mycological research 110 (2006) 1257–1270



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Diverse tulasnelloid fungi form mycorrhizas                                                                               ...
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Diverse tulasnelloid fungi form mycorrhizas                                                                            126...
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Diverse tulasnelloid fungi form mycorrhizas                                                                               ...
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Fig 8 – Phylogenetic placement of Tulasnella sequences, clades A-C, from Stelis hallii, Stelis superbiens, Stelis concinna...
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Diverse tulasnelloid fungi form mycorrhizas                                                                               ...
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Diverse tulasnelloid fungi form mycorrhizas                                                                               ...
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Diverse Tulasnelloid Fungi Form Mycorrhizas With Epiphytic Orchids In An Andean Cloud Forest

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Diverse Tulasnelloid Fungi Form Mycorrhizas With Epiphytic Orchids In An Andean Cloud Forest

  1. 1. mycological research 110 (2006) 1257–1270 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest ´ Juan Pablo SUAREZa,b,*, Michael WEIßb, Andrea ABELEb, Sigisfredo GARNICAb, Franz OBERWINKLERb, Ingrid KOTTKEb a ´cnica Particular de Loja, San Cayetano Alto s/n C.P. 11 01 608, Loja, Ecuador Centro de Biologıa Celular y Molecular, Universidad Te ´ b Eberhard-Karls-Universita Tubingen, Botanisches Institut, Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, ¨t ¨ D-72076 Tubingen, Germany ¨ article info abstract Article history: The mycorrhizal state of epiphytic orchids has been controversially discussed, and the Received 3 May 2006 state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high Received in revised form number of species on stems of trees in the Andean cloud forest, were unknown. Root sam- 7 August 2006 ples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Accepted 12 August 2006 Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ul- Published online 31 October 2006 trastructural evidence of symbiotic interaction was combined with molecular sequencing Corresponding Editor: of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analy- John W. G. Cairney ses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates Keywords: were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from cod- Heterobasidiomycetes ing regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of Molecular phylogeny the internal transcribed spacers, obtained directly from the roots and from the fungal iso- Pleurothallidinae lates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were Southern Ecuador restricted to two Tulasnella sequence types while the other orchids were associated with up Tropical mountain rain forest to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct Ultrastructure from known sequences of mycobionts of terrestrial orchids. The results indicate that tulas- nelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Williamson 1972), but a high infection rate was reported from canopy-dwelling orchid species in Florida (Benzing Although most land plants are associated with symbiotic 1982). Different degrees of infection including non-infected fungi forming mycorrhizas or mycorrhiza-like associations, roots were observed in epiphytic orchids in Ecuador (Ber- many epiphytes live without such associations, e.g. mosses, mudes & Benzing 1989). Goh et al. (1992) found high fungal col- many liverworts, bromeliads, and ferns (Kottke 2002). Find- onization in the epiphytic orchid Dendrobium crumenatum from ings on the mycorrhizal state of epiphytic orchids were con- a natural stand in Singapore, but only low or no mycorrhiza- troversial. Only sporadic fungal colonization was found tion in orchids from nurseries. Rivas et al. (1998) and Pereira in a number of epiphytic Malaysian orchids (Hadley & et al. (2005) reported intense colonization of epiphytic orchids * Corresponding author. E-mail address: jpsuarez@utpl.edu.ec 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.08.004
  2. 2. 1258 ´ J. P. Suarez et al. in Costa Rica and Brazil, respectively. All investigators stated minimized. The genera Stelis and Pleurothallis belong to that fungal colonization was restricted to roots attaching to subtribe Pleurothallidinae, the largest subtribe in the tribe the substrate; aerial roots were not colonized. Epidendreae of Orchidaceae (Luer 1986a,b), which is widely dis- Identification of root-associated fungi was mostly achieved tributed in tropical America. These two genera include 485 by isolation on sterile media (Rasmussen 2002). However, the Pleurothallis and 465 Stelis species reported until now for Ecua- distinction between endophytic fungi inhabiting only the ve- dor (L. Endara, pers. comm.). Many of these epiphytes are en- lamen or the root surface and reliably mycorrhiza-forming demic species of Ecuadorian tropical forests. Only a few of fungi colonizing the cortical tissue was mostly unclear (Cur- them are in culture so far. The rapid loss of habitats requires rah et al. 1997; Pridgeon 1987). Xylaria (Ascomycota) was fre- an understanding of the symbiotic relationships in order quently isolated (Bayman et al. 1997, Tremblay et al. 1998), to support