Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest

Diverse tulasnelloid fungi form mycorrhizas with epiphyticorchids in an Andean cloud forest

Juan Pablo SUAREZab Michael WEIszligb Andrea ABELEb Sigisfredo GARNICAbFranz OBERWINKLERb Ingrid KOTTKEb

aCentro de Biologıa Celular y Molecular Universidad Tecnica Particular de Loja San Cayetano Alto sn CP 11 01 608 Loja EcuadorbEberhard-Karls-Universitat Tubingen Botanisches Institut Spezielle Botanik und Mykologie Auf der Morgenstelle 1D-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 58S 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

ordf 2006 The British Mycological Society Published by Elsevier Ltd All rights reserved

Introduction

Although most land plants are associated with symbioticfungi forming mycorrhizas or mycorrhiza-like associationsmany epiphytes live without such associations eg mossesmany liverworts bromeliads and ferns (Kottke 2002) Find-ings on the mycorrhizal state of epiphytic orchids were con-troversial Only sporadic fungal colonization was foundin a number of epiphytic Malaysian orchids (Hadley amp

Williamson 1972) but a high infection rate was reportedfrom canopy-dwelling orchid species in Florida (Benzing1982) Different degrees of infection including non-infectedroots were observed in epiphytic orchids in Ecuador (Ber-mudes amp Benzing 1989) Goh et al (1992) found high fungal col-onization in the epiphytic orchid Dendrobium crumenatum froma natural stand in Singapore but only low or no mycorrhiza-tion in orchids from nurseries Rivas et al (1998) and Pereiraet al (2005) reported intense colonization of epiphytic orchids

Corresponding authorE-mail address jpsuarezutpleduec

ava i lab le at wwwsc ienced i rec t com

journa l homepage wwwe l sev i er com loca te mycres

m y 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 ndash 1 2 7 0

0953-7562$ ndash see front matter ordf 2006 The British Mycological Society Published by Elsevier Ltd All rights reserveddoi101016jmycres200608004

in Costa Rica and Brazil respectively All investigators statedthat fungal colonization was restricted to roots attaching tothe substrate aerial roots were not colonized

Identification of root-associated fungi was mostly achievedby isolation on sterile media (Rasmussen 2002) However thedistinction between endophytic fungi inhabiting only the ve-lamen or the root surface and reliably mycorrhiza-formingfungi 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 beensuccessfully attempted in terrestrial orchids (Warcup amp Talbot1967 1971 1980 Bougoure et al 2005) In cases where sexualstages could be achieved the isolated fungi were determinedas basidiomycetes belonging to the Sebacinales Tulasnellalesor Ceratobasidiales (Warcup 1981 Warcup amp Talbot 19671971 1980) Tulasnella anamorphs (Epulorhiza) were isolatedeg from epiphytic Epidendrum conopseum in Florida (Zettleret al 1998) epiphytic Epidendrum rigidum Polystachia concreta(Pereira et al 2003) and terrestrial Oeceoclades maculata fromBrazil (Pereira et al 2005) DNA sequencing supported thepresence of Tulasnella Sebacinales and Ceratobasidium inCypripedium 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 al2005) or by direct DNA isolation from pelotons (Kristiansenet 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 sequencingdirectly from mycorrhizas (Taylor amp Bruns 1997 1999 Tayloret al 2003 Bidartondo et al 2004 Selosse et al 2004 Julou et al2005) thus widening the previous knowledge on orchidmycobionts

Selosse et al (2004) confirmed their molecular finding of Tu-ber spp (Ascomycota) as orchid mycobionts by ultrastructuraldemonstration of ascomycetous hyphae in the cortical cells ofthe orchid roots Ascomycetes can be discerned from basidio-mycetes by ultrastructure of the cell wall and the septal poreand different groups of basidiomycetes can be distinguishedby the parenthesomes covering the dolipores (Wells amp Ban-doni 2001) tulasnelloid fungi display characteristic slime bod-ies in the cell walls (Bauer 2004) In spite of these diagnosticpossibilities transmission electron microscopy has rarelybeen used in orchid studies addressing fungal identity How-ever the previous work is encouraging (Currah amp Sherburne1992 Andersen 1996) and minimizes errors resulting fromcontamination during isolation of fungi or DNA directly frommycorrhiza samples In our study of the orchid mycobiontsof four epiphytic pleurothallid orchid species in the Andeancloud forest of south Ecuador we therefore combined ultra-structural studies with DNA sequencing and isolation

Stelis concinna S hallii S superbiens and Pleurothallis lilijaeFoldats were selected because of the abundance and frequentflowering of these small orchids in the tropical mountainrainforest of the study area Thus severe violations of theorchid populations in this highly endangered forest could be

minimized The genera Stelis and Pleurothallis belong tosubtribe Pleurothallidinae the largest subtribe in the tribeEpidendreae of Orchidaceae (Luer 1986ab) which is widely dis-tributed in tropical America These two genera include 485Pleurothallis 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 ofthem are in culture so far The rapid loss of habitats requiresan understanding of the symbiotic relationships in orderto support conservation efforts for these orchids Accordingto Hamilton et al (1995) approximately 90 of the NorthernAndean forests have been already destroyed Consequentlythe orchids and their fungi might be lost in the near futureif not taken into culture As the mycorrhizal state and myco-bionts of the epiphytic Pleurothallidinae were unknown noadvice could be given to laboratories interested in orchid cul-turing or to local forest management aiming to rehabilitatethe tropical mountain forest with its epiphytic orchid diversity(see httpwwwbergregenwaldde of which this work is apart) We therefore started with light- and transmission elec-tron microscopic investigation of the selected orchid speciesand continued with DNA isolation and sequencing of themost frequently observed fungal group the Tulasnellales Inparallel isolation of mycelia was carried out yielding severalTulasnella isolates We were especially interested to seewhether the Tulasnellales present as mycobionts of epiphyticorchids in the tropical mountain rain forest were distinctfrom those described for other habitats of the Northern Hemi-sphere and Australia This knowledge would help to decide iflocal or ubiquitous fungal isolates were appropriate for culti-vation of the local orchids and would support evaluation ofloss of local fungi for rehabilitation of orchids in the tropicalmountain forest area

Materials and methods

Study site

The study site is located on the eastern slope of the Cordillera ElConsuelo in the northern Andes of southern Ecuador The areaof about 1000 ha belongs to the Reserva Biologica San Franciscoand borders the Podocarpus National Park in the north halfway between Loja and Zamora Zamora-Chinchipe Province(358 S 7904 W) The tropical mountain rainforest covers thesteep slopes between 1850 and 2700 m asl Characteristicand most frequent trees are Melastomataceae Rubiaceae Laura-ceae and Euphorbiaceae reaching a height of 25 m Crown densityas measured by a spherical densitometer is 94 on averageonly 75 were open canopy (Homeier 2004)

The richness and abundance of epiphytes is due to thesemi- to sub-humid climate with rainfall during ten monthsand even more frequent fog combined with moderate temper-atures (Richter 2003) Mean annual precipitation at 1950 masl is 2200 mm annual mean temperature is 155 C (144-175 C) Precipitation increases with higher elevation andreaches 4000 mm at 2600 m asl Air humidity in two monthsis 96 on average and does not fall below 70 during thedrier season (Noske 2004) The high air humidity is especiallyimportant for stem epiphytes

