Franz OBERWINKLER , Ingrid KOTTKEaCentro de Biologa Celular y Molecular, Universidad Tecnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja, EcuadorbEberhard-Karls-Universitat Tubingen, Botanisches Institut, Spezielle Botanik und Mykologie, Auf der Morgenstelle 1,

D-72076 Tubingen, Germany

a r t i c l e i n f o

Article history:

Received 3 May 2006

Received in revised form

7 August 2006

Accepted 12 August 2006

Published online 31 October 2006

Corresponding Editor:

John W. G. Cairney

Keywords:

Heterobasidiomycetes

Molecular phylogeny

Pleurothallidinae

Southern Ecuador

Tropical mountain rain forest

Ultrastructure

a b s t r a c t

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

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

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

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

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

trastructural evidence of symbiotic interaction was combined with molecular sequencing

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

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

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

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

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

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

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

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

from known sequences of mycobionts of terrestrial orchids. The results indicate that tulas-

nelloid 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,

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 (Ber-Diverse tulasnelloid fungi formorchids in an Andean cloud for

Juan Pablo SUAREZa,b,*, Michael WEIb, Andb b

journa l homepage : www.emany epiphytes live without such associations, e.g. mosses,

many liverworts, bromeliads, and ferns (Kottke 2002). Find-

ings on the mycorrhizal state of epiphytic orchids were con-

troversial. Only sporadic fungal colonization was found

in a number of epiphytic Malaysian orchids (Hadley &

* Corresponding author.E-mail address: jpsuarez@utpl.edu.ec

0953-7562/$ see front matter 2006 The British Mycological Sociedoi:10.1016/j.mycres.2006.08.004mycorrhizas with epiphyticest

rea ABELEb, Sigisfredo GARNICAb,

l sev ier . com/ loca te /mycresava i lab le at www.sc ienced i rec t . com

my c o l o g i c a l r e s e a r c h 1 1 0 ( 2 0 0 6 ) 1 2 5 7 1 2 7 0mudes & Benzing 1989). Goh et al. (1992) found high fungal col-

onization in the epiphytic orchid Dendrobium crumenatum from

a natural stand in Singapore, but only low or no mycorrhiza-

tion in orchids from nurseries. Rivas et al. (1998) and Pereira

et al. (2005) reported intense colonization of epiphytic orchids

ty. Published by Elsevier Ltd. All rights reserved.

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 ve-

lamen or the root surface and reliably mycorrhiza-forming

fungi colonizing the cortical tissue was mostly unclear (Cur-

rah et al. 1997; Pridgeon 1987). Xylaria (Ascomycota) was fre-

quently isolated (Bayman et al. 1997, Tremblay et al. 1998),

but was never proven experimentally or demonstrated by ul-

trastructure to form mycorrhizas with orchids. Fungal isola-

tion 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 iden-

tify 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 ectomycor-

rhiza-forming fungus, was found in Dactylorhiza majalis

(Kristiansen et al. 2001) in addition to Tulasnella. Ectomycor-

rhiza-forming mycobionts were also proven for non-

photosynthetic 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 Tu-

ber spp. (Ascomycota) as orchid mycobionts by ultrastructural

demonstration of ascomycetous hyphae in the cortical cells of

the orchid roots. Ascomycetes can be discerned from basidio-

mycetes 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 & Ban-

doni 2001); tulasnelloid fungi display characteristic slime bod-

ies 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. How-

ever, 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 ultra-

structural 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

1258orchid populations in this highly endangered forest could beminimized. The genera Stelis and Pleurothallis belong to

subtribe Pleurothallidinae, the largest subtribe in the tribe

Epidendreae of Orchidaceae (Luer 1986a,b), which is widely dis-

tributed in tropical America. These two genera include 485

Pleurothallis and 465 Stelis species reported until now for Ecua-

dor (L. Endara, pers. comm.). Many of these epiphytes are en-

demic 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 myco-

bionts of the epiphytic Pleurothallidinae were unknown, no

advice could be given to laboratories interested in orchid cul-

turing 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 elec-

tron 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 Hemi-

sphere and Australia. This knowledge would help to decide if

local or ubiquitous fungal isolates were appropriate for culti-

vation 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

(358 S, 7904 W). The tropical mountain rainforest covers thesteep slopes between 1850 and 2700 m a.s.l. Characteristic

and most frequent trees are Melastomataceae, Rubiaceae, Laura-

ceaeandEuphorbiaceae 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 temper-

atures (Richter 2003). Mean annual precipitation at 1950 m

a.s.l. is 2200 mm, annual mean temperature is 15,5 C (14,4-17,5 C). Precipitation increases with higher elevation andreaches 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

J. P. Suarez et al.important for stem epiphytes.

Sampling

Sampling was carried out at small paths at an altitudinal gra-

dient 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 up-

per part of the mountain ridge where the forest was less

dense, with only 92 % crown density, and exposition to fre-

quent 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 flower-

ing orchids varied between 50 cm and several metres (up to

20 m). Identification of trees was not taken into consider-

ation. 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 pre-

investigation 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).