conservation efforts for these orchids. According but was never proven experimentally or demonstrated by ul- to Hamilton et al. (1995) approximately 90 % of the Northern trastructure to form mycorrhizas with orchids. Fungal isola- Andean forests have been already destroyed. Consequently, tion from pelotons as a more selective approach has been the orchids and their fungi might be lost in the near future successfully attempted in terrestrial orchids (Warcup & Talbot if not taken into culture. As the mycorrhizal state and myco- 1967, 1971, 1980, Bougoure et al. 2005). In cases where sexual bionts of the epiphytic Pleurothallidinae were unknown, no stages could be achieved, the isolated fungi were determined advice could be given to laboratories interested in orchid cul- as basidiomycetes belonging to the Sebacinales, Tulasnellales turing or to local forest management aiming to rehabilitate or Ceratobasidiales (Warcup 1981; Warcup & Talbot 1967, the tropical mountain forest with its epiphytic orchid diversity 1971, 1980). Tulasnella anamorphs (Epulorhiza) were isolated, (see: http://www.bergregenwald.de of which this work is a e.g. from epiphytic Epidendrum conopseum in Florida (Zettler part). We therefore started with light- and transmission elec- et al. 1998), epiphytic Epidendrum rigidum, Polystachia concreta tron microscopic investigation of the selected orchid species, (Pereira et al. 2003), and terrestrial Oeceoclades maculata from and continued with DNA isolation and sequencing of the Brazil (Pereira et al. 2005). DNA sequencing supported the most frequently observed fungal group, the Tulasnellales. In presence of Tulasnella, Sebacinales and Ceratobasidium in parallel, isolation of mycelia was carried out, yielding several Cypripedium spp. from the temperate Northern Hemisphere Tulasnella isolates. We were especially interested to see (Shefferson et al. 2005). Molecular tools were also used to iden- whether the Tulasnellales present as mycobionts of epiphytic tify fungal isolates obtained from pelotons (Bougoure et al. orchids in the tropical mountain rain forest were distinct 2005) or by direct DNA isolation from pelotons (Kristiansen from those described for other habitats of the Northern Hemi- et al. 2001). A taxon distantly related to Laccaria, an ectomycor- sphere and Australia. This knowledge would help to decide if rhiza-forming fungus, was found in Dactylorhiza majalis local or ubiquitous fungal isolates were appropriate for culti- (Kristiansen et al. 2001) in addition to Tulasnella. Ectomycor- vation of the local orchids, and would support evaluation of rhiza-forming mycobionts were also proven for non- loss of local fungi for rehabilitation of orchids in the tropical photosynthetic orchids by DNA isolations and sequencing mountain forest area. directly from mycorrhizas (Taylor & Bruns 1997, 1999; Taylor et al. 2003, Bidartondo et al. 2004, Selosse et al. 2004, Julou et al. 2005), thus widening the previous knowledge on orchid Materials and methods mycobionts. Selosse et al. (2004) confirmed their molecular finding of Tu- Study site ber spp. (Ascomycota) as orchid mycobionts by ultrastructural demonstration of ascomycetous hyphae in the cortical cells of The study site is located on the eastern slope of the Cordillera El the orchid roots. Ascomycetes can be discerned from basidio- Consuelo in the northern Andes of southern Ecuador. The area mycetes by ultrastructure of the cell wall and the septal pore, ´ of about 1000 ha belongs to the Reserva Biologica San Francisco and different groups of basidiomycetes can be distinguished and borders the Podocarpus National Park in the north, half by the parenthesomes covering the dolipores (Wells & Ban- way between Loja and Zamora, Zamora-Chinchipe Province doni 2001); tulasnelloid fungi display characteristic slime bod- ´ ´ (3 58 S, 79 04 W). The tropical mountain rainforest covers the ies in the cell walls (Bauer 2004). In spite of these diagnostic steep slopes between 1850 and 2700 m a.s.l. Characteristic possibilities transmission electron microscopy has rarely and most frequent trees are Melastomataceae, Rubiaceae, Laura- been used in orchid studies addressing fungal identity. How- ceae and Euphorbiaceae reaching a height of 25 m. Crown density ever, the previous work is encouraging (Currah Sherburne as measured by a spherical densitometer is 94 % on average, 1992; Andersen 1996) and minimizes errors resulting from only 7.5 % were open canopy (Homeier 2004). contamination during isolation of fungi or DNA directly from The richness and abundance of epiphytes is due to the mycorrhiza samples. In our study of the orchid mycobionts semi- to sub-humid climate with rainfall during ten months of four epiphytic, pleurothallid orchid species in the Andean and even more frequent fog combined with moderate temper- cloud forest of south Ecuador, we therefore combined ultra- atures (Richter 2003). Mean annual precipitation at 1950 m structural studies with DNA sequencing and isolation. a.s.l. is 2200 mm, annual mean temperature is 15,5 C (14,4- Stelis concinna, S. hallii, S. superbiens, and Pleurothallis lilijae 17,5 C). Precipitation increases with higher elevation and Foldats were selected because of the abundance and frequent reaches 4000 mm at 2600 m asl. Air humidity in two months flowering of these small orchids in the tropical mountain is 96 % on average and does not fall below 70 % during the rainforest of the study area. Thus severe violations of the drier season (Noske 2004). The high air humidity is especially ¨ orchid populations in this highly endangered forest could be important for stem epiphytes.