1258 J P Suarez et al

Sampling

Sampling was carried out at small paths at an altitudinal gra-dient between 1850 and 2100 m asl Stelis hallii was found inthe forest covering the steep slopes between 1800 and1900 m asl while S superbiens and Pleurothallis lilijae werecollected in the forest covering the mountain ridge between1900 and 2100 m asl Stelis concinna was restricted to the up-per part of the mountain ridge where the forest was lessdense with only 92 crown density and exposition to fre-quent and heavy winds

Roots were collected continuously during three yearsfrom 2003 until 2005 from a total of 77 flowering individuals22 of S hallii 17 of S superbiens 25 of S concinna and 13 ofPleurothallis lilijae All selected plants were epiphytes ontrunks or branches of standing trees at 50 cm to 200 cmabove the forest floor Distances between trees with flower-ing orchids varied between 50 cm and several metres (up to20 m) Identification of trees was not taken into consider-ation Roots of one flowering individual orchid per treestem were collected One to four roots per plant individualwere packed in aluminum foil to prevent desiccationand transported to the laboratory the same day As pre-investigation had shown that mycorrhizal fungi colonizedonly roots in contact with the stems best when also coveredby mosses or a minute humus layer later on only such rootswere selected Root samples were processed the day ofcollection as pre-investigation had revealed a fast loss ofvitality in the symbiotic fungi Vouchers of the orchidspecimens were deposited in the Herbarium of UTPL LojaEcuador including flowers fixed in ethanol Vouchers ofthe mycorrhizas were embedded in resin and deposited inthe Herbarium of Tubingen University (TUB)

Light and transmission electron microscopy

Light microscopy was used to select material with fungal coilsTransversal sections were cut from the middle part of eachroot sample by hand using a razor blade Sections werestained by Methyl blue 005 solution (C I 42780 Merck) inlactic acid for 10 min on microscopic slides The sampleswere 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 displayinghigh frequency of vital looking hyphal coils 56 in total andat least ten of each species were fixed in 25 glutaralde-hyde-formaldehyde in Soslashrensen buffer (Karnovsky 1965)post-fixed in 1 osmium tetroxide for 1 h dehydrated in anacetone series and flat embedded in Spurrrsquos resin low viscos-ity longer pot-life formulation (Spurr 1969) Semithin sectionswere cut from the embedded samples stained with 06 neo-fuchsin crystal-violet mounted in Entellan and observed inthe light microscope 20 samples with apparently vitalhyphae originating from different plant individuals wereselected 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 ZeissTEM109

Fungal isolation

Isolation of fungi was initiated the day of sampling Colonizedroot pieces were surface-sterilized Roots were rinsed indistilled water with some drops of liquid soap immersed inethanol (70 ) for 30 s immersed in Ajax chloro 20 (house-hold bleach sodium hypochlorite 525 ) for 10 min andfinally rinsed in sterile distilled water The velamen wasthen removed using a stereo microscope a thin blade and for-ceps Five square sections of 1-3 mm thickness were cut byhand from the middle part of the root and transferred toa plate with MYP media (malt extract 7 g peptone 1 g andagar agar 15 g l1) or MMNC media (modified Melin-NorkransKottke et al 1987 NaCl 0025 g KH2PO4 05 g (NH4)2HPO4 025 gCaCl2 005 g MgSO4 7H2O 015 g FeCl3 (1 ) 1 ml thiamin1 ml malt extract 5 g glucose 10 g caseinhydrolysate 1 gagar 20 g riboflavin 1 ml of 001 solution trace elements10 ml according to Fortin and Piche 1979) No antibioticswere added

DNA extraction PCR and sequencing

Portions of 1-2 cm length of well colonized roots of which thevelamen was removed were collected in cups the same day ordried and kept on silica gel for later DNA isolations DNA wasextracted from the fresh or dried mycorrhizal tissue and fromfungal 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 usinguniversal fungal primer combinations ITS1FITS4 ITS1FNL4NLMW1LR5 NLMW1TW14 and ITS1FTW14 (details con-cerning the primers used are given in the Electronic AppendixA) Several PCR products were obtained and sequenced DNAisolated from fungal cultures was amplified using the primercombination ITS1FNL4 or ITS1NL4 Nested PCR was con-ducted to specifically amplify DNA from tulasnelloid fungias 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 theprimer combination ITS1FTW14 or ITS1TW14 and the sec-ond using template obtained in the first PCR in dilutions of101 102 and 103 with the primer combinations ITS1ITS4-Tul for the internal transcribed spacers (ITS1 58S nu-clear ribosomal gene and ITS2) and NLMW1LR5 ITS4-TulRLR5 and 58S-TulNL4 for the 5rsquo part of the nuclear large sub-unit ribosomal DNA (nucLSU) Primers ITS4-Tul and ITS4-TulR target a Tulasnella-specific sequence at the 3rsquo end ofITS2 The Tulasnella-specific primer 58S-Tul (5rsquo-TCATTCGATGAAGACCGTTGC-3rsquo) designed for this study targets a specificsequence at the 5rsquo end of the 58S rDNA

PCR conditions were as follows initial denaturation at94 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 15 mM MgCl2 200 mM of each dNTP (Life Tech-nologies Eggenstein Germany) 05 mM of each of the primers(MWG-Biotech Ebersberg Germany) 1U Taq polymerase (Life

Diverse tulasnelloid fungi form mycorrhizas 1259

Technologies Eggenstein Germany) with an amplificationbuffer (Life Technologies Eggenstein Germany)

In every PCR a control including PCR mix without DNAtemplate was included Success of the PCR amplificationswas tested in 07 agarose stained in a solution of ethidiumbromide 05 mg ml1 PCR products were purified using theQIAquick protocol (Qiagen) Cycle sequencing was conductedusing BigDye version 31 chemistry and sequencing wasdone on an ABI 3100 Genetic Analyzer (Applied BiosystemsFoster City CA) Both strands of DNA were sequencedSequence editing was performed using Sequencher version45 (Gene Codes Ann Arbor MI) The sequences obtainedin this study are available from GenBank under accessionnumbers 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 werepreviously isolated from Australian orchids and determinedby J H Warcup (Warcup amp Talbot 1967 1971)

Phylogenetic analyses

We used BLAST (Altschul et al 1997) against the NCBI nucleo-tide database (GenBank httpwwwncbinlmnihgov) to de-tect published sequences with a high similarity to the nucLSUsequences obtained from the Ecuadorian epiphytic orchidsFor thorough phylogenetic analysis of the Tulasnella se-quences we analyzed nucLSU and ITS-58S alignments includ-ing the closest BLAST matches together with the sequencesfrom the Warcup Tulasnella reference isolates (see above)and other sequences from Tulasnellaceae and related groupsretrieved from GenBank

