Guilds Of Mycorrhizal Fungi And Their Relation To Trees Ericads Orchids And Liverworts In A Neotropical Mountain Rain Forest

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Basic and Applied Ecology ] (]]]]) ]]]–]]] www.elsevier.de/baae

OF

Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids

and 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 Tu-

bingen, 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
O
CORRECTED PRAbstract

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 Tulasnellas 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 Tulasnellas 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–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.

UNZusammenfassung

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

e front matter r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

ae.2007.03.007

ing author. Tel.: +497071 2976688; fax: +49 7071 295534.

ess: [email protected] (I. Kottke).

s article as: Kottke, I., et al. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical

forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae.2007.03.007

www.elsevier.de/baae

dx.doi.org/10.1016/j.baae.2007.03.007

mailto:[email protected]

dx.doi.org/10.1016/j.baae.2007.03.007

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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 Tulasnella 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; Tulasnella; Andean clade of Ericaceae; Aneuraceae; pleurothallid orchids; nucSSU; nucLSU; fungal

networks; Reserva Biologica San Francisco

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Introduction

Mycorrhizas of vascular plants and mycorrhiza-likeassociations of liverworts and hornworts are integralparts of terrestrial ecosystems. Mycorrhizal fungi do notonly improve the nutrient status and thereby the fitnessof plant individuals (Smith & Read, 1997) but alsoinfluence richness and composition of plant commu-nities (Hartnett & Wilson, 1999). Distinct fungal guildsappear to be present in important plant ecosystems,such as Basidiomycota in ectomycorrhiza-dominatedforests, Ascomycota in ericoid-mycorrhiza-dominatedheathlands and Glomeromycota in arbuscular-mycor-rhiza-dominated grasslands (Francis & Read, 1994;Kottke, 2002) and tropical ecosystems (Alexander &Lee, 2005). Experiments showed higher competitivenessof mycorrhizal versus non-mycorrhizal plants in arbus-cular-mycorrhiza-forming plant communities (Grime,Mackey, Hillier, & Read, 1987), and revealed a positivecorrelation between diversity of arbuscular mycorrhizalfungi and plant species richness (Van der Heijden et al.,1998). Plants, on the other hand, may regulate thecommunity structure and diversity of mycorrhizal fungiby unspecific or specific binding (Johnson, lJdo,Genney, Anderson, & Alexander, 2005). Fungi with abroad host range could even establish functionalmycorrhizal networks to improve nutrient exploitationfrom soil resources, yield interplant carbon transfer,facilitate seedling establishment and influence interplantcompetition (Simard & Durall, 2004). A high number offungal species with differences in functional compat-ibility could, by an additive beneficial effect of each

Please cite this article as: Kottke, I., et al. Guilds of mycorrhizal fungi and

mountain rain forest. Basic and Applied Ecology (2007), doi:10.1016/j.baae

D PROfungal species, also support plant diversity and produc-

tivity (Read, 1998).Recent studies in the species-rich Andean rain forest

revealed 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 investigationsshowed arbuscular mycorrhizas in 112 tree species from53 families on mineral as well as pure organic soils(Kottke, Beck, Oberwinkler, Homeier, & Neill, 2004). Anew type of mycorrhizas formed by Sebacinales(Basidiomycota) with members of the hemiepiphyticAndean clade ericads was documented by ultrastructur-al characters and molecular identification (Setaro,Oberwinkler, & Kottke, 2006a; Setaro, Weiß, Ober-winkler, & Kottke, 2006b). Transmission electronmicroscopical studies proved Sebacinales and Tulasnella

(Basidiomycota) in epiphytic orchids of the subtribePleurothallidinae (Suarez et al., 2006), and Tulasnella inthe thalloid liverwort Aneura pinguis (M. Preußing,unpublished). We hypothesized that fungal networksmight be formed by Glomeromycota between distincttree species of different families, and that identicalSebacinales or Tulasnella species might link epiphyticorchids with hemiepiphytic ericads or with liverworts ofthe Aneuraceae, via mycorrhizal associations. Even if nofunctional, physical fungal networks between epiphyticand terrestrial plants could be expected, a broad hostrange and common fungal guilds would improve thechances of long-term maintenance of both the fungi andthe plants, in such heterogeneous ecosystem. Further‘‘ploughing up the wood wide web’’ (Helgason, Daniell,

their relation to trees, ericads, orchids and liverworts in a neotropical

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Husband, Fitter, & Young, 1998) in order to identify theassociated fungi was, therefore, challenging (Fig. 1).

