ORIGINAL ARTICLE

Defining species in Tulasnella by correlating morphologyand nrDNA ITS-5.8S sequence data of basidiomatafrom a tropical Andean forest

Darío Cruz & Juan Pablo Suárez & Ingrid Kottke &

Meike Piepenbring & Franz Oberwinkler

Received: 14 April 2010 /Accepted: 16 April 2010# German Mycological Society and Springer 2010

Abstract The genus Tulasnella comprises important orchidmycobionts. Molecular phylogenetic studies on nrITS-5.8Ssequences of Tulasnella species previously isolated frommycorrhizas of epiphytic orchids from a tropical Andeanforest showed genomic variability among clones which wasdifficult to interpret as intra- or interspecific variations or tocorrelate with described Tulasnella species. To improve thissituation, we collected basidiomata of Tulasnella in anAndean forest, studied part of the sequences of fungalribosomal genes and correlated molecular data with themorphology of the specimens. Within five basidiomatadisplaying slight morphological variability, we found inter-specimen nrITS1-5.8S-ITS2 variability corresponding toproportional differences of less than 1% except for oneclone with 5.1% divergence. Results indicate that theslightly variable basidiomata should be considered as onespecies, which is morphologically tentatively assigned tothe Tulasnella pruinosa complex. However, comparison ofnrITS1-5.8S-ITS2 sequences, including sequences of T.

pruinosa from other origins, indicate that Tulasnella sp. isonly distantly related to the T. pruinosa specimens includedin the analyses. Sequences of all morphologically similarand taxonomically well-identified species are required todecide whether the basidiomata analyzed in the presentstudy represent a new species. The new sequences arerather similar to sequences obtained previously frommycorrhizae of epiphytic orchids of the same area indicat-ing mycorrhizal potential of this fungus.

Keywords Tulasnella . Morphological characterization .

Orchid mycorrhiza .Molecular phylogeny . Intragenomicpolymorphism . Tropical mountain rain forest .

Southern Ecuador

Introduction

Species of the genus Tulasnella J. Schröt. (1888, in Cohn,Krypt. Fl. Schles. 3: 397) are characterized by basidia withmature basally swollen and apically constricted sterigmataand globose, ellipsoid and allantoid to vermiform orhelicoid basidiospores producing secondary spores byrepetition. Currently, 54 species of Tulasnellaceae are listedby Kirk et al. (2008). Tulasnella species are widespread andhave been reported from many countries of the northernhemisphere (Bourdot and Galzin 1909, 1928; Jülich andJülich 1976; Roberts 1992, 1993a, b, 1994a, b, 1999;Rogers 1932, 1933) but also from Australia (Warcup andTalbot 1967, 1971, 1980) and Tahiti (Olive 1957).Tulasnellaceae are poorly studied in South America(Greslebin and Rajchenberg 2001). Morphological charac-teristics of sexual states of Tulasnella species have oftenbeen documented inadequately, thus creating confusion inspecies delimitation (Roberts 1999).

D. Cruz (*) : J. P. Suárez : I. KottkeCentro de Biología Celular y Molecular,Universidad Técnica Particular de Loja,San Cayetano Alto s/n ,C.P. 11 01 608 Loja, Ecuadore-mail: djcruz@utpl.edu.ec

F. OberwinklerInstitute of Evolution and Ecology, Organismic Botany,Eberhard-Karls-University Tübingen,Auf der Morgenstelle 1,72076 Tübingen, Germany

M. PiepenbringInstitute of Ecology, Evolution and Diversity,Johann Wolfgang Goethe-University Frankfurt am Main,Siesmayerstr. 70-72,60323 Frankfurt am Main, Germany

Mycol ProgressDOI 10.1007/s11557-010-0692-3

In culture, Tulasnella rarely forms sexual states andinduction to form basidiomata has failed so far for SouthAmerican strains (Suárez et al. 2006, Pereira et al. 2003,2005). Warcup and Talbot (1967, 1971, 1980), however,successfully induced fructification of isolates of Tulasnellafrom Australian green terrestrial orchids. In the past,asexual states of Tulasnella isolates have been assigned tothe genus Rhizoctonia, but cultures and ultrastructuralstudies as well as molecular phylogenetic analyses revealedthat Rhizoctonia is an artificial and polyphyletic group(Taylor et al. 2002). Superfluously, asexual states ofTulasnella have been named as Epulorhiza (Bougoure etal. 2005; Ma et al. 2003; Pereira et al. 2003, 2005).

Molecular data of Tulasnella are available for isolatesfrom soil and orchid roots (isolates provided by Warcup andTalbot 1967, 1971, 1980; sequenced by diverse authors, seeNCBI GenBank, and Sharon et al. 2008; Shimura et al.2009) of a few cultures from basidiomata (McCormick etal. 2004), and most frequently by direct PCR amplificationof mycobionts from roots (Kristiansen et al. 2001, 2004;Bidartondo et al. 2003; Suárez et al. 2006; Shefferson et al.2005, 2007, 2008). Molecular phylogeny of the publisheddata revealed inconsistencies in species concepts andtaxonomy of Tulasnella (McCormick et al. 2004; Suárezet al. 2006; Taylor and McCormick 2008).