Sequences were aligned using the G-INS-i or L-INS-i strat-egy as implemented in MAFFT v5667 (Katoh et al 2005) Dueto the heterogeneity of the Tulasnella sequences we had to ex-clude considerable portions of the nucLSU sequences for phy-logenetic analysis Even the 58S ribosomal region consideredas universally conserved exhibited a remarkable heterogene-ity as was already mentioned by Bidartondo et al (2003) Asexpected the ITS1 and ITS2 rDNA could not be aligned overthe whole data set Therefore we used the 58S region to cal-culate phylogenetic trees of a wider phylogenetic spectrumand produced several other phylogenetic analyses includingsubsets of related sequences for which we used portions ofthe ITS1 and ITS2 regions in addition to the 58S sequencesThe alignments used can be obtained from TreeBASE (httpwwwtreebaseorg) under accession number S1629

Neighbour-joining (NJ) and a Bayesian likelihood approachwere used to estimate the phylogenetic relationships Theneighbour-joining analysis was performed in PAUP (Swofford2002) using the BIONJ modification of the NJ algorithm toaccomplish the observed high genetic variability in the se-quences used (Gascuel 1997) DNA substitution models and in-dividual model parameters were estimated using the Akaikeinformation criterion (AIC) as implemented in Modeltest ver-sion 37 (Posada amp Crandall 1998) For the Bayesian approachbased on Markov chain Monte Carlo (MCMC) we usedMrBayes version 30b4 (Huelsenbeck amp Ronquist 2001) Eachdataset was analyzed using the DNA substitution models esti-mated using the Akaike information criterion (AIC) in

MrModeltest version 22 (Nylander 2004) involving fourincrementally heated Markov chains over four million gener-ations and using random starting trees Trees were sampledevery 100 generations resulting in a total of 40000 trees fromwhich the last 24000 were used to compute a 50 majorityrule consensus tree Each analysis was repeated to checkthe 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 75 R K Colwell unpubl)

We determined the proportional differences between se-quences within each clade of the nucLSU D1D2 in order to de-fine sequence types We compared the number of Tulasnellasequence types within single and between different orchidspecies The proportional differences between sequenceswere pooled into five tables (Electronic Appendix B)

Results

Microscopical and ultrastructural features of the mycorrhizas

Fungal pelotons were present in nearly all cross-sections ofroots sampled directly from the tree bark No fungal pelotonswere observed in aerial roots This observation was confirmedby sampling roots of another 65 epiphytic Stelis and Pleurothal-lis orchids indicating that the roots became colonized onlywhere the fungi contacted the bark or the thin humus layerPelotons 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 thesame cells suggesting that cells became re-infected severaltimes According to the light microscopical observationsmany fungal pelotons were found collapsed after the plantshad been kept one night in the laboratory Abundant hyphaecolonized 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 theplant vacuole forming small compartments or a network ofsmall vacuoles (Fig 1) Degenerating hyphae were attachedto collapsed pelotons (Fig 2) Alive hyphae contained abundantglycogen granules (Figs 1 3 and 5) The hyphae formed septaclamps were not observed (Fig 3) The septa showed doliporeswith imperforate dish-shaped parenthesomes with slightlyrecurved margins (Fig 6) These tulasnelloid parenthesomeswere 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 4and 5 arrows) This phenomenon became very prominent inageing cultures and the slime was then strongly osmiophilic(not shown) The combination of this type of parenthesomesand the lsquolsquoslime bodiesrsquorsquo in the cell walls was confirmed forall the investigated mycorrhizas and in the Tulasnella isolatesThe recurved ends of the parenthesome were only detected byserial sectioning since the appearance of the parenthesomesvaried among the sections and may appear flattened or bowedin a steeper angle In three samples we additionally found flatimperforate parenthesomes indicating sebacinoid fungi (Wil-liams amp Thiol 1989 not shown) In one sample a dome-shaped


Table 1 ndash 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-58S

clade GenBank accession no clade GenBank accession no

Pleurothallis lilijae C211 A DQ178035 A DQ178099Pleurothallis lilijae C212 E DQ178067Pleurothallis lilijae C213 A DQ178040 A DQ178100Pleurothallis lilijae C215 E DQ178080Pleurothallis lilijae C217 A DQ178102Pleurothallis lilijae C221 E DQ178079Pleurothallis lilijae C21MN A DQ178034 A DQ178098Pleurothallis lilijae C25MN7 F DQ178047 F DQ178069Pleurothallis lilijae C2MN1 D DQ178063 D DQ178116Pleurothallis lilijae C2MN5 F DQ178049 F DQ178070Pleurothallis lilijae C2MN2 E DQ178068 E DQ178081Pleurothallis lilijae C2MN6 B DQ178045Stelis concinna 76 A DQ178108Stelis concinna 77 A DQ178106Stelis concinna 78 A DQ178091Stelis concinna 7132 A DQ178109Stelis concinna 7133 A DQ178107Stelis concinna 7134 A DQ178110Stelis concinna 7142 A DQ178112Stelis concinna 7183 A DQ178043 A DQ178095Stelis concinna 7184 E DQ178082Stelis concinna 7191 A DQ178094Stelis concinna 7193 A DQ178032 A DQ178093Stelis concinna 7201 A DQ178030 A DQ178096Stelis concinna 7202 A DQ178042 A DQ178088Stelis concinna 7203 A DQ178033 A DQ178090Stelis concinna 7204 A DQ178041 A DQ178089Stelis concinna 7211 E DQ178075Stelis concinna 7212 E DQ178076Stelis concinna 92 A DQ178111Stelis concinna 93 culture G DQ178029 G DQ178029Stelis concinna 96 A DQ178084Stelis concinna 97 A DQ178092Stelis concinna 98 A DQ178038 A DQ178097Stelis concinna 99 A DQ178031 A DQ178086Stelis hallii 11 B DQ178044 B DQ178113Stelis hallii 12 E DQ178065Stelis hallii 12b D DQ178051Stelis hallii 14 G DQ178118Stelis hallii 16 B DQ178114Stelis hallii 17 D DQ178050Stelis hallii 18 A DQ178085Stelis hallii 111 culture E DQ178066 E DQ178066Stelis hallii 115 D DQ178057Stelis hallii 116 D DQ178055Stelis hallii 117 A DQ178103Stelis hallii 118 A DQ178037 A DQ178104Stelis hallii 118b D DQ178053Stelis hallii 119 D DQ178059 E DQ178073Stelis hallii 119b E DQ178071Stelis hallii 121 E DQ178072Stelis hallii 121b E DQ178077Stelis hallii 123 D DQ178060Stelis superbiens C352 culture A DQ178036 A DQ178036Stelis superbiens C353 E DQ178083Stelis superbiens C354 E DQ178078Stelis superbiens C392 A DQ178039 A DQ178087Stelis superbiens C3 MN3 D DQ178058 D DQ178117Stelis superbiens C3MN4 C DQ178046 C DQ178115

(continued on next page)


parenthesome was found that displayed coarse perforationsand might thus putatively be assigned to Ceratobasidium (Cur-rah amp Sherburne 1992 not shown) No simple-pored ascomy-cetes were found in the cortical tissue of the 20 investigatedsamples although they were present in the velamen (notshown)

