Basic and Applied Ecology 9 (2008) 13–23

Guilds of mycorrhizal fungi and their relation to trees, ericads, orchidsand liverworts in a neotropical mountain rain forest

Ingrid Kottkea,!, Ingeborg Hauga, Sabrina Setaroa, Juan Pablo Suarezc, Michael Weißa,Markus Preußingb, Martin Nebelb, Franz Oberwinklera

aEberhard-Karls-Universitat Tubingen, Spezielle Botanik, Mykologie und Botanischer Garten,Auf der Morgenstelle 1, D-72076 Tubingen, GermanybStaatliches Museum fur Naturkunde Stuttgart, Rosenstein 1, D-70191 Stuttgart, GermanycCentro de Biologıa Celular y Molecular, Universidad Tecnica Particular de Loja,San Cayetano Alto s/n C.P. 11 01 608, Loja, Ecuador

Received 9 March 2006; accepted 5 March 2007

Abstract

Mycorrhizas of vascular plants and mycorrhiza-like associations of liverworts and hornworts are integral parts ofterrestrial ecosystems, but have rarely been studied in tropical mountain rain forests. The tropical mountain rain forestarea of the Reserva Biologica San Francisco in South Ecuador situated on the eastern slope of the Cordillera ElConsuelo is exceptionally rich in tree species, ericads and orchids, but also in liverworts. Previous light and electronmicroscopical studies revealed that tree roots are well colonized by structurally diverse Glomeromycota, and thatepiphytic, pleurothallid orchids form mycorrhizas with members of the Tulasnellales and the Sebacinales(Basidiomycota). Sebacinales also occurred in mycorrhizas of hemiepiphytic ericads and Tulasnellales were found inliverworts belonging to the Aneuraceae. On the basis of these findings, we hypothesized that symbiotic fungi with abroad host range created shared guilds or even fungal networks between different plant species and plant families. Totest this hypothesis, molecular phylogenetic studies of the fungi associated with roots and thalli were carried out usingsequences of the nuclear rDNA coding for the small subunit rRNA (nucSSU) of Glomeromycota and the large subunitrRNA (nucLSU) of Basidiomycota. Sequence analyses showed that Sebacinales and Tulasnellales were only sharedwithin but not between ericads and orchids or between liverworts and orchids, respectively. Regarding arbuscular-mycorrhiza-forming trees, however, 18 out of 33 Glomus sequence types were shared by two to four tree speciesbelonging to distinct families. Nearly all investigated trees shared one sequence type with another tree individual. Hostrange and potential shared guilds appeared to be restricted to the plant family level for Basidiomycota, but werecovering diverse plant families in case of Glomeromycota. Given that the sequence types as defined here correspond tofungal species, our findings indicate potential fungal networks between trees.r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Zusammenfassung

Die Mykorrhizen der Gefaßpflanzen und ahnliche Symbiosen von Lebermoosen und Hornmoosen sind wesentliche,funktionale Bestandteile terrestrischer Okosysteme, wurden bisher in tropischen Bergregenwaldern aber kaum

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1439-1791/$ - see front matter r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.doi:10.1016/j.baae.2007.03.007

!Corresponding author. Tel.: +497071 2976688; fax: +49 7071 295534.E-mail address: Ingrid.Kottke@uni-tuebingen.de (I. Kottke).