  3. 3. Diverse tulasnelloid fungi form mycorrhizas 1259 Sampling Fungal isolation Sampling was carried out at small paths at an altitudinal gra- Isolation of fungi was initiated the day of sampling. Colonized dient between 1850 and 2100 m a.s.l. Stelis hallii was found in root pieces were surface-sterilized. Roots were rinsed in the forest covering the steep slopes between 1800 and distilled water with some drops of liquid soap, immersed in 1900 m a.s.l., while S. superbiens and Pleurothallis lilijae were ethanol (70 %) for 30 s, immersed in Ajax chloro 20 % (house- collected in the forest covering the mountain ridge between hold bleach, sodium hypochlorite 5.25 %) for 10 min and 1900 and 2100 m a.s.l. Stelis concinna was restricted to the up- finally rinsed in sterile distilled water. The velamen was per part of the mountain ridge where the forest was less then removed using a stereo microscope, a thin blade and for- dense, with only 92 % crown density, and exposition to fre- ceps. Five square sections of 1-3 mm thickness were cut by quent and heavy winds. hand from the middle part of the root and transferred to Roots were collected continuously during three years a plate with MYP media (malt extract 7 g, peptone 1 g, and from 2003 until 2005 from a total of 77 flowering individuals, agar agar 15 g lÀ1) or MMNC media (modified Melin-Norkrans; 22 of S. hallii, 17 of S. superbiens, 25 of S. concinna, and 13 of Kottke et al. 1987; NaCl 0,025 g, KH2PO4 0,5 g, (NH4)2HPO4 0,25 g, Pleurothallis lilijae. All selected plants were epiphytes on CaCl2 0,05 g, MgSO4 Â 7H2O 0,15 g, FeCl3 (1 %) 1 ml, thiamin trunks or branches of standing trees at 50 cm to 200 cm 1 ml, malt extract 5 g, glucose 10 g, caseinhydrolysate 1 g, above the forest floor. Distances between trees with flower- agar 20 g, riboflavin 1 ml of 0.01 % solution, trace elements ing orchids varied between 50 cm and several metres (up to ´ 10 ml according to Fortin and Piche 1979). No antibiotics 20 m). Identification of trees was not taken into consider- were added. ation. Roots of one flowering individual orchid per tree stem were collected. One to four roots per plant individual were packed in aluminum foil to prevent desiccation DNA extraction, PCR and sequencing and transported to the laboratory the same day. As pre- investigation had shown that mycorrhizal fungi colonized Portions of 1-2 cm length of well colonized roots of which the only roots in contact with the stems, best when also covered velamen was removed were collected in cups the same day or by mosses or a minute humus layer, later on only such roots dried and kept on silica gel for later DNA isolations. DNA was were selected. Root samples were processed the day of extracted from the fresh or dried mycorrhizal tissue and from collection as pre-investigation had revealed a fast loss of fungal mycelium of our own isolates using a Plant Mini Kit vitality in the symbiotic fungi. Vouchers of the orchid (Qiagen, Hilden, Germany). A first attempt to PCR amplify ge- specimens were deposited in the Herbarium of UTPL, Loja, nomic DNA was carried out from mycorrhizal tissue using Ecuador, including flowers fixed in ethanol. Vouchers of universal fungal primer combinations ITS1F/ITS4, ITS1F/NL4, the mycorrhizas were embedded in resin and deposited in NLMW1/LR5, NLMW1/TW14 and ITS1F/TW14 (details con- the Herbarium of Tubingen University (TUB). ¨ cerning the primers used are given in the Electronic Appendix A). Several PCR products were obtained and sequenced. DNA isolated from fungal cultures was amplified using the primer Light and transmission electron microscopy combination ITS1F/NL4 or ITS1/NL4. Nested PCR was con- ducted to specifically amplify DNA from tulasnelloid fungi, Light microscopy was used to select material with fungal coils. as the ultrastructural analysis had revealed these fungi fre- Transversal sections were cut from the middle part of each quently in the cortical tissue of all the orchid species under in- root sample by hand using a razor blade. Sections were vestigation. The first amplification was carried out with the stained by Methyl blue 0.05 % solution (C. I. 42780, Merck) in primer combination ITS1F/TW14 or ITS1/TW14 and the sec- lactic acid for 10 min on microscopic slides. The samples ond, using template obtained in the first PCR in dilutions of were examined in fresh lactic acid at 100- to 1000-fold magni- 10À1, 10À2 and 10À3, with the primer combinations ITS1/ fication (Leitz SM-LUX or Zeiss Axioskop 2). ITS4-Tul for the internal transcribed spacers (ITS1, 5.8S nu- Root pieces of 1 cm length of all the samples displaying clear ribosomal gene and ITS2) and NLMW1/LR5, ITS4-TulR/ high frequency of vital looking hyphal coils, 56 in total and LR5 and 5.8S-Tul/NL4 for the 5’ part of the nuclear large sub- at least ten of each species, were fixed in 2.5 % glutaralde- unit ribosomal DNA (nucLSU). Primers ITS4-Tul and ITS4- hyde-formaldehyde in Sørensen buffer (Karnovsky 1965), TulR target a Tulasnella-specific sequence at the 3’ end of post-fixed in 1 % osmium tetroxide for 1 h, dehydrated in an ITS2. The Tulasnella-specific primer 5.8S-Tul (5’-TCATTCGAT acetone series and flat embedded in Spurr’s resin low viscos- GAAGACCGTTGC-3’) designed for this study targets a specific ity, longer pot-life formulation (Spurr 1969). Semithin sections sequence at the 5’ end of the 5.8S rDNA. were cut from the embedded samples, stained with 0.6 % neo- PCR conditions were as follows: initial denaturation at fuchsin crystal-violet, mounted in Entellan, and observed in 94 C for 3 min; 35 cycles, each cycle consisting of one step the light microscope. 20 samples with apparently vital of denaturation at 94 C for 30 s; annealing depending of the hyphae, originating from different plant individuals, were primer combinations for 45 s and extension at 72 C for selected for ultrathin cutting. Sections were mounted on For- 1 min; a final extension at 72 C for 7 min was performed to mvar-coated copper grids and stained with 1 % uranyl acetate finish the PCR. The PCR reaction volume was 50 ml, with con- (40 min) and lead citrate (12 min). Sections were examined us- centrations of 1.5 mM MgCl2, 200 mM of each dNTP (Life Tech- ing transmission electron microscopes Zeiss TEM 902 or Zeiss nologies, Eggenstein, Germany), 0.5 mM of each of the primers TEM109. (MWG-Biotech, Ebersberg, Germany), 1U Taq polymerase (Life