The ITS1 and ITS2 of the nuclear ribosomal RNA genes,including 5.8S (nrITS-5.8S;) are frequently used for DNA-based studies of fungi due to (1) ease of amplification ofthis multi-copy region even from field material (Iotti andZambonelli 2006); (2) variability on the species level(Nilsson et al. 2008); and (3) the large amount of sequencesavailable for this region in public databases (Ryberg et al.2008). However, undetermined intraspecific variability ofthe region may bias species delimitation. Nilsson et al.(2008) used all 4,185 fully identified fungal speciesrepresented by at least two satisfactory nrITS-5.8S sequen-ces in the International Nucleotide Sequence Database forevaluating intragenomic variability of this genome regionand its implications for molecular species identification. Nounifying and stringent upper limit for intraspecific variabil-ity in fungi was found, cautioning the canonical 3%threshold obtained by weighted average, and calling fordetailed determination of intraspecific nrITS-5.8S variabil-ity of distinct fungi. The genus Tulasnella did not fulfill therequests of the Nilsson et al. (2008) study and was notincluded in their analysis. Mostly, only one nrITS-5.8Ssequence is available in GenBank for one isolate from onespecimen or named species of Tulasnella impeding evalu-ation of intragenomic variability and biasing phylogeneticanalysis (Sharon et al. 2008). We hypothesize that suchmissing data can best be obtained by DNA extraction frombasidiomata of Tulasnella and sequencing of nrITS-5.8S.Simultaneously, molecular data can be correlated with

morphological characters. Here, we present the first resultsobtained from specimens of Tulasnella sampled in tropicalAndean forest.

Materials and methods

Study site and sampling

The study site Reserva Biológica San Francisco (RBSF) islocated on the eastern slope of the Cordillera El Consueloin the northern Andes of southern Ecuador, ZamoraChinchipe province (3°58′S, 79°04′W). The forest isclassified as an evergreen lower and upper montane rainforest (Ceja Andina) characterized by richness of vascularplants, cryptogams and epiphytes (Beck et al. 2008).Sampling of basidiomata was carried out along themountain ridge between 1,900 and 2,500 m a. s. l. duringMarch to June 2007. Resupinate basidiomata with Tulas-nellales characteristics like ceraceous to subgelatinousappearance, violaceous gray or lightly pink were collectedon decayed wood or fallen branches close to epiphyticorchids. The fresh basidiomata, in total 100 resupinatespecimens, were observed by light microscopy to selectTulasnella spp. based on the distinctive generic features.Eight basidiomata (FT8–110; FT-86; DC5; DC10; DC24;DC34; DC42; DC54) were morphologically pre-selectedand studied in detail by light microscopy. Free-handsections were stained with 1% phloxine. Sections wereexamined at ×100 to ×1,000 magnification by use of LeitzSM-LUX or Zeiss Axioskop 2 microscopes. Morphologicalcharacteristics of hymenia, basidia, sterigmata and spores indifferent stages of development were studied. Size values inthe species descriptions are based on at least 25 measure-ments of each structure, 30 measurements were taken fromspores. Illustrations were done at ×1,000 magnificationusing a camera lucida. Vouchers were dried and stored inthe Herbarium of Universidad Técnica Particular de Loja(UTPL), Loja, Ecuador. Small pieces of basidiomata wereused to induce Tulasnella sporulation and further isolationon solid PDA and wood-agar medium. However, isolationof Tulasnella spp. failed.

DNA isolation, PCR, cloning and sequencing

DNAwas extracted from parts of fresh or dried basidiomatausing a Plant Mini Kit (Qiagen) without any pre-treatment.After first attempts to amplify the nrITS-5.8S region frombasidiomata using the tulasnelloid primer combination ITS1(5′-TCCGTAGGTGAACCTGCGG-3′; White et al. 1990)and ITS4Tul (5′-CCG CCAGATTCACACATTGA-3′;Taylor 1997) gave no PCR products, a nested PCR wascarried out using the universal primer combinations ITS1