Fungal isolation and molecular identification of isolates

Fungal growth was observed in only 44 plates out of 108 usedfor fungal isolation each one containing five root pieces Fourfungal cultures were obtained from a total of 13 plates of Stelishallii 15 from 36 plates of Stelis superbiens 22 from 55 plates ofStelis concinna and three from four plates of Pleurothallis lilijaemycorrhizas A preliminary molecular identification of thefungal isolates was carried out by BLAST searches againstthe GenBank nucleotide database retrieving the most similaravailable sequences (data not shown) The isolated fungiwere mainly ascomycetes closest to Xylaria Hypoxylon andCryptosporiopsis and less often basidiomycetes closest to Bjer-kandera Polyporus and Tulasnella Three cultures were identi-fied as Tulasnella These Tulasnella cultures exhibited slow

growth rates contrasting with the relative fast growth rateobserved 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 PCRproducts preliminarily identified by BLAST searches as closestto Cryptosporiopsis Fusarium Trichoderma (Ascomycota) andBjerkandera Antrodiella (Basidiomycota) Tulasnella sequenceswere infrequently obtained only the primer combinationNLMW1LR5 yielding few PCR products Primer combinationsincluding ITS1F and ITS4 failed to amplify Tulasnella DNA aswas already reported by Bidartondo et al (2003) Sequencesof Sebacinales basidiomycetes involved in a broad range ofmycorrhizal associations (Weiszlig et al 2004) were alsodetected but at lower frequence than Tulasnellales sequencesNo 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 the77 orchid individuals Tulasnelloid fungi were detected in 84samples (63 ) including the PCR products obtained by thetulasnelloid specific primer combinations without successfull

Table 1 (continued)



Stelis superbiens C31MN D DQ178056Stelis superbiens C32MN D DQ178052 A DQ178105Stelis superbiens C33MN D DQ178054Stelis superbiens C34MN D DQ178061 A DQ178101Stelis superbiens C34MN4 D DQ178062Stelis superbiens C35MN5 E DQ178064 E DQ178074Stelis superbiens C35MN5b F DQ178048

Fig 1 ndash Ultrastructure of the cortical tissue of Stelis concinnaroot displaying alive hyphae (h) of equal diameter inactive host cell (c) (v) Small compartments of orchid cellvacuoles Bar [ 1 mm

Fig 2 ndash Degenerating hyphae adjoining collapsed hyphae(ch) in an active cortical cell of Stelis concinna rootBar [ 1 mm


sequencing The nested PCR conducted in order to selectivelyamplify Tulasnella DNA using the primer combination ITS1TW14 in the first amplification and the primer combinationsITS1ITS4-Tul for the ITS-58S region and ITS4-TulRLR5 or58S-TulNL4 for a part of the LSU region respectively in thesecond PCR yielded PCR products for the majority of samplesPCR success was higher with DNA extracted from fresh rootsamples and lower with DNA from dried roots

The phylogenetic analyses of nucLSU and ITS-58S se-quences yielded consistent results Seven clades which inthe following we refer to as clades A to G were retrievedfrom the analyses of both ribosomal regions (Fig 7 and Elec-tronic Appendix C) BIONJ (trees not shown) and MCMCyielded similar groupings of Tulasnella clades Only small var-iations were present in the clade support values As men-tioned above the 58S tree (Electronic Appendix C) was lessresolved than the nucLSU tree However the unexpected het-erogeneity displayed by the 58S data set made it difficult tofind a suitable outgroup sequence Therefore we rooted the

58S overview tree (Electronic Appendix C) in such a waythat we obtained best consistency with the rooted LSU tree(Fig 7) Portions of ITS1 and ITS2 were added to the 58S align-ment where phylogenetic analysis was restricted to suitablesubsets of sequences detected in the 58S analysis finallyresulting in an increase of phylogenetic resolution for thesesubsets (Figs 8 and 9)

Our analysis of proportional differences between se-quences within each clade of the nucLSU D1D2 yielded 13Tulasnella sequence types We treated sequences as belonging

Fig 3 ndash Branched hypha displaying septa (arrow) withoutclamp formation in root cortical tissue of Stelis concinnaBar [ 1 mm

Fig 4 ndash Square section of active hypha of Tulasnella dis-playing mitochondria (m) glycogen rosettes and fibrillarslime between cell wall layers (arrowheads) Bar [ 1 mm

Fig 5 ndash Hypha in cortical root tissue of Stelis concinnadisplaying fibrillar slime between cell wall layers(arrowhead) and a doliporus with imperforate slightlydish-shaped parenthesomes (arrow) Bar [ 05 mm

Fig 6 ndash Close-up of a median section through the doliporusThe parenthesomes consist of two electron-densemembranes bordering an internal electron transparentzone and show slightly recurved borders (arrows)Bar [ 03 mm


Fig 7 ndash Phylogenetic placement of Tulasnella sequences from Stelis hallii Stelis superbiens Stelis concinna and Pleurothallislilijae inferred by MCMC analysis of nuclear rDNA coding for the 5rsquo terminal domain of the large ribosomal subunit (nucLSU)Numbers on branches designate neighbor-joining bootstrap values MCMC estimates of posterior probabilities (only valuesexceeding 50 are shown) Note that genetic distances cannot be directly correlated to branch lengths in the tree sincehighly diverse alignment regions were excluded for tree construction The tree was rooted with Multiclavula mucidaAF287875


Fig 8 ndash Phylogenetic placement of Tulasnella sequences clades A-C from Stelis hallii Stelis superbiens Stelis concinna andPleurothallis lilijae inferred by MCMC analysis of nuclear ITS-58S rDNA Numbers on branches designate neighbor-joiningbootstrap 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 fortree construction The tree was rooted with Tulasnella sequences from clade D from the analysis of 58S rDNA (ElectronicAppendix C)

Fig 9 ndash Phylogenetic placement of Tulasnella sequences clades E and F from Stelis hallii Stelis superbiens Stelis concinnaand Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-58S Numbers on branches designate neighbor-joiningbootstrap values MCMC estimates of posterior probabilities (only values exceeding 50 are shown) Note that geneticdistances cannot be directly correlated to branch lengths in the tree since highly diverse alignment regions wereexcluded for tree construction The tree was rooted with the Tulasnella sequence from the Warcup isolateT violea DQ520097


to the same sequence type when proportional differenceswere lt1 Clades D and E comprised four sequence typeswhile the other clades consisted only of one sequence typeeach (Fig 7 Electronic Appendix B) A direct comparison be-tween 58S-ITS and nucLSU phylogenies was hampered bythe fact that publicly available sequence data were restrictedto either one or the other of these two DNA regions for thevast majority of specimens studied so far (Table 1)

Mycobionts of the same sequence type were shared amongorchid species eg sequence types 2 6 and 12 Tulasnella se-quences from all four studied orchid species were present insequence type 1 (Fig 7) Tulasnellas belonging to different se-quence types were detected in mycorrhizas from the sameplant and even from the same root piece (Table 1) Six differentTulasnella sequence types were found in mycorrhizas of S halliiand S superbiens and five in P lilijae while only two sequencetypes were detected in mycorrhizas of S concinna (Fig 7)