  4. 4. 1260 ´ J. P. Suarez et al. Technologies, Eggenstein, Germany), with an amplification MrModeltest, version 2.2 (Nylander 2004) involving four buffer (Life Technologies, Eggenstein, Germany). incrementally heated Markov chains over four million gener- In every PCR a control including PCR mix without DNA ations and using random starting trees. Trees were sampled template was included. Success of the PCR amplifications every 100 generations resulting in a total of 40000 trees from was tested in 0.7 % agarose, stained in a solution of ethidium which the last 24000 were used to compute a 50 % majority bromide 0.5 mg mlÀ1. PCR products were purified using the rule consensus tree. Each analysis was repeated to check QIAquick protocol (Qiagen). Cycle sequencing was conducted the reproducibility of the results (Huelsenbeck et al. 2002). using BigDye version 3.1 chemistry, and sequencing was An accumulation curve of clades vs number of collected indi- done on an ABI 3100 Genetic Analyzer (Applied Biosystems, viduals from the four orchid species was computed with Esti- Foster City, CA). Both strands of DNA were sequenced. mateS (Version 7.5, R. K. Colwell, unpubl.). Sequence editing was performed using Sequencher version We determined the proportional differences between se- 4.5 (Gene Codes, Ann Arbor, MI). The sequences obtained quences within each clade of the nucLSU D1/D2 in order to de- in this study are available from GenBank under accession fine sequence types. We compared the number of Tulasnella numbers DQ178029-DQ178118 (Table 1). sequence types within single and between different orchid We also included in this study sequence data from Tulas- species. The proportional differences between sequences nella reference strains kindly provided by the National Insti- were pooled into five tables (Electronic Appendix B). tute of Agrobiological Sciences (NIAS), Japan, which were previously isolated from Australian orchids and determined by J. H. Warcup (Warcup Talbot 1967, 1971). Results Phylogenetic analyses Microscopical and ultrastructural features of the mycorrhizas We used BLAST (Altschul et al. 1997) against the NCBI nucleo- Fungal pelotons were present in nearly all cross-sections of tide database (GenBank; http://www.ncbi.nlm.nih.gov/) to de- roots sampled directly from the tree bark. No fungal pelotons tect published sequences with a high similarity to the nucLSU were observed in aerial roots. This observation was confirmed sequences obtained from the Ecuadorian epiphytic orchids. by sampling roots of another 65 epiphytic Stelis and Pleurothal- For thorough phylogenetic analysis of the Tulasnella se- lis orchids, indicating that the roots became colonized only quences we analyzed nucLSU and ITS-5.8S alignments includ- where the fungi contacted the bark or the thin humus layer. ing the closest BLAST matches together with the sequences Pelotons were distributed throughout the cortex, with no dif- from the Warcup Tulasnella reference isolates (see above) ference between cortical layers. Vital, blue staining and col- and other sequences from Tulasnellaceae and related groups lapsed, slightly yellow coloured pelotons were visible in the retrieved from GenBank. same cells suggesting that cells became re-infected several Sequences were aligned using the G-INS-i or L-INS-i strat- times. According to the light microscopical observations, egy as implemented in MAFFT v5.667 (Katoh et al. 2005). Due many fungal pelotons were found collapsed after the plants to the heterogeneity of the Tulasnella sequences we had to ex- had been kept one night in the laboratory. Abundant hyphae clude considerable portions of the nucLSU sequences for phy- colonized the velamen. logenetic analysis. Even the 5.8S ribosomal region, considered TEM observations confirmed the known fungal-root inter- as universally conserved, exhibited a remarkable heterogene- action in orchid mycorrhizas. Hyphae of more or less equal di- ity as was already mentioned by Bidartondo et al. (2003). As ameter were surrounded by the plant plasma membrane, the expected, the ITS1 and ITS2 rDNA could not be aligned over plant vacuole forming small compartments or a network of the whole data set. Therefore, we used the 5.8S region to cal- small vacuoles (Fig 1). Degenerating hyphae were attached culate phylogenetic trees of a wider phylogenetic spectrum to collapsed pelotons (Fig 2). Alive hyphae contained abundant and produced several other phylogenetic analyses including glycogen granules (Figs 1, 3 and 5). The hyphae formed septa, subsets of related sequences, for which we used portions of clamps were not observed. (Fig 3). The septa showed dolipores the ITS1 and ITS2 regions in addition to the 5.8S sequences. with imperforate, dish-shaped parenthesomes with slightly The alignments used can be obtained from TreeBASE (http:// recurved margins (Fig 6). These tulasnelloid parenthesomes www.treebase.org/) under accession number S1629. were observed in all the 20 mycorrhizas analyzed by TEM. Oc- Neighbour-joining (NJ) and a Bayesian likelihood approach casionally, the hyphal walls were split into two layers and a fi- were used to estimate the phylogenetic relationships. The brillar or slimy mass appeared between the two layers (Figs 4 neighbour-joining analysis was performed in PAUP* (Swofford and 5, arrows). This phenomenon became very prominent in 2002) using the BIONJ modification of the NJ algorithm to ageing cultures and the slime was then strongly osmiophilic accomplish the observed high genetic variability in the se- (not shown). The combination of this type of parenthesomes quences used (Gascuel 1997). DNA substitution models and in- and the ‘‘slime bodies’’ in the cell walls was confirmed for dividual model parameters were estimated using the Akaike all the investigated mycorrhizas and in the Tulasnella isolates. information criterion (AIC) as implemented in Modeltest, ver- The recurved ends of the parenthesome were only detected by sion 3.7 (Posada Crandall 1998). For the Bayesian approach serial sectioning, since the appearance of the parenthesomes based on Markov chain Monte Carlo (MCMC) we used varied among the sections and may appear flattened or bowed MrBayes, version 3.0b4 (Huelsenbeck Ronquist 2001). Each in a steeper angle. In three samples we additionally found flat, dataset was analyzed using the DNA substitution models esti- imperforate parenthesomes, indicating sebacinoid fungi (Wil- mated using the Akaike information criterion (AIC) in liams Thiol 1989; not shown). In one sample a dome-shaped
  5. 5. Diverse tulasnelloid fungi form mycorrhizas 1261 Table 1 – List of sampled individuals from which tulasnelloid sequences were obtained. Letters and numbers behind the species names correspond to species, orchid individual, and root (superscript). Superscript b marks a second sequence obtained from the same root sample. Clades A-G correspond to the MCMC phylogenetic analysis. The two rDNA regions from the same root listed in each line originate from a single PCR amplicon Orchid species nucLSU nrDNA ITS-5.8S clade GenBank accession no. clade GenBank accession no. Pleurothallis lilijae C2.1.1 A DQ178035 A DQ178099 Pleurothallis lilijae C2.1.2 E DQ178067 Pleurothallis lilijae C2.1.3 A DQ178040 A DQ178100 Pleurothallis lilijae C2.15 E DQ178080 Pleurothallis lilijae C2.17 A DQ178102 Pleurothallis lilijae C2.21 E DQ178079 Pleurothallis lilijae C2.1MN A DQ178034 A DQ178098 Pleurothallis lilijae C2.5MN7 F DQ178047 F DQ178069 Pleurothallis lilijae C2.MN1 D DQ178063 D DQ178116 Pleurothallis lilijae C2.MN5 F DQ178049 F DQ178070 Pleurothallis lilijae C2MN2 E DQ178068 E DQ178081 Pleurothallis lilijae C2MN6 B DQ178045 Stelis concinna 7.6 A DQ178108 Stelis concinna 7.7 A DQ178106 Stelis concinna 7.8 A DQ178091 Stelis concinna 7.13.2 A DQ178109 Stelis concinna 7.13.3 A DQ178107 Stelis concinna 7.13.4 A DQ178110 Stelis concinna 7.14.2 A DQ178112 Stelis concinna 7.18.3 A DQ178043 A DQ178095 Stelis concinna 7.18.4 E DQ178082 Stelis concinna 7.19.1 A DQ178094 Stelis concinna 7.19.3 A DQ178032 A DQ178093 Stelis concinna 7.20.1 A DQ178030 A DQ178096 Stelis concinna 7.20.2 A DQ178042 A DQ178088 Stelis concinna 7.20.3 A DQ178033 A DQ178090 Stelis concinna 7.20.4 A DQ178041 A DQ178089 Stelis concinna 7.21.1 E DQ178075 Stelis concinna 7.21.2 E DQ178076 Stelis concinna 9.2 A DQ178111 Stelis concinna 9.3 culture G DQ178029 G DQ178029 Stelis concinna 9.6 A DQ178084 Stelis concinna 9.7 A DQ178092 Stelis concinna 9.8 A DQ178038 A DQ178097 Stelis concinna 9.9 A DQ178031 A DQ178086 Stelis hallii 1.1 B DQ178044 B DQ178113 Stelis hallii 1.2 E DQ178065 Stelis hallii 1.2b D DQ178051 Stelis hallii 1.4 G DQ178118 Stelis hallii 1.6 B DQ178114 Stelis hallii 1.7 D DQ178050 Stelis hallii 1.8 A DQ178085 Stelis hallii 1.11 culture E DQ178066 E DQ178066 Stelis hallii 1.15 D DQ178057 Stelis hallii 1.16 D DQ178055 Stelis hallii 1.17 A DQ178103 Stelis hallii 1.18 A DQ178037 A DQ178104 Stelis hallii 1.18b D DQ178053 Stelis hallii 1.19 D DQ178059 E DQ178073 Stelis hallii 1.19b E DQ178071 Stelis hallii 1.21 E DQ178072 Stelis hallii 1.21b E DQ178077 Stelis hallii 1.23 D DQ178060 Stelis superbiens C3.5.2 culture A DQ178036 A DQ178036 Stelis superbiens C3.5.3 E DQ178083 Stelis superbiens C3.5.4 E DQ178078 Stelis superbiens C3.9.2 A DQ178039 A DQ178087 Stelis superbiens C3 MN3 D DQ178058 D DQ178117 Stelis superbiens C3.MN4 C DQ178046 C DQ178115 (continued on next page)
  6. 6. 1262 ´ J. P. Suarez et al. Table 1 (continued) Orchid species nucLSU nrDNA ITS-5.8S clade GenBank accession no. clade GenBank accession no. Stelis superbiens C3.1MN D DQ178056 Stelis superbiens C3.2MN D DQ178052 A DQ178105 Stelis superbiens C3.3MN D DQ178054 Stelis superbiens C3.4MN D DQ178061 A DQ178101 Stelis superbiens C3.4MN4 D DQ178062 Stelis superbiens C3.5MN5 E DQ178064 E DQ178074 Stelis superbiens C3.5MN5b F DQ178048 parenthesome was found that displayed coarse perforations growth rates contrasting with the relative fast growth rate and might thus putatively be assigned to Ceratobasidium (Cur- observed in the fungi isolated. Ascomycetes closest to Crypto- rah Sherburne 1992; not shown). No simple-pored ascomy- sporiopsis were the most frequently isolated fungi. cetes were found in the cortical tissue of the 20 investigated samples, although they were present in the velamen (not Molecular identification and phylogenetic analysis shown). of mycorrhiza-associated fungi Fungal isolation and molecular identification of isolates The combinations of