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and TW14 (5 ′-GCTATCCTGAGGGAAACTTC-3 ′;Cullings 1994) for the first PCR, and the tulasnelloidprimer combination ITS1 and ITS4Tul for the second PCR.The product obtained in the first PCR was diluted 1:10, 1:100or 1:1,000. PCR conditions were as follows: initial denaturationat 98°C for 30 s, 30 cycles, each cycle consisting of one step ofdenaturation at 98°C for 10 s, annealing for both primercombinations at 60°C for 20 s, and extension at 72°C for 30 s.A final extension at 72°C for 10 min was performed to finishthe PCR. The PCR reaction volume was 20 μl withconcentrations of 0.2 pmol of each of the primers, 10% BovineSerum Albumin (BSA-SIGMA) with final concentration of0.8 μg μl−1 (Iotti and Zambonelli 2006) and 1U Finnzymes’Phusion™ High-Fidelity DNA Polymerase®. In every PCR, anegative control including PCR mix without DNA templateand a positive control including DNA of Tulasnella spp.obtained by Suárez et al. (2006) were included. The successof each PCR was tested in 0.7% agarose stained in a solutionof ethidium bromide 0.5 mg ml−1. Products obtained from thesecond PCR were cloned using Zero Blunt® TOPO® PCRCloning Kit (Invitrogen). The colonies grew on solidmedium LB AGAR, MILLER (Difco™). In order to verifythe DNA insert, 11 growing colonies for each sample(numbered C01–C11) where selected by random anddirect PCR was carried out with M13F and M13R primersmodified according to Krüger et al. (2009). The PCRconditions were as follows: 34 cycles with initial denatur-ation at 95°C for 5 min, 35 cycles each with one step ofdenaturation at 94°C for 30 s, annealing 65°C for 30 s,extension at 72°C for 2 min and final extension at 72°Cfor 10 min. Success of PCR was tested as mentionedbefore. The colonies were purified following S.N.A.P.™

miniprep kit (Invitrogen) protocol. Sequencing was carriedout using primer M13F. The sequences obtained in thisstudy are available from GenBank under accessionnumbers GU732298–GU732345.

Phylogenetic analysis

The sequence chromatograms were verified using thesoftware Sequencher 4.6 (Gene Codes, Ann Arbor, MI,USA). The new sequences were added to a previousalignment of Suárez et al. (2006), together with publishedsequences of Tulasnella available in the NCBI nucleotidedatabase (GenBank; http://www.ncbi.nlm.nih.gov/).Sequences were aligned following the “multiple analysismethod” (Lee 2001). The nrITS-5.8S region was alignedusing the G-INS-i strategy as implemented in MAFFTv5.667 (Katoh et al. 2002) and POA (Lee et al. 2002)without further manual corrections. Due to the heterogene-ity of the Tulasnella sequences (Sharon et al. 2008), thesole 5.8S region was used to calculate a first phylogenetictree of a wider phylogenetic spectrum (Fig. 1). Based on

this result, we selected a subset of closely related sequencesof the entire nrITS-5.8S (567 bp) to obtain a reliablealignment, as was previously shown by Suárez et al. (2006)and Taylor and McCormick (2008).

The phylogenetic trees were calculated using threedifferent approaches. A neighbor-joining (NJ) analysiswas performed in PAUP* (Swofford 2002) using theBIONJ modification of the NJ algorithm (Gascuel 1997).Bayesian analysis based on Markov chain Monte Carlo(MCMC) was implemented in MrBayes version v3.1.2(Huelsenbeck and Ronquist 2001), including two runs eachinvolving four incrementally heated Markov chains over 4million generations and using random starting trees. Treeswere sampled every 100 generations resulting in a total of40,000 trees from which the last 30,000 were used tocompute a 50% majority rule consensus tree. Each datasetwas analyzed using the DNA substitution model GTR+I+G(see Swofford et al. 1996). Finally, a maximum likelihood(ML) calculation as implemented in RAxML version 7.0.4software (Stamatakis 2006) was used with 1,000 bootstrapreplicates (Felsenstein 1985) using the GTRMIX algorithmto perform a tree inference and search for a good topology.Support values from both bootstrapping runs were mappedon the globally best tree using the -f a option of RAxMLand -x 12345 as a random seed to invoke the novel rapidbootstrapping algorithm.

Intra- and intergenomic variation

The proportional differences of the complete nrITS-5.8Sregion (567 bp) between the clonal sequences of theindividual basidiomata and between the specimens werecalculated.

Results

Phylogenetic analysis

In total, 88 sequences were obtained from the eightbasidiomata after cloning. The majority of sequences fromsamples FT8-110, FT-86, DC10 had poor quality and wereexcluded from the analysis. A total of 48 sequences wereincluded in the analysis, 10 of samples DC5, DC24 andDC42 and 9 of samples DC34 and DC54, respectively.Clones obtained from specimens DC5, DC24 and DC54contained only one intragenomic polymorphism (IGP)corresponding to 0.2% proportional differences. The se-quence DC34 C10 exhibited a single nucleotide insertion.Only sequence DC42 C02 was quite different containing 29IGPs (including several indels) resulting in a 5.1%proportional difference when compared to all DC42 clones.All detected IGPs were unique, with variation not displayed