Discussion

The importance of orchid mycorrhizal fungi and their role inorchid seed germination are known since Bernardrsquos observa-tions (Bernard 1909) In natural conditions the protocormdoes not develop further unless it receives an exogenous car-bohydrate supply from a suitable mycorrhizal fungus (Smithamp Read 1997) Although not demonstrated in nature so farthis dependence will also account for germination of epiphyticorchids Our finding of constant colonization of roots in con-tact with the bark supports the view of Benzing (1982) thatthe role of the fungi may be crucial also for the adult epiphyticorchids The most important benefit for the orchid may be toretain the fungus in order to assure further seed germinationSaprotrophic capabilities documented for several Tulasnellaspecies (Roberts 1999) could explain the growth of Tulasnellaon tree bark including the capacity of fruiting close to colo-nized roots (JPS pers obs) and promotion of seed germina-tion We observed decline of vitality of hyphae after only onenight of plant storage in the laboratory independent ofwhether or not the samples were cooled The decline of vital-ity was first indicated by loss of stainability of the pelotonswhich appeared yellow instead of blue in the light microscopeTEM revealed very rare occurrences of vital pelotons butabundant collapsed hyphae in this material No isolates ofTulasnellales were obtained from the stored samples and PCRamplification success was very low although only fully tur-gescent roots were processed It is rather unlikely that thefast decline of hyphal vitality was linked to carbon shortageas plenty of starch grains were visible in the root tissue andlarge amounts of glycogen were found in the vital hyphae Dis-ruption of the extraradical mycelium or increase of plantdefense reaction could be involved (Hadley 1982) So far thereason for this fast decline is unclear but it might erroneouslyimply the impression of low hyphal colonization rate

Our combination of ultrastructural research and DNA se-quencing revealed that Tulasnella species were regularly asso-ciated with the epiphytic Stelis and Pleurothallis species Wesuccessfully isolated three of the associated Tulasnellarsquosidentity proven by DNA sequences and septal porus typeThe flat bell-shaped imperforate parenthesomes with slightly

recurved margins consist of two electron-dense membranesbordering an internal electron transparent zone as was al-ready shown by Andersen (1996) for Rhizoctonia repens NBer-nard$ Epulorhiza repens (syn) the anamorph of Tulasnelladeliquescens (syn T calospora sensu Warcup amp Talbot 1967 fideRoberts 1999) The combination of this parenthesome typeand the slime bodies in the cell walls was previously describedfrom mycorrhiza-like associations of the liverwort Aneura pin-guis housing Tulasnella (Ligrone et al 1993) and apparently isthe best structural indication of Tulasnella (Bauer 2004) Theascomycetes that we isolated from the roots were not foundin the cortical tissue of the orchids but colonized only the ve-lamen Therefore these ascomycetes cannot be considered asmycorrhiza-forming fungi Without specific primers Tulasnellais nearly undetectable (Bidartondo et al 2003) The use of bothTulasnella-specific primers ITS4-Tul and 58S-Tul that targetITS2 and 58S respectively increased the success of Tulasnellaamplification but a complete dataset of all tulasnelloid fungiassociated with the investigated orchids can currently not beguaranteed The accumulation curve of clades vs number ofcollected individuals was not saturated (Electronic Appendix D)

Tulasnella was confirmed as a genus with a range of orchidmycorrhiza forming species (Kristiansen et al 2004 Ma et al2003 McCormick et al 2004 Shefferson et al 2005 Warcup1971 1981 1985 Warcup amp Talbot 1967 1971 1980) Tulasnellacalospora was proposed by Hadley (1970) as an universal orchidsymbiont considering the capacity to establish in vitro symbi-otic associations with a broad range of orchid species Thereare however taxonomic problems concerning the speciesconcept in Tulasnella (Roberts 1999) The T calospora strains(T deliquescens fide Roberts 1999) isolated by Warcup from ter-restrial Australian orchids (Warcup amp Talbot 1967) belong totwo separate clades according to our nucLSU tree (Fig 7) Thestrains are even more clearly distinguished in the 58S-ITStree (Fig 8) clustering with Tulasnellarsquos detected in terrestrialorchids from a wide range of localities such as Spathaglottisplicata from Singapore and Epipactis gigantea from CaliforniaOur molecular analyses indicate that T calospora is likely tocomprise several distinct species Taxonomic problems ac-count also for T violea and T asymmetrica as T violea accessionAY293216 appears close to T pruinosa AF518662 in the nucLSUphylogenetic tree while the Warcup T violea isolate from theorchid Thelymitra sp clustered separately the WarcupT asymmetrica strains (T pinicola fide Roberts 1999) isolatedfrom the orchid Thelymitra sp fall into two groups (Fig 7) Onthe other hand Tulasnellarsquos with quite different trophic strat-egies are displayed as closely related in the nucLSU tree egthe mycobiont of the myco-heterotrophic liverwort Cryptothal-lus mirabilis (AY192482) that also forms ectomycorrhizas withPinus and Betula (Bidartondo et al 2003) and T irregularis(AY243519) a Warcup isolate from Dendrobium dicuphum anAustralian orchid Attempts are currently undertaken to col-lect and describe Tulasnellarsquos occurring on the bark of thesampled trees in our research area and to induce basidia for-mation in the isolate cultures Morphological descriptionand determination combined with sequence typing appearedas promising tools to further clarify relationships betweenthese poorly studied mycobionts

Irrespective of the taxonomic problems available se-quence data from nucLSU and ITS-58S made it possible to


compare the seven Tulasnella clades of symbionts of epiphyticorchids studied here with named Tulasnella species and my-corrhizal Tulasnellarsquos mostly from terrestrial orchids Tulas-nellarsquos of the studied epiphytic orchid species were distinctfrom 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 nucLSUphylogeny (Fig 7) In the 58S nrDNA phylogeny the tulasnel-loid fungi associated with Dactylorhiza majalis (AY634130)Orchis purpurea (AJ549121) Spathaglottis plicata (AJ313457)Ophrys sphegodes (AJ549122) (all subfamily Orchidoideae) andCypripedium fasciculatum (AY966883) appeared in a basal posi-tion relative to Tulasnella sequence types from Stelis and Pleu-rothallis (Electronic Appendix C) Molecular phylogeneticanalyses of the family Orchidaceae are consistent in respectto a basal position of Cypripedioideae and Orchidoideae com-pared to Epidendroideae (eg Cameron et al 1999) Switchesfrom terrestrial to epiphytic habit or back were found to bemajor driving forces in radiation and specialization of orchids(Cameron 2002 2005) and beside pollinator relationships my-corrhizal interactions are now recognized crucial for orchidevolution (Taylor et al 2003) However more species need tobe 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 Tulasnellasymbionts associated with one orchid species Six Tulasnellasequence types were associated with one individual orchidspecies of S hallii and S superbiens five in P lilijae but onlytwo 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 orchidspecimen and roots will be examined However preferencesfor fungal partners have been demonstrated in other epiphyticorchids (Otero et al 2002 2004) In several cases we found thatone orchid individual was associated with Tulasnellarsquos frommore than one clade even in the same root segment Obvi-ously diverse Tulasnellarsquos form mycorrhizas with the greenepiphytic pleurothallid orchids in the Andean cloud forestWhether these distinct fungi are crucial for seed germinationneeds to be verified experimentally In case of the lsquolsquoRhizocto-niasrsquorsquo seed germination was stimulated by non-optimal myco-bionts but symbionts that were not fully compatible resultedin high seedling mortality (Rasmussen 2002) Our analysesindicate that efficient rehabilitation of epiphytic orchids innature and recruitment in the nursery probably requires theusage of distinct Tulasnella species as orchid mycobionts