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 magni-

fication (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 % glutaralde-

hyde-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 viscos-

ity, longer pot-life formulation (Spurr 1969). Semithin sections

were cut from the embedded samples, stained with 0.6 % neo-

fuchsin 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 For-

mvar-coated copper grids and stained with 1 % uranyl acetate

(40 min) and lead citrate (12 min). Sections were examined us-

ing transmission electron microscopes Zeiss TEM 902 or Zeiss

Diverse tulasnelloid fungi form mycorrhizasTEM109.Fungal isolation

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 % (house-

hold 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 for-

ceps. 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, thiamin1 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.

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 ge-

nomic DNA was carried out from mycorrhizal tissue using

universal fungal primer combinations ITS1F/ITS4, ITS1F/NL4,

NLMW1/LR5, NLMW1/TW14 and ITS1F/TW14 (details con-

cerning 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 con-

ducted to specifically amplify DNA from tulasnelloid fungi,

as the ultrastructural analysis had revealed these fungi fre-

quently in the cortical tissue of all the orchid species under in-

vestigation. The first amplification was carried out with the

primer combination ITS1F/TW14 or ITS1/TW14 and the sec-

ond, 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 nu-

clear ribosomal gene and ITS2) and NLMW1/LR5, ITS4-TulR/

LR5 and 5.8S-Tul/NL4 for the 5 part of the nuclear large sub-

unit ribosomal DNA (nucLSU). Primers ITS4-Tul and ITS4-

TulR 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 stepof denaturation at 94 C for 30 s; annealing depending of theprimer combinations for 45 s and extension at 72 C for1 min; a final extension at 72 C for 7 min was performed tofinish the PCR. The PCR reaction volume was 50 ml, with con-

centrations of 1.5 mM MgCl2, 200 mM of each dNTP (Life Tech-

nologies, Eggenstein, Germany), 0.5 mM of each of the primers

1259(MWG-Biotech, Ebersberg, Germany), 1U Taq polymerase (Life

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 theQIAquick 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 Tulas-

nella reference strains kindly provided by the National Insti-

tute of Agrobiological Sciences (NIAS), Japan, which were

previously isolated from Australian orchids and determined

by J. H. Warcup (Warcup & Talbot 1967, 1971).

Phylogenetic analyses

We used BLAST (Altschul et al. 1997) against the NCBI nucleo-

tide database (GenBank; http://www.ncbi.nlm.nih.gov/) to de-

tect published sequences with a high similarity to the nucLSU

sequences obtained from the Ecuadorian epiphytic orchids.

For thorough phylogenetic analysis of the Tulasnella se-

quences we analyzed nucLSU and ITS-5.8S alignments includ-

ing 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 strat-

egy as implemented in MAFFT v5.667 (Katoh et al. 2005). Due

to the heterogeneity of the Tulasnella sequences we had to ex-

clude considerable portions of the nucLSU sequences for phy-

logenetic analysis. Even the 5.8S ribosomal region, considered

as universally conserved, exhibited a remarkable heterogene-

ity 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 cal-

culate 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 se-

quences used (Gascuel 1997). DNA substitution models and in-

dividual model parameters were estimated using the Akaike

information criterion (AIC) as implemented in Modeltest, ver-

sion 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 esti-

1260mated using the Akaike information criterion (AIC) inMrModeltest, version 2.2 (Nylander 2004) involving four

incrementally heated Markov chains over four million gener-

ations 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 indi-

viduals from the four orchid species was computed with Esti-

mateS (Version 7.5, R. K. Colwell, unpubl.).

We determined the proportional differences between se-

quences within each clade of the nucLSU D1/D2 in order to de-

fine 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).