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DC24 C01 GU732325DC24 C02 GU732326DC5 C02 GU732316DC24 C07 GU732331DC24 C10 GU732334DC5 C08 GU732322DC42 C09 GU732313DC54 C06 GU732339DC42 C05 GU732309DC34 C07 GU732304DC42 C01 GU732306DC24 C04 GU732328DC34 C02 GU732299DC42 C03 GU732307DC42 C06 GU732310DC5 C03 GU732317DC5 C09 GU732323DC24 C05 GU732329DC54 C07 GU732340DC5 C01 GU732315DC24 C03 GU732327DC24 C06 GU732330DC34 C08 GU732305DC34 C01 GU732298DC54 C09 GU732342DC54 C08 GU732341DC5 C07 GU732321DC54 C02 GU732336 DC5 C06 GU732320DC34 C05 GU732302DC54 C03 GU732337DC42 C10 GU732314DC54 C01 GU732335DC34 C10 GU732344DC5 C04 GU732318DC34 C03 GU732300DC24 C08 GU732332DC42 C07 GU732311DC54 C04 GU732338DC24 C09 GU732333DC42 C08 GU732312DC34 C06 GU732303DC42 C02 GU732345DC42 C04 GU732308DC34 C04 GU732301DC5 C10 GU732324DC5 C05 GU732319DC54 C10 GU732343Epulorhiza sp from Arundina graminifolia Ag3b AJ313436from Arundina graminifolia AJ313435Epulorhiza sp from Diuris corymbosa Kings Park Dm01 EF160068Tulasnella calospora CBS 326 47 AY373298from Epipactis gigantea 6009 AY634123Tulasnella deliquescens DAOM 47 8 AY373291Tulasnella calospora Chunxin 25 CG15X3 EF393631Tulasnella calospora MAFF P305805 DQ388045Tulasnella calospora CBS 573 83 EU218888 2From Anacamptis laxiflora AL1 2 FJ809762Tulasnella calospora H1401 12 FJ594924Tulasnella calospora HUI0101 8 FJ613262Tulasnella calospora MAFF P305802 DQ388042from Stelis superbiens C3 2MN DQ178105Epulorhiza sp from Cymbidium fabery CH02X2 1 EF393625Tulasnella calospora MAFF P305804 DQ388044Tulasnella calospora C0103 43 FJ613226Tulasnella calospora F0501 1 FJ613256Tulasnella calospora H0402 42 FJ613196Tulasnella calospora H0502 5 FJ613197Tulasnella calospora H1401 21 FJ594928Tulasnella calospora HUI0101 12 FJ613266Tulasnella calospora MAFF P305803 DQ388043Tulasnella calospora H0302 19 FJ613176Tulasnella calospora H0302 8 FJ613169Tulasnella calospora H0601 2 FJ613198Tulasnella calospora H0701 3 FJ613199Tulasnella calospora Shen CF07 EF393621Tulasnella bifrons BPI 724849 AY373290Tulasnella calospora H0201 50 FJ613150from Goodyera pubescens AY373270from Goodyera pubescens 245 AY373282from Goodyera pubescens AY373274from Goodyera pubescens AY373264from Goodyera pubescens 179 AY373275from Spathaglotis plicata AJ313453from Pleurothallis lilijae C2 1MN DQ178098from Stelis hallii 1 18 DQ178104from Stelis concinna 7 13 2 DQ178108from Stelis concinna 9 6 DQ178084from Stelis concinna 9 7 DQ178092from Stelis superbiens C3 4MN DQ178101from Stelis concinna 9 2 DQ178111from Stelis superbiens C3 5 2 culture DQ178036from Stelis hallii 1 17 DQ178103from Pleurothallis lilijae C2 1 3 DQ178100from Stelis concinna 13 4 DQ178110from Stelis concinna 7 13 2 DQ178109from Spathaglotis plicata AJ313452

Tulasnella calospora CZ446 FJ755217Epulorhiza sp from Oncidium varimyce x Oncidium nona Onv4 3 AJ313449

83/-/-

63/73/63

-/-/74

aFig. 1 Phylogenetic placementof clonal sequences from fivespecimens of Tulasnella sp.obtained directly from the basi-diomata DC5, DC24, DC34,DC42, and DC54 (in bold)illustrated in Fig. 3 and all theavailable sequences of Tulas-nella spp. downloaded fromGenBank. C01 to C10 corre-spond to clone numbers. Treeinferred from 5.8S region usinga maximum likelihood analysisimplemented in RAxML withGTRMIX algorithm. Numberson branches designate ML andBayesian MCMC estimates ofposterior probabilities andBIONJ values (only values ex-ceeding 50% are shown). Thetree was rooted with Multicla-vula corynoides U66440. Thesequences from this study fallinto a clade with consistentalignment and support by –/51/96%, respectively, the cladeincludes several stets of orchidmycobionts from the same studysite (Suárez et al. 2006) andseveral sets of the long-spored T.calospora. The globose andellipsoid-spored T. albida, T.violea, T. pruinosa, T. eichleri-ana and T. asymmetrica clusterin separate clades. T. irregularisCBS 574.83 EU218889 (isolatefrom Dendrobium dicuphu JHWarcup, 0632, 1980) corre-sponds to the holotype strain(Taylor and McCormick 2008).Tulasnella asymmetrica MAFFP305806 DQ388046 (isolatefrom Thelymitra luteocilium JHWarcup, 085, 1967) and T.asymmetrica MAFF P305808DQ388047 (isolate from Thely-mitra luteocilium JH Warcup,0302, 1967) and T. asymmetricaAFTOL ID 1678 DQ520101strain MAFF305807 (isolatefrom blue orchid JH Warcup,0277, 1967), (Warcup andTalbot 1967, Warcup 1973) areT. pinicola fide Roberts 1999

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in other sequences. Only one clone per basidioma displayedthe respective variation (DC5 C10, DC24 C03, DC34 C10,DC42 C02 and DC54 C08). Proportional differencebetween the five basidiomata were 0,4%, but 5.3% in caseDC42 was included due to the sequence DC42 C02.