untersucht. Das Gebiet der Reserva Biologica San Francisco im tropischen Bergregenwald von Sudecuador, amOsthang der Cordillera El Consuelo gelegen, zeichnet sich durch eine sehr hohe Artenzahl bei Baumen, Ericaceen undOrchideen, aber auch bei Lebermoosen aus. Vorausgehende licht- und elektronenmikroskopische Untersuchungenhatten gezeigt, dass die arbuskularen Mykorrhizen der Baume von strukturell unterschiedlichen Glomeromycetengebildet wurden. Epiphytische, pleurothallide Orchideen waren von Tulasnellales und Vertretern der Sebacinales(Basidiomycota) mykorrhiziert, wobei Sebacinales auch an den Ericaceen und Tulasnellales auch an Lebermoosen derFamilie Aneuraceae auftraten. Aus diesen Beobachtungen ergab sich die Frage, ob die symbiotischen Pilze ein breitesWirtsspektrum haben und moglicherweise pilzliche Netzwerke zwischen unterschiedlichen Pflanzen bilden konnen.Molekularphylogenetische Untersuchungen der Mykobionten unter Verwendung der nucSSU der Glomeromycetenund der nucLSU der Basidiomyceten ergaben ein differenziertes Bild. Identische oder nahezu identische Sequenzen derBasidiomyceten wurden nur innerhalb der Familien, aber nicht zwischen Orchideen und Ericaceen oder Orchideen undAneuraceen gefunden. Bei den Glomeromyceten hingegen kamen 18 von 33 Sequenztypen der Formgattung Glomus inzwei bis vier verschiedenen Baumarten unterschiedlicher Familien vor. Nahezu alle untersuchten Baume hatten einengemeinsamen Sequenztyp mit einem anderen Baumindividuum. Pilzliche Netzwerke von Glomeromyceten zwischenBaumen unterschiedlicher Familienzugehorigkeit waren demnach moglich, vorausgesetzt die hier definiertenSequenztypen entsprechen gemeinsamen Pilzarten.r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Keywords: Glomus; Sebacinales; Tulasnellales; Andean clade of Ericaceae; Aneuraceae; pleurothallid orchids; nucSSU; nucLSU;fungal networks; Reserva Biologica San Francisco

Introduction

Mycorrhizas of vascular plants and mycorrhiza-like-associations of liverworts and hornworts are integralparts of terrestrial ecosystems. Mycorrhizal fungi do notonly improve the nutrient status and thereby the fitness ofplant individuals (Smith & Read, 1997) but also influencerichness and composition of plant communities (Hartnett& Wilson, 1999). Distinct fungal guilds appear to bepresent in important plant ecosystems, such as Basidio-mycota in ectomycorrhiza-dominated forests, Ascomy-cota in ericoid-mycorrhiza-dominated heathlands andGlomeromycota in arbuscular-mycorrhiza-dominatedgrasslands (Francis & Read, 1994; Kottke, 2002) andtropical ecosystems (Alexander & Lee, 2005). Experi-ments showed higher competitiveness of mycorrhizalversus non-mycorrhizal plants in arbuscular-mycorrhiza-forming plant communities (Grime, Mackey, Hillier, &Read, 1987), and revealed a positive correlation betweendiversity of arbuscular mycorrhizal fungi and plantspecies richness (Van der Heijden et al., 1998). Plants,on the other hand, may regulate the community structureand diversity of mycorrhizal fungi by unspecific orspecific binding (Johnson, lJdo, Genney, Anderson, &Alexander, 2005). Fungi with a broad host range couldeven establish functional mycorrhizal networks to im-prove nutrient exploitation from soil resources, yieldinterplant carbon transfer, facilitate seedling establish-ment and influence interplant competition (Simard &Durall, 2004). A high number of fungal species withdifferences in functional compatibility could, by anadditive beneficial effect of each fungal species, alsosupport plant diversity and productivity (Read, 1998).

Recent studies in the species-rich Andean rain forestrevealed the co-occurrence of many mycorrhizal typeswithin short local distances, including some previouslyunknown ones (Beck, Kottke, & Oberwinkler, 2005;Haug et al., 2004; Haug, Weiß, Homeier, Oberwinkler, &Kottke, 2005). Light microscopical investigations showedarbuscular mycorrhizas in 112 tree species from 53families on mineral as well as pure organic soils (Kottke,Beck, Oberwinkler, Homeier, & Neill, 2004). A new typeof mycorrhizas formed by Sebacinales (Basidiomycota)with members of the hemiepiphytic Andean clade ericadswas documented by ultrastructural characters andmolecular identification (Setaro, Oberwinkler, & Kottke,2006a; Setaro, Weiß, Oberwinkler, & Kottke, 2006b).Transmission electron microscopical studies provedSebacinales and Tulasnellales (Basidiomycota) in epiphy-tic orchids of the subtribe Pleurothallidinae (Suarez et al.,2006), and Tulasnellales in the thalloid liverwort Aneurapinguis (M. Preußing, unpublished). We hypothesizedthat fungal networks might be formed by Glomeromy-cota between distinct tree species of different families,and that identical Sebacinales or Tulasnellales speciesmight link epiphytic orchids with hemiepiphytic ericadsor with liverworts of the Aneuraceae, via mycorrhizalassociations. Even if no functional, physical fungalnetworks between epiphytic and terrestrial plants couldbe expected, a broad host range and common fungalguilds would improve the chances of long-term main-tenance of both the fungi and the plants, in suchheterogeneous ecosystem. Further ‘‘ploughing up thewood wide web’’ (Helgason, Daniell, Husband, Fitter, &Young, 1998) in order to identify the associated fungiwas, therefore, challenging (Fig. 1).