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 carried out.The sequence types (ribosomal genotypes), at thepresent stage of knowledge, can rarely be relatedprecisely to either morphological or biological species.However, the amount of information derived from thesequences of the ribosomal genes appeared to bemeaningful in previous ecological studies on arbuscularmycorrhizas (Helgason et al., 2002; Husband et al.,2002; Sanders, 2004b) as well as on mycorrhiza-formingBasidiomycota (Bidartondo, Bruns, Weiß, Sergio, &Read, 2003; Bidartondo, Burghardt, Gebauer, Bruns, &Read, 2004; Weiß, Selosse, Rexer, Urban, & Oberwink-ler, 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 mycorrhizal-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.
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UNCORMaterials 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. Home-



ED PROOF

ier). The trees were found within 15 plots of 400m2 each,on a mountain ridge along the altidudinal gradientbetween 1920 and 2100ma.s.l. and on the steep slope ofthe close ravine. Additional root samples were collectedfrom four indigenous tree species in a nearby afforesta-tion area and from three indigenous tree species in anursery. Five cups with three fine roots (1 cm in length)were collected from each tree individual. Humus grownroots were sampled of 11 ericads in the pristine forest onthe mountain ridge within or close to the tree plots,along road sides and in the bushy paramo. Ten cupscontaining one mycorrhiza (0.5 cm in length) werecollected from each ericad individual. Roots of fourpleurothallid orchid species were removed from stand-ing tree stems along the mountain ridge close to the treeplots and sometimes 1–2m away from the sampledericads or Aneuraceae. One–four roots (1–2 cm inlength) per orchid individual that had close contact tothe substrate were sampled in separate cups. Threespecies of Aneuraceae (liverworts) were collected fromrotten wood or, less frequently, from wet rocks. Oneslice of the thallus (0.5 cm thick) was sampled from eachliverwort individual. Details of the sampled individualsare presented in Appendix A, Supplementary Table 1.All the roots and thalli were cleaned under tap water thesame day. The velamen was afterwards removed fromorchid roots. Orchid roots and thalli of the liverwortswere controlled for hyphal colonization by light micro-scopy from sections. Tree and ericad roots werecontrolled for mycorrhization using standard stainingmethods (Haug et al., 2004; Setaro et al., 2006a). All thefurther proceeded samples were highly colonized bymycorrhizal fungi. Roots and liverworts slices weredried in 1.5ml tubes and kept on silica gel for DNAisolation.

Processing of fungal DNA sequences

DNA was isolated from the dried mycorrhizalsamples using the DNAeasy Plant Mini Kit (Qiagen,Hilden, Germany). One–four cups per plant individualwere processed, the numbers of cups yielding sequencesare given in Appendix A, Supplementary Table 1. Partof the nuclear rDNA coding for the small subunit rRNA(nucSSU) of the arbuscular fungi of the tree mycor-rhizas, and part of the large subunit rRNA (nucLSU) ofthe putative Sebacinales and Tulasnellales in orchids,ericads and liverworts were amplified by the polymerasechain reaction (PCR). Primers and PCR design are givenin Appendix A, Supplementary Tables 2 and 3, thetargeted plants are given in Supplementary Table 1 (seeAppendix A, Supplementary Table 1). For detailedinformation see Appendix A: Processing of fungalsequences.


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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 mountain

rain forest of South Ecuador. Diverse trees as indicated by different leaves form mycorrhizas with Glomeromycota. The potential

fungal network as found on the basis of Glomus sequences is indicated by spores and hyphal connections in the humus layer. The

liana-like Cavendishia forms ectendomycorrhizas with Sebacinales, the epiphytic orchid Stelis forms endomycorrhizas with

Sebacinales and Tulasnellas, and the thalloid liverwort Aneura growing on a rotten stem forms mycorrhiza-like associations with

Tulasnellas. The mycorrhizal types of ericads (left), orchids (middle) and Aneuraceae (right) are displayed by sections in which

hyphae were stained by methyl blue. No common fungal guilds were found among the ericads and the epiphytic orchids (suspected

for Sebacinales) and among the epiphytic orchids and the Aneuraceae (suspected for Tulasnellas).


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

Tulasnella mycobionts in orchids and liverworts

The phylogenetic analysis of the nucLSU sequences ofTulasnella yielded eight clades of epiphytic orchidmycobionts and nine clades of Aneura and Riccardia

mycobionts (Fig. 2). Tulasnella 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 similarities



Dof more than 99%; A. pinguis and Riccardia smaragdina

in 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 andcan 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.