Phylogenetic calculations based only on the 5.8S regionshowed all clones from basidiomata from this study

clustering in one poorly supported clade –/–/74% (ML,Bayesian MCMC and BIONJ, respectively) in the phylo-genetic trees based on MAFFT and –/–/76% (ML, BayesianMCMC and BIONJ, respectively) in the phylogenetic treesbased on POA alignment. Only the phylogenetic treeresulting from MAFFT alignment is presented (Fig. 1a, b).Based on the 5.8S tree, a subset supported with –/51/96%

Epulorhiza anaticula UAMH 5428 EU218891Epulorhiza sp from Disa bracteata Kings Park D34 EF176481Epulorhiza sp from Disa bracteata Kings Park D35 EF176482

Epulorhiza sp from Pyrorchis nigricans Kings Park P35 EF176472from Stelis superbiens C3 MN4 DQ178115Tulasnella danica AY373297

Epulorhiza sp from Arachnis maggie oei Am8 AJ313442Epulorhiza sp from Arachnis maggie oei Am9 AJ313444Epulorhiza sp Spathaglotis plicata Sp1a AJ313437

from Vanda miss “Joaquin” AJ313443Epulorhiza sp from Spathaglotis plicata S1 AJ313439Epulorhiza sp from Spathaglotis plicata B1 AJ313445Epulorhiza sp from Spathaglotis plicata Eb3c AJ313450from Serapias vomeracea SV5 5 FJ809764Tulasnella calospora MAFF P305801 DQ388041from Dendrobium crumenatun AJ313438

from Epipactis gigantea 4022 AY634124from Serapias vomeracea SV3 2 FJ809763from Liparis lilifolia 258 AY373289Uncultured Tulasnella OF1 10 FJ809771from Orchis purpurea OP4 6 FJ809770from Stelis hallii 1 1 DQ178113from Stelis hallii 1 6 DQ178114from Cryptothallus mirabilis 3028 6 AY192476from Cryptothallus mirabilis AY192483from Pinus muricata AY192461from Cryptothallus mirabilis AY192482

Tulasnella irregularis CBS 574 83 EU218889from Cypripedium californicum P543 DQ925493from Cypripedium californicum P566 DQ925496

from Tipularia discolor 184 AY373306from Tipularia discolor 218 AY373312from Tipularia discolor 254 AY373316

from Platanthera chlorantha AY634131from Tipularia discolor AY373310from Tipularia discolor 170 AY373302from Tipularia discolor 175 AY373304from Pleurothallis lilijae C2MN5 DQ178070

from Tipularia discolor 252 AY373315from Pleurothallis lilijae C2MN2 DQ178081from Pleurothallis lilijae C2 21 DQ178079Tulasnella albida KC 110 AY373294Tulasnella violea AFTOL 621 DQ457643Tulasnella violea DAOM 22200 AY373303Tulasnella pruinosa AFTOL 610 DQ457642Tulasnella pruinosa DAOM 17641 AY373295from Stelis concinna 7 18 4 DQ178082from Stelis superbiens C3 5MN5 DQ178074from Stelis superbiens C3 5 4 DQ178078from Stelis concinna 7 21 1 DQ178075from Stelis superbiens C3 5 3 DQ178083from Stelis hallii 1 19 DQ178073from Stelis hallii 1 21 DQ178072from Stelis hallii 1 11 culture DQ178066

Tulasnella asymmetrica AFTOL ID 1678 DQ520101Tulasnella asymmetrica MAFF P305806 DQ388046

Tulasnella asymmetrica MAFF P305808 DQ388047Tulasnella violea AFTOL 1879 DQ520097

from Pleurothallis lilijae C2 MN1 DQ178116from Stelis superbiens C3MN3 DQ178117

from Stelis concinna 9 3 culture DQ178029from Stelis hallii 1 4 DQ178118Tulasnella eichleriana KC 852 AY373292Tulasnella violea KC 851 AY373293Tulasnella tomaculum KC 429 AY373296

from Cypripedium californicum P246 DQ925501from Cypripedium fasciculatum P383 DQ925500Epulorhiza sp from Spathaglotis plicata H21 AJ313459from Cypripedium guttatum 005 1 A4 EF433955from Cypripedium guttatum 005 4 D4 EF433954Epulorhiza sp UAMH 5430 EU218890from Ophrys sphegodes OpS03P AJ549122from Orthilia secunda Bas1 AM181390from Pyrola rotundifolia KR41 EU668955

from Cypripedium reginae 868 DQ925591from Cypripedium reginae 874 DQ925592from Cypripedium calceolus 438 DQ925558from Cypripedium calceolus 449 DQ925570Multiclavula corynoides U66440