universal fungal primers yielded PCR products preliminarily identified by BLAST searches as closest Fungal growth was observed in only 44 plates out of 108 used to Cryptosporiopsis, Fusarium, Trichoderma (Ascomycota) and for fungal isolation, each one containing five root pieces. Four Bjerkandera, Antrodiella (Basidiomycota). Tulasnella sequences fungal cultures were obtained from a total of 13 plates of Stelis were infrequently obtained, only the primer combination hallii, 15 from 36 plates of Stelis superbiens, 22 from 55 plates of NLMW1/LR5 yielding few PCR products. Primer combinations Stelis concinna, and three from four plates of Pleurothallis lilijae including ITS1F and ITS4 failed to amplify Tulasnella DNA as mycorrhizas. A preliminary molecular identification of the was already reported by Bidartondo et al. (2003). Sequences fungal isolates was carried out by BLAST searches against of Sebacinales, basidiomycetes involved in a broad range of the GenBank nucleotide database retrieving the most similar mycorrhizal associations (Weiß et al. 2004), were also available sequences (data not shown). The isolated fungi detected, but at lower frequence than Tulasnellales sequences. were mainly ascomycetes closest to Xylaria, Hypoxylon and No cultures of Sebacinales were obtained from root samples Cryptosporiopsis and less often basidiomycetes closest to Bjer- (Kottke et al. 2007). kandera, Polyporus and Tulasnella. Three cultures were identi- The total number of investigated roots was 134, consider- fied as Tulasnella. These Tulasnella cultures exhibited slow ing that three or four roots were collected from each of the 77 orchid individuals. Tulasnelloid fungi were detected in 84 samples (63 %), including the PCR products obtained by the tulasnelloid specific primer combinations without successfull Fig 1 – Ultrastructure of the cortical tissue of Stelis concinna root displaying alive hyphae (h) of equal diameter in Fig 2 – Degenerating hyphae adjoining collapsed hyphae active host cell (c). (v) Small compartments of orchid cell (ch) in an active cortical cell of Stelis concinna root. vacuoles. Bar [ 1 mm. Bar [ 1 mm.
  7. 7. Diverse tulasnelloid fungi form mycorrhizas 1263 Fig 3 – Branched hypha displaying septa (arrow) without Fig 5 – Hypha in cortical root tissue of Stelis concinna clamp formation in root cortical tissue of Stelis concinna. displaying fibrillar slime between cell wall layers Bar [ 1 mm. (arrowhead) and a doliporus with imperforate, slightly dish-shaped parenthesomes (arrow). Bar [ 0.5 mm. sequencing. The nested PCR conducted in order to selectively amplify Tulasnella DNA using the primer combination ITS1/ 5.8S overview tree (Electronic Appendix C) in such a way TW14 in the first amplification and the primer combinations that we obtained best consistency with the rooted LSU tree ITS1/ITS4-Tul for the ITS-5.8S region and ITS4-TulR/LR5 or (Fig 7). Portions of ITS1 and ITS2 were added to the 5.8S align- 5.8S-Tul/NL4 for a part of the LSU region, respectively, in the ment where phylogenetic analysis was restricted to suitable second PCR yielded PCR products for the majority of samples. subsets of sequences detected in the 5.8S analysis, finally PCR success was higher with DNA extracted from fresh root resulting in an increase of phylogenetic resolution for these samples and lower with DNA from dried roots. subsets (Figs 8 and 9). The phylogenetic analyses of nucLSU and ITS-5.8S se- Our analysis of proportional differences between se- quences yielded consistent results. Seven clades, which in quences within each clade of the nucLSU D1/D2 yielded 13 the following we refer to as clades A to G, were retrieved Tulasnella sequence types. We treated sequences as belonging from the analyses of both ribosomal regions (Fig 7 and Elec- tronic Appendix C). BIONJ (trees not shown) and MCMC yielded similar groupings of Tulasnella clades. Only small var- iations were present in the clade support values. As men- tioned above, the 5.8S tree (Electronic Appendix C) was less resolved than the nucLSU tree. However, the unexpected het- erogeneity displayed by the 5.8S data set made it difficult to find a suitable outgroup sequence. Therefore, we rooted the Fig 6 – Close-up of a median section through the doliporus. The parenthesomes consist of two electron-dense Fig 4 – Square section of active hypha of Tulasnella, dis- membranes bordering an internal electron transparent playing mitochondria (m), glycogen rosettes, and fibrillar zone and show slightly recurved borders (arrows). slime between cell wall layers (arrowheads). Bar [ 1 mm. Bar [ 0,3 mm.
  8. 8. 1264 ´ J. P. Suarez et al. Fig 7 – Phylogenetic placement of Tulasnella sequences from Stelis hallii, Stelis superbiens, Stelis concinna and Pleurothallis lilijae inferred by MCMC analysis of nuclear rDNA coding for the 5’ terminal domain of the large ribosomal subunit (nucLSU). Numbers on branches designate neighbor-joining bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic distances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for tree construction. The tree was rooted with Multiclavula mucida AF287875.
  9. 9. Fig 8 – Phylogenetic placement of Tulasnella sequences, clades A-C, from Stelis hallii, Stelis superbiens, Stelis concinna and Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S rDNA. Numbers on branches designate neighbor-joining bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic dis- tances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for tree construction. The tree was rooted with Tulasnella sequences from clade D from the analysis of 5.8S rDNA (Electronic Appendix C).