Acknowledgements

This research was generously supported by the Deutsche For-schungsgemeinschaft (DFG project FOR 402) We thank theFundacion Cientıfica San Francisco for providing research fa-cilities Lorena Endara for help in orchid identification andPaulo 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 foundin the online version at 101016jmycres200608004

r e f e r e n c e s

Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller WLipman DJ 1997 Gapped BLAST and PSI-Blast a new genera-tion of protein database search programs Nucleic Acids Re-search 25 3389ndash3402

Andersen TF 1996 A comparative taxonomic study of Rhizocto-nia sensu lato employing morphological ultrastructural andmolecular methods Mycological Research 100 117ndash128

Bauer R 2004 Basidiomycetous interfungal cellular interactions ndasha synopsis In Agerer R Piepenbring M Blanz P (eds) Frontiersin Basidiomycote Mycology IHW-Verlag Eching Germany pp325ndash337

Bayman P Lebron LL Tremblay RL Lodge DJ 1997 Variation inendophytic fungi from roots and leaves of Lepanthes (Orchida-ceae) New Phytologist 135 143ndash149

Bermudes D Benzing DH 1989 Fungi in neotropical epiphyteroots BioSystems 23 65ndash73

Bernard N 1909 Lrsquoevolution dans la symbiose Les orchidees etleur champignons commenseaux Annales des Sciences Nature-lles Botanique Paris 9 1ndash196

Benzing DH 1982 Mycorrhizal infections of epiphytic orchidsin southern Florida American Orchid Society Bulletin 51618ndash622

Bidartondo MI Bruns TD Weiszlig M Sergio C Read D 2003 Spe-cialized cheating of the ectomycorrhizal symbiosis by an epi-parasitic liverwort Proceedings of the Royal Society of LondonSeries B Biological Sciences 270 835ndash842

Bidartondo MI Burghardt B Gebauer G Bruns TD Read DJ 2004Changing partners in the dark isotopic and molecular evi-dence of ectomycorrhizal liaisons between forest orchidsand trees Proceedings of the Royal Society of London Series BBiological Sciences 271 1799ndash1806

Bougoure JJ Bougoure DS Cairney WG Dearnaley DW 2005 ITS-RFLP and sequence analysis of endophytes from AcianthusCaladenia and Pterostylis (Orchidaceae) in southeasternQueensland Mycological Research 109 452ndash460

Cameron KM Chase MW Whitten WM Kores PJ Jarrell DCAlbert VA Yukawa T Hills HG Goldman DH 1999 Aphylogenetic analysis of the Orchidaceae evidence fromrbcL nucleotide sequences American Journal of Botany 86208ndash224

Cameron KM 2002 Intertribal relationships within Orchidaceaeas inferred from analyses of five plastid genes In Abstracts ofBotany 2002 Madison WI USA abstract 116 Also available atwebsite http wwwbotany2002orgsection12abstracts33shtml

Cameron KM 2005 Leave it to the leaves a molecular phyloge-netic study of Malaxideae (Epidendroideae Orchidaceae) AmericanJournal of Botany 96 1025ndash1032

Currah RS Sherburne R 1992 Septal ultrastructure of somefungal endophytes from boreal orchid mycorrhizas Mycologi-cal Research 96 583ndash587

Currah RS Zelmer CD Hambleton S Richardson KA 1997 Fungifrom orchid mycorrhizas In Arditti J Pridgeon AM (eds) Or-chid Biology Reviews and Perspectives VII Kluwer AcademicPublishers Dordrecht pp 117ndash170


Fortin JA Piche Y 1979 Cultivation of Pinus strobus root-hypocotylexplants for synthesis of ectomycorrhiza New Phytologist 83109ndash119

Gascuel O 1997 BIONJ An improved version of the NJ algorithmbased on a simple model of sequence data Molecular Biologyand Evolution 14 685ndash695

Goh CJ Sim AA Lim G 1992 Mycorrhizal associations in sometropical orchids Lindleyana 7 13ndash17

Hadley G 1970 Non-specificity of symbiotic infection in orchidmycorrhiza New Phytologist 69 1015ndash1023

Hadley G 1982 Orchid Mycorrhiza In Arditti J (ed) Orchid BiologyReviews and Perspectives II Cornell University Press Ithaca NYpp 83ndash118

Hadley G Williamson B 1972 Features of mycorrhizal infectionin some Malayan orchids New Phytologist 71 1111ndash1118

Hamilton LS Juvik JO Scatena FN 1995 The Puerto Rico tropicalcloud forest symposium ndashIntroduction and workshop synthe-sis Ecological Studies 110 1ndash19

Homeier J 2004 Baumdiversitat Waldstruktur und Wachstums-dynamik zweier tropischer Bergregenwalder in Ecuador undCosta Rica Dissertationes Botanicae 391 indashv 1ndash207

Huelsenbeck JP Ronquist FR 2001 MrBayes Bayesian inferenceof phylogenetic trees Bioinformatics 17 754ndash755

Huelsenbeck JP Larget B Miller RE Ronquist F 2002 Potentialapplications and pitfalls of Bayesian inference of phylogenySystematic Biology 51 673ndash688

Julou T Burghardt B Gebauer G Berveilleir D Damesin CSelosse MA 2005 Mixotrophy in orchids insight from a com-parative study of green individuals and non-photosynthesticindividuals of Cephanlanthera damasonium New Phytologist 166639ndash653

Karnovsky MJ 1965 A formaldehyde glutaraldehyde fixation ofhigh osmolarity for use in electron microscopy Journal of CellBiology 27 137ndash138

Katoh K Kuma K Toh H Miyata T 2005 MAFFT version 5 im-provement in accuracy of multiple sequence alignmentNucleic Acids Research 33 511ndash518

Kottke I Guttenberger M Hampp R Oberwinkler F 1987 An invitro method for establishing mycorrhizae on coniferous treeseedlings Trees 1 191ndash194

Kottke I 2002 Mycorrhizae ndash Rhizosphere determinants ofplant communities In Waisel Y Eshel A Kafkafi U (eds)Plant Roots The Hidden Half 3rd edn Marcel DekkerNew York pp 919ndash932

Kottke I Haug I Preuszliging M Setaro S Suarez JP Weiszlig MNebel M Oberwinkler F 2007 Guilds of mycorrhizal fungiand their relation to trees ericads orchids and liverworts ina neotropical mountain rain forest Basic and Applied Ecologyin press

Kristiansen KA Freudenstein JV Rasmussen FN Rasmussen HN2004 Molecular identification of mycorrhizal fungi in Neuwie-dia veratrifolia (Orchidaceae) Molecular Phylogenetics and Evolution33 251ndash258