0.005 substitutions/site

91/96/91

65/63/62

94/99/74

87/100/75

76/-/63

93/97/99

72/100/52

98/100/10098/-/94

99/63/99100/100/71

100/69/97

85/88/55

74/97/53

71/82/-

73/95/-

-/99/100

63/75/56

63/97/-

60/95/75

98/100/100

-/51/96

88/98/98b

Fig. 1 (continued)

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(ML, Bayesian MCMC and BIONJ, respectively, Fig. 1a, b)was selected for phylogenic calculations including thewhole ITS1-5.8S-ITS2 region. Phylogenetic calculations

of the nrITS-5.8S region showed slight variation intopology results when three different methods (ML,Bayesian MCMC, and BIONJ) where applied. The clade

DC34 C01 GU732298DC5 C03 GU732317DC34 C02 GU732299DC5 C05 GU732319DC24 C08 GU732332DC54 C01 GU732335DC34 C06 GU732303DC42 C07 GU732311DC24 C02 GU732326DC24 C05 GU732329DC24 C09 GU732333DC5 C06 GU732320DC54 C03 GU732337DC42 C01 GU732306DC42 C09 GU732313DC24 C07 GU732331DC54 C02 GU732336DC54 C10 GU732343DC42 C10 GU732314DC5 C09 GU732323DC24 C06 GU732330DC24 C01 GU732325DC42 C06 GU732310DC54 C04 GU732338DC54 C07 GU732340DC5 C01 GU732315DC42 C04 GU732308DC34 C08 GU732305DC34 C10 GU732344DC42 C03 GU732307DC42 C05 GU732309DC54 C09 GU732342DC5 C04 GU732318DC5 C08 GU732322DC24 C10 GU732334DC5 C07 GU732321DC54 C08 GU732341

DC24 C04 GU732328DC54 C06 GU732339DC34 C03 GU732300DC34 C07 GU732304DC42 C08 GU732312DC34 C04 GU732301DC34 C05 GU732302DC5 C02 GU732316DC5 C10 GU732324DC24 C03 GU732327

DC42 C02 GU732345from Stelis hallii 1 6 DQ178114

from Stelis hallii 1 1 DQ178113from Stelis concinna 9 2 DQ178111

from Stelis concinna 13 4 DQ178110from Stelis concinna 7 13 2 DQ178109

from Stelis concinna 7 13 2 DQ178108from Pleurothallis lilijae C2 1MN DQ178098from Pleurothallis lilijae C2 1 3 DQ178100from Stelis concinna 9 6 DQ178084from Stelis concinna 9 7 DQ178092from Stelis superbiens C3 4MN DQ178101

from Stelis superbiens C3 2MN DQ178105from Stelis hallii 1 17 DQ178103from Stelis hallii 1 18 DQ178104

Tulasnella calospora MAFF P305803 DQ388043Tulasnella calospora Shen CF07 EF393621

Tulasnella calospora MAFF P305804 DQ388044from Goodyera pubescens AY373264

from Goodyera pubescens AY373274from Goodyera pubescens AY373270

Tulasnella bifrons BPI 724849 AY373290from Stelis superbiens C3 MN4 DQ178115

Tulasnella danica AY373297Epulorhiza sp from Spathaglotis plicata Sp1a AJ313437

from Spathaglotis plicata AJ313452from Spathaglotis plicata AJ313453

Epulorhiza sp from Spathaglotis plicata S1 AJ313439from Epipactis gigantea 4022 AY634124

from Dendrobium crumenatun AJ313438from Vanda miss “Joaquin” AJ313443

Tulasnella calospora CBS 326 47 AY373298Tulasnella deliquescens DAOM 47 8 AY373291

Tulasnella calospora MAFF P305801 DQ388041from Arundina graminifolia AJ313435

Epulorhiza sp from Spathaglotis plicata Eb3c AJ313450Tulasnella calospora MAFF P305805 DQ388045

Tulasnella calospora MAFF P305802 DQ388042Tulasnella calospora CBS 573.83 EU218888.2