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The identification of the mycorrhiza-forming fungi inthe forest could not be done by sampling spores orfruiting bodies, because these methods would have onlyyielded a very narrow spectrum of the fungal commu-nities (Husband, Herre, Turner, Gallery, & Young, 2002;Sanders, 2004a). Instead, rDNA sequencing of theassociated fungi from the plant material was carriedout. The sequence types (ribosomal genotypes), at thepresent stage of knowledge, can rarely be related preciselyto either morphological or biological species. However,the amount of information derived from the sequences ofthe ribosomal genes appeared to be meaningful inprevious ecological studies on arbuscular mycorrhizas(Helgason et al., 2002; Husband et al., 2002; Sanders,2004b) as well as on mycorrhiza-forming Basidiomycota(Bidartondo, Bruns, Weiß, Sergio, & Read, 2003;Bidartondo, Burghardt, Gebauer, Bruns, & Read, 2004;Weiß, Selosse, Rexer, Urban, & Oberwinkler, 2004).

In order to test the hypothesis that fungal networks orat least common fungal guilds could potentially beformed in the tropical mountain rain forest betweentrees of diverse families, between ericads and orchids, orbetween orchids and the aneuracean liverworts (Fig. 1),we sequenced fungal rDNA directly from the mycor-rhizas of 21 tree species, 11 species of the Ericaceae andfour species of the Orchidaceae. We also sequenced thefungal rDNA from the mycorrhiza like associations ofthree aneuracean liverwort species. Most sampling wascarried out within an area of about 12 ha. The DNAsequences of the fungi suspected to form shared guilds,the Glomeromycota, the Sebacinales and the Tulasnel-lales, were analyzed by molecular phylogenetic methods.Identical or closely related sequences were identified inorder to evaluate the potential of fungal networks orfungal guilds within and between the plant families.

Materials and methods

Site and sampling

The investigations were carried out in South Ecuadorduring 2001–2005. Study sites lay in a tropical mountainrain forest area on the eastern slope of the Cordillera ElConsuelo at 1850–2300ma.s.l. and in the nearby bushyparamo at 2700–3000ma.s.l. The pristine forest isexceptionally rich in tree species, ericads and orchids,and also in number of liverworts (Homeier, Dalitz, &Breckle, 2002; Parolly, Kurschner, Schafer-Verwimp, &Gradstein, 2004).

Roots were sampled from 17 different tree species inthe pristine forest (identified and marked by J. Homeier).The trees were found within 15 plots of 400m2 each, on amountain ridge along the altidudinal gradient between1920 and 2100ma.s.l. and on the steep slope of the closeravine. Additional root samples were collected from four

indigenous tree species in a nearby afforestation area andfrom three indigenous tree species in a nursery. Five cupswith three fine roots (1 cm in length) were collected fromeach tree individual. Humus grown roots were sampledof 11 ericads in the pristine forest on the mountain ridgewithin or close to the tree plots, along road sides and inthe bushy paramo. Ten cups containing one mycorrhiza(0.5 cm in length) were collected from each ericadindividual. Roots of four pleurothallid orchid specieswere removed from standing tree stems along themountain ridge close to the tree plots and sometimes1–2m away from the sampled ericads or Aneuraceae.One to four roots (1–2 cm in length) per orchidindividual that had close contact to the substrate weresampled in separate cups. Three species of Aneuraceae(liverworts) were collected from rotten wood or, lessfrequently, from wet rocks. One slice of the thallus(0.5 cm thick) was sampled from each liverwort indivi-dual. Details of all the sampled individuals are presentedin Appendix A, Supplementary Table 1. All the rootsand thalli were cleaned under tap water the same day.The velamen was afterwards removed from orchid roots.Orchid roots and thalli of the liverworts were controlledfor hyphal colonization by light microscopy fromsections. Tree and ericad roots were controlled formycorrhization using standard staining methods (Hauget al., 2004; Setaro et al., 2006a). All the furtherproceeded samples were highly colonized by mycorrhizalfungi. Roots and liverworts slices were dried in 1.5mltubes and kept on silica gel for DNA isolation.