Kristiansen KA Taylor DL Kjoller HN Rasmusen NRosendahl S 2001 Identification of mycorrhizal fungi fromsingle pelotons of Dactylorhiza majalis (Orchidaceae) usingsingle-strand conformation polymorphism and mitochon-drial ribosomal large subunit DNA sequences Molecular Ecol-ogy 10 2089ndash2093

Ligrone R Pocock K Duckett JG 1993 A comparative ultrastruc-tural study of endophytic basidiomycetes in the parasiticachlorophyllous hepatic Cryptothallus mirabilis and the closelyrelated allied photosynthetic species Aneura pinguis (Metzger-iales) Canadian Journal of Botany 71 666ndash679

Luer CA 1986a Icones Pleurothallidinarum I Systematics ofPleurothallidinae (Orchidaceae) Monographs in Systematic Bot-any 15 1ndash81

Luer CA 1986b Icones Pleurothallidinarum III Systematics ofPleurothallis (Orchidaceae) Monographs in Systematic Botany 201ndash109

Ma M Tan TK Wong SM 2003 Identification and molecularphylogeny of Epulorhiza isolates from tropical orchids Myco-logical Research 107 1041ndash1049

McCormick MK Whigham DF OrsquoNeill J 2004 Mycorrhizal diver-sity in photosynthetic terrestrial orchids New Phytologist 163425ndash438

Noske N 2004 Effekte anthropogener Storung auf die Diversitatkryptogamischer Epiphyten (Flechten Moose) in einemBergregenwald in Sudecuador Dissertation Gottingen

Nylander JAA 2004 MrModeltest 22 Program distributedby the author Evolutionary Biology Centre UppsalaUniversity

Otero JT Ackerman JD Bayman P 2002 Diversity and hostspecificity of endophytic Rhizoctonia-like fungi from tropicalorchids American Journal of Botany 89 1852ndash1858

Otero JT Ackerman JD Bayman P 2004 Differences in mycor-rhizal preferences between two tropical orchids MolecularEcology 13 2393ndash2404

Pereira OL Rollemberg CL Borges AC Matsuoka KKasuya MCM 2003 Epulorhiza epiphytica sp nov isolatedfrom mycorrhizal roots of epiphytic orchids in BrazilMycoscience 44 153ndash155

Pereira OL Kasuya MCM Borges AC Fernandes de Araujo E 2005Morphological and molecular characterization of mycorrhizalfungi isolated from neotropical orchids in Brazil CanadianJournal of Botany 83 54ndash65

Posada D Crandall KA 1998 Modeltest testing the model of DNAsubstitution Bioinformatics 14 817ndash818

Pridgeon AM 1987 The velamen and exodermis of orchid rootsIn Arditti J (ed) Orchid Biology Reviews and Perspectives IVCornell University Press Ithaca NY pp 140ndash192

Rasmussen HN 2002 Recent developments in the study of orchidmycorrhiza Plant and Soil 244 149ndash163

Richter M 2003 Using epiphytes and soil temperatures for eco-climatic interpretations in Southern Ecuador Erdkunde 57161ndash181

Rivas M Warner J Bermudez M 1998 Presencia de micorrizas enorquıdeas de un jardın botanico neotropical Revista de BiologıaTropical 46 211ndash216

Roberts P 1999 Rhizoctonia-forming Fungi a taxonomic guide RoyalBotanic Gardens Kew

Selosse M-A Faccio A Scappaticci G Bonfante P 2004 Chloro-phyllous and achlorophyllous specimens of Epipactis micro-phylla (Neottieae Orchidaceae) are associated withectomycorrhizal septomycetes including truffles MicrobialEcology 47 416ndash426

Shefferson RP Weiszlig M Kull T Taylor L 2005 High specificitygenerally characterizes mycorrhizal association in rare ladyrsquosslipper orchids genus Cypripedium Molecular Ecology 14613ndash626

Smith SE Read DJ 1997 Mycorrhizal Symbiosis 2nd edn AcademicPress San Diego California

Spurr AR 1969 A low viscosity epoxy resin embedding mediumfor electron microscopy Journal of Ultrastructure Research 2631ndash43

Swofford DL 2002 PAUP40 Phylogenetic Analysis UsingParsimony (and Other Methods) Sinauer AssociatesSunderland MA

Taylor DL Bruns TD 1997 Independent specialized invasions ofectomycorrhizal mutualism by two nonphotosynthetic or-chids Proceedings of the National Academy of Sciences USA 944510ndash4515

Taylor DL Bruns TD 1999 Population habitat and genetic cor-relates of mycorrhizal specialization in the lsquocheatingrsquo orchids


Corallorhiza maculata and C mertensiana Molecular Ecology 81719ndash1732

Taylor DL Bruns TD Szaro TM Hodges SA 2003 Divergence inmycorrhizal specialization with Hexalectris spicata (Orchida-ceae) a nonphotosynthetic desert orchid American Journal ofBotany 90 1168ndash1179

Tremblay RL Zimmerman JK Lebron L Bayman P Sastre IAxelrod F Alers-Garcıa J 1998 Host specificity andlower reproductive success in the rare endemic PuertoRican orchid Lepanthes caritensis Biological Conservation 85295ndash304

Warcup JH 1971 Specificity of mycorrhizal association in someAustralian terrestrial orchids New Phytologist 70 41ndash46

Warcup JH 1981 The mycorrhizal relationships of Australianorchids New Phytologist 87 371ndash381

Warcup JH 1985 Rhizanthella gardneri (Orchidaceae) its Rhizoctoniaendophyte and close association with Melaleuca uncinata(Myrtaceae) in western Australia New Phytologist 99 273ndash280

Warcup JH Talbot PHB 1967 Perfect states of Rhizoctoniasassociated with orchids I New Phytologist 66 631ndash641

Warcup JH Talbot PHB 1971 Perfect states of Rhizoctoniasassociated with orchids II New Phytologist 70 35ndash40

Warcup JH Talbot PHB 1980 Perfect states of Rhizoctonias as-sociated with orchids III New Phytologist 86 267ndash272

Weiszlig M Selosse M-A Rexer K Urban A Oberwinkler F 2004Sebacinales a hitherto overlooked cosm of heterobasidiomy-cetes with a broad mycorhizal potential Mycological Research108 1003ndash1010

Wells K Bandoni R 2001 Heterobasidiomycetes InMcLaughlin DJ McLaughlin EG Lemke PA (eds) The MycotaVol VII Part B Systematics and Evolution Springer VerlagHeidelberg pp 85ndash120

Williams PG Thiol E 1989 Ultrastructural evidence for theidentity of some multinucleate Rhizoctonias New Phytologist112 513ndash518

Zettler LW Delaney TW Sunley JA 1998 Seed propagation of theepiphytic green-fly orchid Epidendrum conopseum Selbyana19 249ndash253

f u r t h e r r e a d i n g

Cullings KW 1994 Molecular phylogeny of the Monotropoideae(Ericaceae) with a note on the placement of the PyroloideaeJournal of Evolutionary Biology 7 501ndash516