0.005 substitutions/site

-/-/91

81/-/64

97/100/70100/100/100

97/-/-

-/100/5485/92/94

75/98/-

67/100/56

95/-/5250/81/7293/100/-

92/100/100

62/93/72

85/99/8565/65/-

100/100/100

100/100/97

100/100/98

98/100/99

99/100/100

92/100/-

-/87/62

-/65/-

-/59/-

Fig. 2 Phylogenetic placementof clonal sequences from fivespecimens of Tulasnella sp.obtained directly from the basi-diomata DC5, DC24, DC34,DC42, and DC54. C01 to C10correspond to clone numbers.Tree inferred from ITS1-5.8S-ITS2 region using a maximumlikelihood analysis implementedin RAxML with the GTRMIXalgorithm. Numbers on branchesdesignate ML and BayesianMCMC estimates of posteriorprobabilities and BIONJ values(only values exceeding 50% areshown). The tree was midpointrooted. The clonal sequencesfrom this study are cited in bold.Sequences from mycobionts ofthe epiphytic orchids Stelis hal-lii, Stelis superbiens, Stelis con-cinna and Pleurothallis lilijaesampled in the same study site(Suárez et al. 2006) appear asthe most closely related but notidentical with the basidiomatasequences. The tree shows twoclades with Tulasnella calo-spora sequences of isolates fromAustralian orchids. The cladeincludes T. calospora MAFFP305802 DQ388042, (isolatefrom Diuris sp., JH Warcup,0388, 1967), T. calosporaMAFF P305805 DQ388045(isolate, JH Warcup, 0689), T.calospora CBS 573 83EU218888 2 (isolate from Cal-adenia reticulata JH Warcup,062, 1966) and T. calosporaDQ388041 (isolate from Acian-thus exsertus Warcup, O7, 1966,Warcup and Talbot 1967,Warcup 1973), the latter two areconsidered T. deliquescens byRoberts (1999)

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containing all clones from basidiomata from this study wassupported by 81/–/64% (ML, Bayesian MCMC and BIONJ,respectively) in the phylogenetic trees based on MAFFTalignment and 77/–/86% (ML, Bayesian MCMC andBIONJ, respectively) when based on POA alignment. Onlythe phylogenetic tree resulting from MAFFT alignment is

presented (Fig. 2). The clade containing all clones frombasidiomata also included sequences of orchid mycobiontsfrom pleurothalloid orchids of the same study site.Proportional differences between the sequences of Tulas-nella spp. from basidiomata and the sequences of the orchidmycorrhizal fungi from Stelis hallii DQ178114 and

Fig. 3 Illustrations of different specimens of Tulasnella sp. fromtropical mountain forest in the northern Andes of southern Ecuador. aDC5. b DC24. c DC34. d DC42. e DC54. Variability regarding

basidia, sterigmata and spores shape and size can be observed.Drawings without internal fluids like lipids. Bar 10 μm

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DQ178113 were 9.7–10.7%, respectively. Proportionaldifferences for a clade appearing as sister group toTulasnella sp. (–/100/54% support values for ML, BayesianMCMC and BIONJ, respectively) comprising S. hallii, S.superbiens, S. concinna and P. lilijae mycobionts were 5.2–6.3% (Fig. 2).

Morphology

Tulasnella sp

The following description is based on five specimens (DC5;DC24; DC34; DC42; DC54), illustrated in Fig. 3a–e.Measurements obtained from the mean value ±SD withextreme values in parenthesis of the five specimens areshown in Table 1. Three further specimens (FT8-110, FT-86,DC10) were not illustrated and not included in the measure-ments because no mature basidia and spores could be found.

Studied specimens showed waxy pruinose basidiomatafresh samples looking gray, violaceous gray or sometimesslightly pinkish, often inconspicuous to the unaided eye indry material. The hyphae are loosely connected, hyaline,without clamps, diameter of 2–3(–4) μm almost invariableamong specimens, branching “T” shaped, sometimes con-stricted at the base of the ramification, forming irregularand rarely compact layers (Fig. 3a–e). Cystidia absent.Basidia 7–10(–11)×5–7 μm somewhat variable in differentspecimens (Table 1), mostly clavate, sometimes subgloboseto oblong. Sterigmata 4 per basidium, in mature statepyriform, 6–9(–10)×4–6 μm. Spores hyaline, smooth,subcylindrical to weakly allantoid, (4–)5–7(–8)×(2–)2.5–4(–5) μm, the quotient of length to width 1.5–2 (–2.7).Basidiospores produce secondary spores by repetition.Differences in the size of spores and sterigmata are evidentin different specimens (Table 1).

Specimens examined

Ecuador, Zamora-Chinchipe Province, Reserva BiológicaSan Francisco: on decayed wood 25 Apr 2007 D. Cruz 5(UTPL); on fallen branch with orchids 21 May 2007 D.Cruz 24 (UTPL); on decayed wood 21 May 2007 D. Cruz34 (UTPL); on trunk with orchids 12 June 2007 D. Cruz 42

(UTPL); on fallen branch with orchids 19 June 2007 D.Cruz 54 (UTPL).

Discussion

Intra- and inter-specimen variability of the nrITS-5.8Sregion was rather low, 0.2% and 0.4% for all clones exceptfor DC42 C02 with 5.1%. We therefore argue that all ourspecimens belong to one genotype or species despite somemorphological variability of the basidiomata. In the case ofDC42 CO2, we suspect that divergence is due to thepresence of hyphae or spores of another Tulasnella spp.intermixed with this specimen. McCormick et al. (2006)suggested sequence differences around 2% in the nrITS-5.8S region to separate pure culture isolates of Tulasnellaand our results do not contradict this assumption. Relyingon nrITS-5.8S to delimitate species would thus besupported, but further examination of intra- and interspe-cific variability of this region in Tulasnella spp. isnecessary to corroborate reliability for delimitation ofspecies in this genus.