Processing of fungal DNA sequences

DNA was isolated from the dried mycorrhizal samplesusing the DNAeasy Plant Mini Kit (Qiagen, Hilden,Germany). One to four cups per plant individual wereprocessed, the numbers of cups yielding sequences aregiven in Appendix A, Supplementary Table 1. Part of thenuclear rDNA coding for the small subunit rRNA(nucSSU) of the arbuscular fungi of the tree mycorrhizas,and part of the large subunit rRNA (nucLSU) of theputative Sebacinales and Tulasnellales in orchids, ericadsand liverworts were amplified by the polymerase chainreaction (PCR). Primers and PCR design are given inAppendix A, Supplementary Tables 2 and 3, the targetedplants are given in Supplementary Table 1 (see AppendixA, Supplementary Table 1). For detailed information seeAppendix A: Processing of fungal sequences.

Phylogenetic analysis

Sequence similarities were determined using theBLAST sequence similarity search tool (Altschul et al.,1997) provided by NCBI (www.ncbi.nlm.nih.gov).Sequence alignments were done with MAFFT and

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subsequent analyses were conducted with PAUP usingthe BIONJ algorithm. For details see Appendix A:Phylogenetic analysis.

Results

Tulasnellales mycobionts in orchids and liverworts

The phylogenetic analysis of the nucLSU sequences ofTulasnellales yielded eight clades of epiphytic orchidmycobionts and nine clades of Aneura and Riccardiamycobionts (Fig. 2). Tulasnellales sequences were notshared between orchid and liverwort species. However,an analysis of proportional differences between se-quences of the nucLSU D1/D2 regions showed thatdifferent orchid species shared sequences with simila-rities more than 99% in clades A, B, D and G. The threeliverwort species also shared sequences with similaritiesof more than 99%; A. pinguis and Riccardia smaragdinain clade 2, A. pinguis and Riccardia sp. in clade 6. All themycobiont sequences are new to science.

Sebacinales mycobionts in ericads and orchids

All the sebacinalean sequences obtained from theericad and orchid mycorrhizas are new to science and

can be assigned to Sebacinales (Fig. 3) as defined byWeiß et al. (2004). The order Sebacinales so farcontains, amongst others, Sebacina vermifera isolatesfrom Australian orchids (Warcup, 1988; Warcup &Talbot, 1967; Weiß et al., 2004) and fungal sequences ofmycorrhizas of Canadian Gaultheria shallon (Ericaceae;Berch, Allen, & Berbee, 2002; Fig. 3). Neither sebaci-nalean mycobionts of the epiphytic pleurothallid orchidsof Ecuador and the terrestrial orchids of Australia, norsebacinalean mycobionts of ericads from Ecuador andCanada showed identical sequences. The mycobionts oforchids and ericads of the study site clustered in separateclades. Sebacinales sequences from both orchid andericad mycorrhizas were not included in shared well-supported terminal clades. Thus, our analysis suggeststhe occurrence of different fungal guilds as mycobiontsof ericads and orchids, respectively.

The Sebacinales sequences from pleurothallid orchidswere separated into six clades (Fig. 3, clades 1–6; clade 6consisting of a single sequence). No identical Sebaci-nales sequences were found in different orchid speciesfrom the study site. Sequence similarity, for example inthe well-supported clade 4, was only 89% amongmycobionts of Stelis hallii and Pleurothallis lilijae.

Identical Sebacinales sequences were, however, foundin different ericad species. Cavendishia nobilis collectedfrom the pristine forest shared a sequence with

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Cavendishia

Tulasnella

Stelis

Aneura

Glomus

Sebacina

Sebacina

Aneura

Stelis

Tulasnella

Cavendishia

rootsV. Uhle-Schneider

Fig. 1. Scheme of the plants and their mycorrhizas assumed to form common fungal guilds or networks in the tropical mountainrain forest of South Ecuador. Diverse trees as indicated by different leaves form mycorrhizas with Glomeromycota. The potentialfungal network as found on the basis of Glomus sequences is indicated by spores and hyphal connections in the humus layer. Theliana-like Cavendishia forms ectendomycorrhizas with Sebacinales, the epiphytic orchid Stelis forms endomycorrhizas withSebacinales and Tulasnellales, and the thalloid liverwort Aneura growing on a rotten stem forms mycorrhiza-like associations withTulasnellales. The mycorrhizal types of ericads (left), orchids (middle) and Aneuraceae (right) are displayed by sections in whichhyphae were stained by methyl blue. No common fungal guilds were found among the ericads and the epiphytic orchids (suspectedfor Sebacinales) and among the epiphytic orchids and the Aneuraceae (suspected for Tulasnellales).