  10. 10. 1266 ´ J. P. Suarez et al. Fig 9 – Phylogenetic placement of Tulasnella sequences, clades E and F, from Stelis hallii, Stelis superbiens, Stelis concinna and Pleurothallis lilijae inferred by MCMC analysis of nuclear ITS-5.8S. Numbers on branches designate neighbor-joining bootstrap values / MCMC estimates of posterior probabilities (only values exceeding 50 % are shown). Note that genetic distances cannot be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for tree construction. The tree was rooted with the Tulasnella sequence from the Warcup isolate T. violea DQ520097.
  11. 11. Diverse tulasnelloid fungi form mycorrhizas 1267 to the same sequence type when proportional differences recurved margins consist of two electron-dense membranes were 1 %. Clades D and E comprised four sequence types, bordering an internal electron transparent zone as was al- while the other clades consisted only of one sequence type ready shown by Andersen (1996) for Rhizoctonia repens N.Ber- each (Fig 7, Electronic Appendix B). A direct comparison be- nard ¼ Epulorhiza repens (syn.), the anamorph of Tulasnella tween 5.8S-ITS and nucLSU phylogenies was hampered by deliquescens (syn. T. calospora sensu Warcup Talbot 1967 fide the fact that publicly available sequence data were restricted Roberts 1999). The combination of this parenthesome type to either one or the other of these two DNA regions for the and the slime bodies in the cell walls was previously described vast majority of specimens studied so far (Table 1). from mycorrhiza-like associations of the liverwort Aneura pin- Mycobionts of the same sequence type were shared among guis housing Tulasnella (Ligrone et al. 1993), and apparently is orchid species, e.g. sequence types 2, 6, and 12. Tulasnella se- the best structural indication of Tulasnella (Bauer 2004). The quences from all four studied orchid species were present in ascomycetes that we isolated from the roots were not found sequence type 1 (Fig 7). Tulasnellas belonging to different se- in the cortical tissue of the orchids, but colonized only the ve- quence types were detected in mycorrhizas from the same lamen. Therefore these ascomycetes cannot be considered as plant and even from the same root piece (Table 1). Six different mycorrhiza-forming fungi. Without specific primers Tulasnella Tulasnella sequence types were found in mycorrhizas of S. hallii is nearly undetectable (Bidartondo et al. 2003). The use of both and S. superbiens and five in P. lilijae, while only two sequence Tulasnella-specific primers ITS4-Tul and 5.8S-Tul that target types were detected in mycorrhizas of S. concinna (Fig 7). ITS2 and 5.8S, respectively, increased the success of Tulasnella amplification, but a complete dataset of all tulasnelloid fungi associated with the investigated orchids can currently not be Discussion guaranteed. The accumulation curve of clades vs. number of collected individuals was not saturated (Electronic Appendix D). The importance of orchid mycorrhizal fungi and their role in Tulasnella was confirmed as a genus with a range of orchid orchid seed germination are known since Bernard’s observa- mycorrhiza forming species (Kristiansen et al. 2004; Ma et al. tions (Bernard 1909). In natural conditions, the protocorm 2003; McCormick et al. 2004; Shefferson et al. 2005; Warcup does not develop further unless it receives an exogenous car- 1971, 1981, 1985; Warcup Talbot 1967, 1971, 1980). Tulasnella bohydrate supply from a suitable mycorrhizal fungus (Smith calospora was proposed by Hadley (1970) as an universal orchid Read 1997). Although not demonstrated in nature so far, symbiont considering the capacity to establish in vitro symbi- this dependence will also account for germination of epiphytic otic associations with a broad range of orchid species. There orchids. Our finding of constant colonization of roots in con- are, however, taxonomic problems concerning the species tact with the bark supports the view of Benzing (1982) that concept in Tulasnella (Roberts 1999). The T. calospora strains the role of the fungi may be crucial also for the adult epiphytic (T. deliquescens fide Roberts 1999) isolated by Warcup from ter- orchids. The most important benefit for the orchid may be to restrial Australian orchids (Warcup Talbot 1967) belong to retain the fungus in order to assure further seed germination. two separate clades according to our nucLSU tree (Fig 7). The Saprotrophic capabilities, documented for several Tulasnella strains are even more clearly distinguished in the 5.8S-ITS species (Roberts 1999), could explain the growth of Tulasnella tree (Fig 8), clustering with Tulasnella’s detected in terrestrial on tree bark, including the capacity of fruiting close to colo- orchids from a wide range of localities, such as Spathaglottis nized roots (J.P.S., pers. obs.), and promotion of seed germina- plicata from Singapore and Epipactis gigantea from California. tion. We observed decline of vitality of hyphae after only one Our molecular analyses indicate that T. calospora is likely to night of plant storage in the laboratory, independent of comprise several distinct species. Taxonomic problems ac- whether or not the samples were cooled. The decline of vital- count also for T. violea and T. asymmetrica, as T. violea accession ity was first indicated by loss of stainability of the pelotons, AY293216 appears close to T. pruinosa AF518662 in the nucLSU which appeared yellow instead of blue in the light microscope. phylogenetic tree, while the Warcup T. violea isolate from the TEM revealed very rare occurrences of vital pelotons, but orchid Thelymitra sp. clustered separately; the Warcup abundant collapsed hyphae in this material. No isolates of T. asymmetrica