The different specimens of Tulasnella sp. from tropicalmountain forest in the northern Andes of southern Ecuadorare basically similar but slightly variable in morphologicalfeatures such as substrate hyphal network, size of basidia,sterigmata and spores (Table 1). Variability of spore size isknown for other species of Tulasnella, e.g., in Tulasnellapinicola and Tulasnella eichleriana, taxa that are consid-ered as species complexes by Roberts (1994b). Ourspecimens are morphologically slightly similar to Tulas-nella pruinosa Bourdot and Galzin (1928) (re-examinationRoberts 1994b) when using the key provided by Roberts(1999). However, T. pruinosa described by Roberts(1994b) show ellipsoid to oblong spores often taperedtowards the apiculus and are only occasionally ventrallydepressed while the spores in our material are subcylin-drical to weakly allantoid. Another difference in speci-mens studied by Roberts are globose to ellipsoidsterigmata becoming clavate, fusiform or mitriform, ca.4 μm across at the widest point, while in our specimensthe sterigmata are pyriform and 4–6 μm wide whenmature. T. pruinosa is considered an uncertain and ill-

Specimen Hyphae Basidia Sterigmata Spores Q of spores

DC5 3(–4) 8–9×6–7 8×5–6 6–7(–8)×3–4 1.5–2 (–2.7)

DC24 (2–)3 9–10×6–7 8–9(–10)×(4–)5 5–7(–8)×3–4(–5) (1.5–)1.6–2 (–2.3)

DC34 2–3(–4) 9–10(–11)×5–6 (6–)7–8×4(–5) 5–6(–7)×3 (–3.5) 1.6–2 (–2.3)

DC42 3 8–9(–10)×5–6 8–9(–10)×5 5–7 (–8)×3–4 1.6–2 (–2.3)

DC54 3 7–8×5–6 6–7×4 (4–)5–6×(2–)2.5–3 1.5–2 (–2.5)

Table 1 Tulasnella sp.Microscopic measurements(μm) of different cellular struc-tures of different specimens

Ranges of measurements and Q(quotient) of spores wereobtained from mean ± SD withextreme values in parentheses

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defined species by Roberts (1994b). Further comparativeanalyses of herbarium material of correctly named speciesare needed to determine whether our specimens are relatedto previously described species of Tulasnella.

Phylogenetic calculation of the 5.8S region showed allclones from basidiomata of this study clustering in one,although poorly supported, clade. The subset selected forphylogenic calculations comprising the whole ITS1-5.8S-ITS2 region included sequences of mycobionts frompleurothalloid orchids of the same study site, several T.calospora sequences, and further sequences of orchidmycobionts from other parts of the world but no sequencesof T. pruinosa (Fig. 2). T. pruinosa (DAOM 17641AY373295 and AFTOL 610 DQ457642) falls into anotherclade (63/75/56% ML, Bayesian MCMC and BIONJ,respectively, Fig. 1b) including T. violea, T. albida andother orchid mycobionts of pleurothalloid orchids from ourstudy site. The selected subset is consistent with ourprevious study (Suárez et al 2006: fig. 8) and similar toTaylor and McCormick (2008: S1), except for sequencesfrom S. hallii DQ178113 and DQ178114 included in Taylorand McCormick (2008) as subset S3. The clade containingT. pruinosa, T. albida and T. violea in our phylogeneticcalculation of the 5.8S region is also consistent with ourprevious studies (Suárez et al. 2006: fig. 9) and with Taylorand McCormick (2008: subset S2). While the spore shapeof our specimens is subcylindrical to weakly allantoidaccording to the terminology of Roberts (1994a, b), T.bifrons has suballantoid spores, T. calospora and T.deliquescens have long spores (Roberts 1994a), whileT. pruinosa, T. violea, and T. albida have globose andellipsoid spores (Roberts 1994b). The shape of basidio-spores apparently is indicative of systematic relationships inthe genus Tulasnella. Therefore, we suggest that ourspecimens from the Andean tropical forest might belongto a so far undescribed Tulasnella species. Our phyloge-netic calculations of the nrITS-5.8S region showed allclones from basidiomata clustering together with sequencesof orchid mycobionts from pleurothalloid orchids of thesame study site indicating a mycorrhizal potential of thisfungus.

Direct PCR amplification from basidiomata as describedhere appears as a suitable approach to solve the manifoldtaxonomic problems in Tulasnella. Without doubt, speciesdefinition in this genus needs more information derivedfrom molecular data in addition to morphological character-istics. As suggested by DeSalle et al. (2005) and exempli-fied by Simon and Weiß (2008), distance values andmolecular characteristics should be included for improvedcircumscriptions of Tulasnella spp. We agree with Nilssonet al. (2008) that more conspecific sequences are neededfrom distinct localities to encompass the genetic variabilityof this and other Tulasnella species.

Acknowledgement The research was funded by the DeutscheForschungsgemeinschaft (DFG RU 816). We thank Markus Gökerfor valuable advice on molecular phylogeny and Nature and CultureInternational (NCI) for providing research facilities at RBSF.

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