I. Kottke et al. / Basic and Applied Ecology 9 (2008) 13–2316

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Fig. 2. Phylogenetic relationships of the detected Tulasnellales mycobionts of pleurothallid orchids (clades A–H) and Aneuraceae(clades 1–9) in the tropical mountain rain forest area of South Ecuador. BIONJ analysis of an alignment of nuclear DNA sequencescoding for the D1/D2 region of the large ribosomal subunit (nucLSU). The tree was rooted with Auricularia auricula-judae.Numbers on branches designate BIONJ bootstrap values (only values exceeding 50% are shown). Note that genetic distances cannotbe directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for phylogeneticreconstruction. The Tulasnellales sequence from Aneura pinguis AY298949, marked with a diamond, is not from the study site.

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Fig. 3. Phylogenetic relationships of the detected Sebacinales mycobionts of ericads (dotted line) and pleurothallid orchids (clades1–6) in the tropical mountain rain forest area of South Ecuador. BIONJ analysis of an alignment of nuclear DNA sequences codingfor the D1/D2 region of the large ribosomal subunit (nucLSU). The tree was rooted with Sebacina allantoidea, Sebacina epigaea,Sebacina incrustans, Tremellodendron pallidum, Efibulobasidium rolleyi and Craterocolla cerasi. Numbers on branches designateBIONJ bootstrap values (only values exceeding 50% are shown). Asterisks, dots and triangles mark identical sequences. AUS,Australia; CAN, Canada; ECU, Ecuador; FRA, France; GER, Germany; IND, India.

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Fig. 4. Phylogenetic relationships of the glomeralean mycobionts of 38 arbuscular mycorrhizal trees in the tropical mountain rainforest: BIONJ analysis of an alignment of nuclear DNA sequences coding for the small ribosomal subunit (nucSSU; 1108characters). The tree was rooted with seven sequences of the Gigasporaceae. Glomeralean sequences which clustered together andshowed sequence similarities higher than 99% were regarded as one sequence type. Numbers on branches designate BIONJbootstrap values (only values exceeding 50% are shown). Nineteen sequence types which were found at different trees werenumbered continuously, sequence types found at only one tree are indicated by black circles.

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Gaultheria erecta collected from the road side (Fig. 3,marked by asterisks), Psammisia guianensis shared asequence with Disterigma microphyllum (Fig. 3, markedby dots) and C. nobilis one with Semiramisia speciosa(Fig. 3, marked by triangles), all collected in the pristineforest.

Glomeraceae of tree mycorrhizas

The molecular investigation using primer GLOM1310rcrevealed 33 sequence types of arbuscular mycorrhizal fungiin the 34 trees that belonged to 21 species out of 12 families

(see Appendix A, Supplementary Table 1). Thirty-twosequence types belonged to Glomus group A, one sequencetype belonged to Glomus group B as defined by Schußler,Schwarzott, and Walker (2001) (Fig. 4, numbered lines andpoints). Eighteen sequence types occurred at least with twoand at most with four tree species. Fifteen sequence typeswere found in one tree individual only (Fig. 4, points). Oneto three sequence types were found with one tree individual(Table 1). Thirty-two tree individuals shared fungalsequence types with other trees, two tree individuals(Tabebuia chrysantha 2 A., Guarea pterorhachis F.) hadno common fungal sequence type with another tree.Different individuals of the same tree species showed

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Table 1. Occurrence of shared (x) and single (a) Glomus sequence types in the arbuscular mycorrhizas of 34 trees from 21 species