Gardes M Bruns TD 1993 ITS primers with enhanced specificityfor basidiomycetes application to the identification of my-corrhizae and rusts Molecular Ecology 2 113ndash118

Sampaio JP Weiszlig M Gadanho M Bauer R 2002 New taxa in theTremellales Bulleribasidium oberjochense gen et sp nov Papil-iotrema bandonii gen et sp nov and Fibulobasidium murrhard-tense sp nov Mycologia 94 873ndash887

Taylor DL 1997 The evolution of myco-heterotrophy and specificityin some North American orchids PhD thesis University ofCalifornia at Berkeley CA

Vilgalys R Hester M 1990 Rapid genetic identification and map-ping of enzymatically amplified ribosomal DNA from severalCryptococcus species Journal of Bacteriology 172 4238ndash4246

White TJ Bruns TD Lee SB Taylor JW 1990 Amplification anddirect sequencing of fungal ribosomal RNA genes for phylo-genetics In Innis MA Gelfand H Sninsky JS White TJ (eds)PCR-Protocols and applications A Laboratory Manual AcademicPress San Diego pp 315ndash322


in Costa Rica and Brazil respectively All investigators statedthat fungal colonization was restricted to roots attaching tothe substrate aerial roots were not colonized

Identification of root-associated fungi was mostly achievedby isolation on sterile media (Rasmussen 2002) However thedistinction between endophytic fungi inhabiting only the ve-lamen or the root surface and reliably mycorrhiza-formingfungi 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 beensuccessfully attempted in terrestrial orchids (Warcup amp Talbot1967 1971 1980 Bougoure et al 2005) In cases where sexualstages could be achieved the isolated fungi were determinedas basidiomycetes belonging to the Sebacinales Tulasnellalesor Ceratobasidiales (Warcup 1981 Warcup amp Talbot 19671971 1980) Tulasnella anamorphs (Epulorhiza) were isolatedeg from epiphytic Epidendrum conopseum in Florida (Zettleret al 1998) epiphytic Epidendrum rigidum Polystachia concreta(Pereira et al 2003) and terrestrial Oeceoclades maculata fromBrazil (Pereira et al 2005) DNA sequencing supported thepresence of Tulasnella Sebacinales and Ceratobasidium inCypripedium 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 al2005) or by direct DNA isolation from pelotons (Kristiansenet 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 sequencingdirectly from mycorrhizas (Taylor amp Bruns 1997 1999 Tayloret al 2003 Bidartondo et al 2004 Selosse et al 2004 Julou et al2005) thus widening the previous knowledge on orchidmycobionts

Selosse et al (2004) confirmed their molecular finding of Tu-ber spp (Ascomycota) as orchid mycobionts by ultrastructuraldemonstration of ascomycetous hyphae in the cortical cells ofthe orchid roots Ascomycetes can be discerned from basidio-mycetes by ultrastructure of the cell wall and the septal poreand different groups of basidiomycetes can be distinguishedby the parenthesomes covering the dolipores (Wells amp Ban-doni 2001) tulasnelloid fungi display characteristic slime bod-ies in the cell walls (Bauer 2004) In spite of these diagnosticpossibilities transmission electron microscopy has rarelybeen used in orchid studies addressing fungal identity How-ever the previous work is encouraging (Currah amp Sherburne1992 Andersen 1996) and minimizes errors resulting fromcontamination during isolation of fungi or DNA directly frommycorrhiza samples In our study of the orchid mycobiontsof four epiphytic pleurothallid orchid species in the Andeancloud forest of south Ecuador we therefore combined ultra-structural studies with DNA sequencing and isolation

Stelis concinna S hallii S superbiens and Pleurothallis lilijaeFoldats were selected because of the abundance and frequentflowering of these small orchids in the tropical mountainrainforest of the study area Thus severe violations of theorchid populations in this highly endangered forest could be

minimized The genera Stelis and Pleurothallis belong tosubtribe Pleurothallidinae the largest subtribe in the tribeEpidendreae of Orchidaceae (Luer 1986ab) which is widely dis-tributed in tropical America These two genera include 485Pleurothallis 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 ofthem are in culture so far The rapid loss of habitats requiresan understanding of the symbiotic relationships in orderto support conservation efforts for these orchids Accordingto Hamilton et al (1995) approximately 90 of the NorthernAndean forests have been already destroyed Consequentlythe orchids and their fungi might be lost in the near futureif not taken into culture As the mycorrhizal state and myco-bionts of the epiphytic Pleurothallidinae were unknown noadvice could be given to laboratories interested in orchid cul-turing or to local forest management aiming to rehabilitatethe tropical mountain forest with its epiphytic orchid diversity(see httpwwwbergregenwaldde of which this work is apart) We therefore started with light- and transmission elec-tron microscopic investigation of the selected orchid speciesand continued with DNA isolation and sequencing of themost frequently observed fungal group the Tulasnellales Inparallel isolation of mycelia was carried out yielding severalTulasnella isolates We were especially interested to seewhether the Tulasnellales present as mycobionts of epiphyticorchids in the tropical mountain rain forest were distinctfrom those described for other habitats of the Northern Hemi-sphere and Australia This knowledge would help to decide iflocal or ubiquitous fungal isolates were appropriate for culti-vation of the local orchids and would support evaluation ofloss of local fungi for rehabilitation of orchids in the tropicalmountain forest area

Materials and methods

Study site

The study site is located on the eastern slope of the Cordillera ElConsuelo in the northern Andes of southern Ecuador The areaof about 1000 ha belongs to the Reserva Biologica San Franciscoand borders the Podocarpus National Park in the north halfway between Loja and Zamora Zamora-Chinchipe Province(358 S 7904 W) The tropical mountain rainforest covers thesteep slopes between 1850 and 2700 m asl Characteristicand most frequent trees are Melastomataceae Rubiaceae Laura-ceae and Euphorbiaceae reaching a height of 25 m Crown densityas measured by a spherical densitometer is 94 on averageonly 75 were open canopy (Homeier 2004)

The richness and abundance of epiphytes is due to thesemi- to sub-humid climate with rainfall during ten monthsand even more frequent fog combined with moderate temper-atures (Richter 2003) Mean annual precipitation at 1950 masl is 2200 mm annual mean temperature is 155 C (144-175 C) Precipitation increases with higher elevation andreaches 4000 mm at 2600 m asl Air humidity in two monthsis 96 on average and does not fall below 70 during thedrier season (Noske 2004) The high air humidity is especiallyimportant for stem epiphytes


Sampling






Fungal isolation















Results


















Table 1 (continued)























Discussion









Acknowledgements



Supplementary data


r e f e r e n c e s

















































































Sampling






Fungal isolation















Results


















Table 1 (continued)























Discussion









Acknowledgements



Supplementary data


r e f e r e n c e s


























































































Results


















Table 1 (continued)























Discussion









Acknowledgements



Supplementary data


r e f e r e n c e s






























































































Table 1 (continued)























Discussion









Acknowledgements



Supplementary data


r e f e r e n c e s

































































































Discussion









Acknowledgements



Supplementary data


r e f e r e n c e s



















































































Acknowledgements



Supplementary data


r e f e r e n c e s

















































































Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest

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