Results

Microscopical and ultrastructural features of the mycorrhizas

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 Pleurothal-

lis 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 dif-

ference between cortical layers. Vital, blue staining and col-

lapsed, 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 inter-

action in orchid mycorrhizas. Hyphae of more or less equal di-

ameter 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. Oc-

casionally, the hyphal walls were split into two layers and a fi-

brillar 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 (Wil-

J. P. Suarez et al.liams & Thiol 1989; not shown). In one sample a dome-shaped

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

Orchid species nucLSU nrDNA ITS-5.8S

clade GenBank accession no. clade GenBank accession no.

Pleurothallis lilijae C2.1.1 A DQ178035 A DQ178099

Pleurothallis lilijae C2.1.2 E DQ178067

Pleurothallis lilijae C2.1.3 A DQ178040 A DQ178100

Pleurothallis lilijae C2.15 E DQ178080

Pleurothallis lilijae C2.17 A DQ178102

Pleurothallis lilijae C2.21 E DQ178079

Pleurothallis lilijae C2.1MN A DQ178034 A DQ178098

Pleurothallis lilijae C2.5MN7 F DQ178047 F DQ178069

Pleurothallis lilijae C2.MN1 D DQ178063 D DQ178116

Pleurothallis lilijae C2.MN5 F DQ178049 F DQ178070

Pleurothallis lilijae C2MN2 E DQ178068 E DQ178081

Pleurothallis lilijae C2MN6 B DQ178045

Stelis concinna 7.6 A DQ178108

Stelis concinna 7.7 A DQ178106

Stelis concinna 7.8 A DQ178091

Stelis concinna 7.13.2 A DQ178109

Stelis concinna 7.13.3 A DQ178107

Stelis concinna 7.13.4 A DQ178110

Stelis concinna 7.14.2 A DQ178112

Stelis concinna 7.18.3 A DQ178043 A DQ178095

Stelis concinna 7.18.4 E DQ178082

Stelis concinna 7.19.1 A DQ178094

Stelis concinna 7.19.3 A DQ178032 A DQ178093

Stelis concinna 7.20.1 A DQ178030 A DQ178096

Stelis concinna 7.20.2 A DQ178042 A DQ178088

Stelis concinna 7.20.3 A DQ178033 A DQ178090

Stelis concinna 7.20.4 A DQ178041 A DQ178089

Stelis concinna 7.21.1 E DQ178075

Stelis concinna 7.21.2 E DQ178076

Stelis concinna 9.2 A DQ178111

Stelis concinna 9.3 culture G DQ178029 G DQ178029

Stelis concinna 9.6 A DQ178084

Stelis concinna 9.7 A DQ178092

Stelis concinna 9.8 A DQ178038 A DQ178097

Stelis concinna 9.9 A DQ178031 A DQ178086

Stelis hallii 1.1 B DQ178044 B DQ178113

Stelis hallii 1.2 E DQ178065

Stelis hallii 1.2b D DQ178051

Stelis hallii 1.4 G DQ178118

Stelis hallii 1.6 B DQ178114

Stelis hallii 1.7 D DQ178050

Stelis hallii 1.8 A DQ178085

Stelis hallii 1.11 culture E DQ178066 E DQ178066

Stelis hallii 1.15 D DQ178057

Stelis hallii 1.16 D DQ178055

Stelis hallii 1.17 A DQ178103

Stelis hallii 1.18 A DQ178037 A DQ178104

Stelis hallii 1.18b D DQ178053

Stelis hallii 1.19 D DQ178059 E DQ178073

Stelis hallii 1.19b E DQ178071

Stelis hallii 1.21 E DQ178072

Stelis hallii 1.21b E DQ178077

Stelis hallii 1.23 D DQ178060

Stelis superbiens C3.5.2 culture A DQ178036 A DQ178036

Stelis superbiens C3.5.3 E DQ178083

Stelis superbiens C3.5.4 E DQ178078

Stelis superbiens C3.9.2 A DQ178039 A DQ178087

Stelis superbiens C3 MN3 D DQ178058 D DQ178117

Stelis superbiens C3.MN4 C DQ178046 C DQ178115

Diverse tulasnelloid fungi form mycorrhizas 1261(continued on next page)

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

Fig 2 Degenerating hyphae adjoining collapsed hyphae

(ch) in an active cortical cell of Stelis concinna root.parenthesome was found that displayed coarse perforations

and might thus putatively be assigned to Ceratobasidium (Cur-

rah & Sherburne 1992; not shown). No simple-pored ascomy-

cetes 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 Bjer-

kandera, Polyporus and Tulasnella. Three cultures were identi-

fied as Tulasnella. These Tulasnella cultures exhibited slow

Table 1 (continued)