strains (T. pinicola fide Roberts 1999) isolated Tulasnellales were obtained from the stored samples and PCR from the orchid Thelymitra sp. fall into two groups (Fig 7). On amplification success was very low, although only fully tur- the other hand Tulasnella’s with quite different trophic strat- gescent roots were processed. It is rather unlikely that the egies are displayed as closely related in the nucLSU tree, e.g. fast decline of hyphal vitality was linked to carbon shortage the mycobiont of the myco-heterotrophic liverwort Cryptothal- as plenty of starch grains were visible in the root tissue and lus mirabilis (AY192482) that also forms ectomycorrhizas with large amounts of glycogen were found in the vital hyphae. Dis- Pinus and Betula (Bidartondo et al. 2003), and T. irregularis ruption of the extraradical mycelium or increase of plant (AY243519), a Warcup isolate from Dendrobium dicuphum, an defense reaction could be involved (Hadley 1982). So far, the Australian orchid. Attempts are currently undertaken to col- reason for this fast decline is unclear but it might erroneously lect and describe Tulasnella’s occurring on the bark of the imply the impression of low hyphal colonization rate. sampled trees in our research area and to induce basidia for- Our combination of ultrastructural research and DNA se- mation in the isolate cultures. Morphological description quencing revealed that Tulasnella species were regularly asso- and determination combined with sequence typing appeared ciated with the epiphytic Stelis and Pleurothallis species. We as promising tools to further clarify relationships between successfully isolated three of the associated Tulasnella’s, these poorly studied mycobionts. identity proven by DNA sequences and septal porus type. Irrespective of the taxonomic problems, available se- The flat bell-shaped, imperforate parenthesomes with slightly quence data from nucLSU and ITS-5.8S made it possible to
  12. 12. 1268 ´ J. P. Suarez et al. compare the seven Tulasnella clades of symbionts of epiphytic strains by the National Institute of Agrobiological Sciences orchids studied here with named Tulasnella species and my- (NIAS), Japan, is also acknowledged. corrhizal Tulasnella’s mostly from terrestrial orchids. Tulas- nella’s of the studied epiphytic orchid species were distinct Supplementary data from so far known Tulasnellas associated with terrestrial or- chids. Tulasnelloid fungi associated with the terrestrial tem- Supplementary data associated with this article can be found, perate orchids Cypripedium spp. (subfamily Cypripedioideae) in the online version, at 10.1016/j.mycres.2006.08.004. and Dactylorhiza majalis (AY634130) (subfamily Orchidoideae) were displayed in a basal position with respect to the Tulas- nella sequence types from Stelis and Pleurothallis in the nucLSU references phylogeny (Fig 7). In the 5.8S nrDNA phylogeny, the tulasnel- loid fungi associated with Dactylorhiza majalis (AY634130), Orchis purpurea (AJ549121), Spathaglottis plicata (AJ313457), Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, ¨ Ophrys sphegodes (AJ549122) (all subfamily Orchidoideae) and Lipman DJ, 1997. Gapped BLAST and PSI-Blast: a new genera- Cypripedium fasciculatum (AY966883) appeared in a basal posi- tion of protein database search programs. Nucleic Acids Re- search 25: 3389–3402. tion relative to Tulasnella sequence types from Stelis and Pleu- Andersen TF, 1996. A comparative taxonomic study of Rhizocto- rothallis (Electronic Appendix C). Molecular phylogenetic nia sensu lato employing morphological, ultrastructural and analyses of the family Orchidaceae are consistent in respect molecular methods. Mycological Research 100: 117–128. to a basal position of Cypripedioideae and Orchidoideae com- Bauer R, 2004. Basidiomycetous interfungal cellular interactions – pared to Epidendroideae (e.g. Cameron et al. 1999). Switches a synopsis. In: Agerer R, Piepenbring M, Blanz P (eds), Frontiers from terrestrial to epiphytic habit or back were found to be in Basidiomycote Mycology. IHW-Verlag, Eching, Germany, pp. major driving forces in radiation and specialization of orchids 325–337. ´ Bayman P, Lebron LL, Tremblay RL, Lodge DJ, 1997. Variation in (Cameron 2002, 2005), and beside pollinator relationships my- endophytic fungi from roots and leaves of Lepanthes (Orchida- corrhizal interactions are now recognized crucial for orchid ceae). New Phytologist 135: 143–149. evolution (Taylor et al. 2003). However, more species need to Bermudes D, Benzing DH, 1989. Fungi in neotropical epiphyte be sampled including terrestrial orchids of the study site to ar- roots. BioSystems 23: 65–73. rive at convincing conclusions about coevolution between or- Bernard N, 1909. L’evolution dans la symbiose. Les orchidees et´ chids and their mycobionts. leur champignons commenseaux. Annales des Sciences Nature- Our results show differences in the number of Tulasnella lles Botanique, Paris 9: 1–196. Benzing DH, 1982. Mycorrhizal infections of epiphytic orchids symbionts associated with one orchid species. Six Tulasnella in southern Florida. American Orchid Society Bulletin 51: sequence types were associated with one individual orchid 618–622. species of S. hallii and S. superbiens, five in P. lilijae, but only ´ Bidartondo MI, Bruns TD, Weiß M, Sergio C, Read D, 2003. Spe- two sequence types were found with S. concinna. We cannot ex- cialized cheating of the ectomycorrhizal symbiosis by an epi- clude the possibility that the differences in numbers of Tulas- parasitic liverwort. Proceedings of the Royal Society of London, nella symbionts will vanish when a higher number of orchid Series B, Biological Sciences 270: 835–842. 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