Sequence type 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a b

Tree speciesTabebuia chrysantha 1 A x 1Tabebuia chrysantha 2 A 1 1Clethra revoluta F x x 2Clusia elliptica F x 1 1Vismia tomentosa F x x x 3Hyeronima asperifolia F x 1 2Hyeronima moritziana F x x 1 3Hyeronima oblonga F x 1Hyeronima sp. F x 1 2Juglans neotropica 1 A x x 2Juglans neotropica 2 A x x 1 3Nectandra laevis 1 F x x 1 3Nectandra laevis 2 F x x 1 3Graffenrieda emarginata 1 F x x 1 3Graffenrieda emarginata 2 F x x 2Graffenrieda emarginata 3 F x x 2Cedrela montana 1 N x 1Cedrela montana 2 A x x 1 3Cedrela montana 3 A x x 2Cedrela sp. F x x 2Guarea kunthiana F x x 2Guarea cf. kunthiana F x x x 3Guarea pterorhachis F 2 2Guarea sp. F x x 1 3Inga acreana 1 F x 1Inga acreana 2 F x 1Inga acreana 3 F x 1Inga acreana 4 F x 1Podocarpus oleifolius F x 1 2Cinchona officinalis N x 1Heliocarpus americanus 1 N x 1Heliocarpus americanus 2 A x 1Heliocarpus americanus 3 A x 1 2Heliocarpus americanus 4 F x x 2Occurrence of sequence types N A F F A A N F F F F F A F F F F F N

A F A FF

N, nursery; A, afforestation; F, pristine forest. For numbers of sequence types, see Fig. 4.aNumber of sequence types found only with this tree individual.bTotal number of sequence types per tree individual.

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different fungal sequence types in most cases (Table 1).In cases were common fungal sequence types occurred intwo tree individuals of the same species, e.g. type 15 and 16in Graffenrieda emarginata, this sequence type was alsofound in other tree species (Table 1). Thus, no tree-specificfungal sequence types were found. Four sequence typescould be linked with sequences of identified morphospecies:sequence type 1 with Glomus intraradices, type 2 withGlomus vesiculiferum, type 5 with Glomus proliferum andtype 19 with Glomus mosseae (Fig. 4).

Discussion

The investigated mycorrhizal associations occurred atthe study site frequently in close vicinity. We thereforeexpected that few identical or closely related fungi mightbe the main mycorrhizal associates. The results did notsupport this simple hypothesis but gave a fairly complexpicture of the associations. An unexpectedly highnumber of sequence types were detected in Glomeraceaefrom Glomus group A and in Basidiomycota fromTulasnellales and Sebacinales. Furthermore, overlap ofthe sequence types belonging to Sebacinales andTulasnellales was restricted to the family level in theEricaceae, Orchidaceae and Aneuraceae, respectively,but was detected in about 55% of the AM fungicolonizing trees of distinct families. These findings arebased on a rather narrow concept of sequence types thatmight intrinsically relate to a concept of species.Traditionally, studies on biodiversity and host specificitywere based on morphologically defined species. No suchapproach was feasible in the case of the mycobionts inour study as the fungi did not display sufficientstructural differences in the mycorrhizas for delimitationof morphospecies. The analysis of host range of themycobionts from field samples using DNA sequences,however, also poses problems. Firstly, results are limitedby the available primers (Husband et al., 2002) and theamount of material that can be analyzed in appropriatetime. The number of fungi that were detected during thisstudy is, therefore, far from being complete for theexceptional species-rich area. We restricted the investi-gation to the Glomeraceae, the Sebacinales and theTulasnellales because these were either the only asso-ciated fungal guilds (Aneuraceae; M. Preußing unpub-lished) or the dominating fungi (trees, Ericaceae,Orchidaceae; Setaro et al., 2006a, b; Suarez et al.,2006, I. Haug unpublished). Secondly, problems re-sulted from the observation that the ribosomal genescan show intraspecific variation especially in case of theGlomeromycota (Lloyd-Macgilp et al., 1996; Sanders,2004b; Sanders, Alt, Groppe, Boller, & Wiemken, 1995).These facts pose general, unresolved challenges to aspecies concept based on meaningful levels of geneticdiversity (Husband et al., 2002).