Orchid species nucLSU

clade GenBank a

Stelis superbiens C3.1MN D DQ1

Stelis superbiens C3.2MN D DQ1

Stelis superbiens C3.3MN D DQ1

Stelis superbiens C3.4MN D DQ1

Stelis superbiens C3.4MN4 D DQ1

Stelis superbiens C3.5MN5 E DQ1

Stelis superbiens C3.5MN5b F DQ1

1262vacuoles. Bar[ 1 mm.growth rates contrasting with the relative fast growth rate

observed in the fungi isolated. Ascomycetes closest to Crypto-

sporiopsis were the most frequently isolated fungi.

Molecular identification and phylogenetic analysisof 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, consider-

ing 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

nrDNA ITS-5.8S

ccession no. clade GenBank accession no.

78056

78052 A DQ178105

78054

78061 A DQ178101

78062

78064 E DQ178074

78048

J. P. Suarez et al.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 se-

quences 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 Elec-

tronic Appendix C). BIONJ (trees not shown) and MCMC

yielded similar groupings of Tulasnella clades. Only small var-

iations were present in the clade support values. As men-

tioned above, the 5.8S tree (Electronic Appendix C) was less

resolved than the nucLSU tree. However, the unexpected het-

erogeneity displayed by the 5.8S data set made it difficult to

find a suitable outgroup sequence. Therefore, we rooted the

Fig 3 Branched hypha displaying septa (arrow) without

clamp formation in root cortical tissue of Stelis concinna.

Bar[ 1 mm.

Fig 4 Square section of active hypha of Tulasnella, dis-

Diverse tulasnelloid fungi form mycorrhizasplaying mitochondria (m), glycogen rosettes, and fibrillar

slime between cell wall layers (arrowheads). Bar[ 1 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 align-

ment 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 se-

quences within each clade of the nucLSU D1/D2 yielded 13

Tulasnella sequence types. We treated sequences as belonging

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.

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

The parenthesomes consist of two electron-dense

membranes bordering an internal electron transparent

1263zone and show slightly recurved borders (arrows).

Bar[ 0,3 mm.

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.

1264 J. P. Suarez et al.

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 dis-

tances 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).

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.

1266 J. P. Suarez et al.

to the same sequence type when proportional differences

were

compare the seven Tulasnella clades of symbionts of epiphytic

orchids studied here with named Tulasnella species and my-

corrhizal Tulasnellas mostly from terrestrial orchids. Tulas-

nellas of the studied epiphytic orchid species were distinct

from so far known Tulasnellas associated with terrestrial or-

chids. Tulasnelloid fungi associated with the terrestrial tem-

perate orchids Cypripedium spp. (subfamily Cypripedioideae)

and Dactylorhiza majalis (AY634130) (subfamily Orchidoideae)

were displayed in a basal position with respect to the Tulas-

nella sequence types from Stelis and Pleurothallis in the nucLSU

phylogeny (Fig 7). In the 5.8S nrDNA phylogeny, the tulasnel-

loid 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 posi-

tion relative to Tulasnella sequence types from Stelis and Pleu-

rothallis (Electronic Appendix C). Molecular phylogenetic

analyses of the family Orchidaceae are consistent in respect

to a basal position of Cypripedioideae and Orchidoideae com-

pared 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 my-

corrhizal 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 ar-

rive at convincing conclusions about coevolution between or-

chids 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 ex-

clude the possibility that the differences in numbers of Tulas-

nella 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. Obvi-

ously, 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 Rhizocto-

nias, seed germination was stimulated by non-optimal myco-

bionts, 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 For-

schungsgemeinschaft (DFG project FOR 402). We thank the

Fundacion Cientfica San Francisco for providing research fa-

cilities, Lorena Endara for help in orchid identification, and

1268Paulo Herrera for help in laboratory work. The supply of fungalstrains 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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Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forestIntroductionMaterials and methodsStudy siteSamplingLight and transmission electron microscopyFungal isolationDNA extraction, PCR and sequencingPhylogenetic analyses

ResultsMicroscopical and ultrastructural features of the mycorrhizasFungal isolation and molecular identification of isolatesMolecular identification and phylogenetic analysis of mycorrhiza-associated fungi

DiscussionAcknowledgementsSupplementary dataReferencesFurther reading