AJ549121), Spathaglottis plicata (AJ313457),
Ophrys sphegodes (AJ549122) (all subfamily Orchidoideae) and
Cypripedium fasciculatum (AY966883) appeared in a basal position relative to Tulasnella sequence types from Stelis and Pleurothallis (Electronic Appendix C). Molecular phylogenetic
analyses of the family Orchidaceae are consistent in respect
to a basal position of Cypripedioideae and Orchidoideae compared to Epidendroideae (e.g. Cameron et al. 1999). Switches
from terrestrial to epiphytic habit or back were found to be
major driving forces in radiation and specialization of orchids
(Cameron 2002, 2005), and beside pollinator relationships mycorrhizal interactions are now recognized crucial for orchid
evolution (Taylor et al. 2003). However, more species need to
be sampled including terrestrial orchids of the study site to arrive at convincing conclusions about coevolution between orchids and their mycobionts.
Our results show differences in the number of Tulasnella
symbionts associated with one orchid species. Six Tulasnella
sequence types were associated with one individual orchid
species of S. hallii and S. superbiens, five in P. lilijae, but only
two sequence types were found with S. concinna. We cannot exclude the possibility that the differences in numbers of Tulasnella symbionts will vanish when a higher number of orchid
specimen and roots will be examined. However, preferences
for fungal partners have been demonstrated in other epiphytic
orchids (Otero et al. 2002, 2004). In several cases we found that
one orchid individual was associated with Tulasnellas from
more than one clade even in the same root segment. Obviously, diverse Tulasnellas form mycorrhizas with the green,
epiphytic, pleurothallid orchids in the Andean cloud forest.
Whether these distinct fungi are crucial for seed germination
needs to be verified experimentally. In case of the Rhizoctonias, seed germination was stimulated by non-optimal mycobionts, but symbionts that were not fully compatible resulted
in high seedling mortality (Rasmussen 2002). Our analyses
indicate that efficient rehabilitation of epiphytic orchids in
nature and recruitment in the nursery probably requires the
usage of distinct Tulasnella species as orchid mycobionts.

Acknowledgements
This research was generously supported by the Deutsche Forschungsgemeinschaft (DFG project FOR 402). We thank the
Fundacion Cientfica San Francisco for providing research facilities, Lorena Endara for help in orchid identification, and
Paulo Herrera for help in laboratory work. The supply of fungal

strains by the National Institute of Agrobiological Sciences

(NIAS), Japan, is also acknowledged.

Supplementary data
Supplementary data associated with this article can be found,
in the online version, at 10.1016/j.mycres.2006.08.004.

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further reading

Cullings KW, 1994. Molecular phylogeny of the Monotropoideae

(Ericaceae) with a note on the placement of the Pyroloideae.
Journal of Evolutionary Biology 7: 501516.
Gardes M, Bruns TD, 1993. ITS primers with enhanced specificity
for basidiomycetes: application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113118.
Sampaio JP, Wei M, Gadanho M, Bauer R, 2002. New taxa in the
Tremellales: Bulleribasidium oberjochense gen. et sp. nov., Papiliotrema bandonii gen. et sp. nov. and Fibulobasidium murrhardtense sp. nov. Mycologia 94: 873887.
Taylor DL, 1997. The evolution of myco-heterotrophy and specificity
in some North American orchids. PhD thesis, University of
California at Berkeley, CA.
Vilgalys R, Hester M, 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several
Cryptococcus species. Journal of Bacteriology 172: 42384246.
White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and
direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand H, Sninsky JS, White TJ (eds),
PCR-Protocols and applications: A Laboratory Manual. Academic
Press, San Diego, pp. 315322.