We decided to use the nucSSU in the case ofGlomeromycota because primers are available, thisDNA region could be well aligned, and many sequenceshave already been deposited in GenBank. The nucSSU hasbeen widely used in ecological studies (Russel & Bulman,2004; Saito, Suyama, Sato, & Sugawara, 2004; Scheublin,Ridgway, Young, & van der Heijden, 2004; Wubet, Weiß,Kottke, Teketay, & Oberwinkler, 2006), and was the basisof a new classification system of Glomeromycota based onsequences assigned to species by spore morphology(Schußler et al., 2001). Our phylogenetic analysis, includ-ing these spore-based sequences from GenBank detectedgroups where inter- and intraspecific differences wereoverlapping (AY635833 Glomus mosseae/U96139 Glomusmosseae proportion of differing sites 2/1025 ! 0.2%;Y17653 G. caledonium/AJ276085 G. fragilistratum 2/1079 ! 0.2%). Nearly every sequenced clonal insertshowed at least 0.2% differences to other clonal insertsof the same PCR product (data not shown). We decided tounite sequences with differences o1% into one sequencetype and evaluated diversity and host preferences of theGlomus mycobionts at this level. Several authors regardedclustering with a high bootstrap value and sequencedifferences o2.5% to define sequence types (Helgason etal., 2002; Husband et al., 2002; Vandenkoornhuyse et al.,2002). We chose a lower sequence difference level in ordernot to overestimate the number of fungal species shared bydifferent plants. It cannot be excluded that even withinthese sequence types several species were joined together,which would mean that we still overestimated the numberof shared fungi. Many AM fungi that are clearly separatedby morphology and e.g. ITS sequences cannot beseparated on the SSU base (A. Schußler pers. commu-nication). We found that 55% of the sequence types wereshared by different tree species of distinct families. Nearlyall the investigated trees shared a sequence type withanother tree. Seven tree species were represented by morethan one individual, but no species-specific sequence typewas recognized. Many sequence types were only isolatedonce, but further investigations may show a widerdistribution of these sequence types resulting in a broaderrange of shared species. Our conclusion that a functionalnetwork might potentially be formed between the highlydiverse trees in the tropical mountain forest appears so farwell supported. The conclusion is in general agreementwith the conceptual model of AM fungal grasslandcommunities (Johnson et al., 2005 and literature therein)which considers the possibility that a small number ofhost-specific arbuscular mycorrhizal species occur along-side many non-specific species.

Up to now, the nucLSU is the only useful marker formolecular studies on Sebacinales and Tulasnellales. Nowell-supported terminal clade was found containingSebacinales sequences from neotropical ericads andfrom orchids, thus no shared fungal guilds appearedfor the investigated ericads and orchids. However,

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Sebacinales were shared between different ericad species.In case of Tulasnellales, we assumed that sequences ofX99% similarity of the D1/D2 region of the nucLSUmay represent one species (Suarez et al., 2006). On thisbasis, no Tulasnellales sequences were shared among theepiphytic orchids and the liverworts from rotten woodor rocks, but identical Tulasnellales sequences werefound among different orchid species and among theAneuraceae. Thus, common guilds of Basidiomycotalikely only exist among plants of the same family. Noneof the sequences detected in this study were identical ornear-identical to a sequence obtained from terrestrialorchids, however, only sequences obtained from myco-bionts of terrestrial orchids from Australia or thenorthern hemisphere were available for this study.Further studies should investigate the mycobionts ofterrestrial orchids in the study site to examine possiblelinkages with ericads or liverworts.

If identical and nearly identical sequences (X99%identity) were regarded as belonging to the same species,a large number of distinct fungal species with rDNAsequences new to science were involved in the symbiosiswith orchids, ericads and liverworts in the tropicalmountain rain forest area. A similarly narrow conceptof identity was assumed here for the AM fungi (99%sequence similarity) and an unexpected and so farunknown high diversity of sequence types was found.The high diversity of fungal symbionts in an area of onlyabout 12 ha points to high radiation and a lowextinction rate of fungal species in the tropical mountainrain forest which was under low climatic stress duringgeological times (Van der Hammen, 1989). The speciesrichness of fungi may by itself support plant speciesrichness and productivity and vice versa as shownexperimentally for grassland communities (Van derHeijden et al., 1998; Van der Heijden, Wiemken, &Sanders, 2003). The associated fungi may show differ-ences in functional compatibility, which was postulatedas a prerequisite for maintenance of the floristic diversityfor grasslands and boreal forests (Helgason et al., 2002;Read, 1998), but this needs further evaluation for theAndean tropical mountain rain forest.

Appendix A. Supplementary materials

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.baae.2007.03.007.

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