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Fig. 2. Phylogenetic relationships of the detected Tulasnella 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 sequences

coding 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 cannot

be directly correlated to branch lengths in the tree, since highly diverse alignment regions were excluded for phylogenetic

reconstruction. The Tulasnella sequence from Aneura pinguis AY298949, marked with a diamond, is not from the study site.


UNCORRECTED PROOF

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from Cavendishia nobilis AY825047 ECU




from Ceratostema reginaldii DQ352049 ECU


from Gaultheria erecta DQ352045 ECU


from Disterigma alaternoides DQ352070 ECU

from Disterigma microphyllum DQ352066 ECU




from Gaultheria erecta DQ352068 ECU

from Sphyrospermum cordifolium DQ352061 ECU


from Psammisia guianensis DQ352046 ECU


from Stelis superbiens DQ358067 ECU


from Diogenesia cf. floribunda DQ352058 ECU

from Cavendishia bracteata DQ352051 ECU







from Semiramisia speciosa DQ352053 ECU



from Psammisia guianensis DQ352069 ECU





from Pleurothallis lilijae DQ358070 ECU



from Pleurothallis sp DQ358064 ECU

Sebacina vermifera from Eriochilus scaber AY505548 AUS

Sebacina vermifera from Microtis uniflora AY505554 AUS



from Stelis superbiensDQ358059 ECU





from Stelis hallii DQ358055 ECU




from Gaultheria shallon AF300777 CAN

from Gaultheria shallon AY112930 CAN


















from Cavendishia nobilisAY825059 ECU




from Ceratostema oellgaardii DQ352064 ECU


from Macleania benthamiana DQ352047 ECU







Sebacina vermifera from Cyrtostylis reniformis AF291366 AUS

Sebacina vermifera from Phyllanthus calycinus AY505552 AUS

Sebacina vermifera from Eriochilus scaber AY505549 AUS

Sebacina vermifera from Microtis uniflora AY505555 AUS

from Lophozia incisa AY298847 SPA

from Calypogeia muelleriana AY298948 FRA

from Lophozia sudetica AY298946 SPA



Multinucleate Rhizoctonia AY505556 AUS

Piriformospora indica AY505557 IND

Sebacina vermifera from Caladenia catenata AY505553 AUS

Sebacina vermifera from Caladenia catenata AY505550 AUS

Sebacina vermifera from Glossodia minor AY505551 AUS

from Sebacina vermifera DQ352060 ECU

Sebacina epigaea AF291267 GER

Sebacina incrustans AF291365 GER

Tremellodendron pallidum AF384862 CAN

Efibulobasidium rolleyi AF291317 CAN

Craterocolla cerasi AY505542 GER

Sebacina allantoidea AF291367 GER

0.005 substitutions/site

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Fig. 3. Phylogenetic relationships of the detected Sebacinales mycobionts of ericads (dotted line) and pleurothallid orchids (clades

1–6) in the tropical mountain rain forest area of South Ecuador. BIONJ analysis of an alignment of nuclear DNA sequences coding

for 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 designate

BIONJ 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.


UNCORRECTED PROOF

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Fig. 4. Phylogenetic relationships of the glomeralean mycobionts of 38 arbuscular mycorrhizal trees in the tropical mountain rain

forest: BIONJ analysis of an alignment of nuclear DNA sequences coding for the small ribosomal subunit (nucSSU; 1108

characters). The tree was rooted with seven sequences of the Gigasporaceae. Glomeralean sequences which clustered together and

showed sequence similarities higher than 99% were regarded as one sequence type. Numbers on branches designate BIONJ

bootstrap values (only values exceeding 50% are shown). Nineteen sequence types which were found at different trees were

numbered continuously, sequence types found at only one tree are indicated by black circles.


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Table 1. Occurrence of 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 species

Tabebuia chrysantha 1 A x 1

Tabebuia chrysantha 2 A 1 1

Clethra revoluta F x x 2

Clusia elliptica F x 1 1

Vismia tomentosa F x x x 3

Hyeronima asperifolia F x 1 2

Hyeronima moritziana F x x 1 3

Hyeronima oblonga F x 1

Hyeronima sp. F x 1 2

Juglans neotropica 1 A x x 2

Juglans neotropica 2 A x x 1 3

Nectandra laevis 1 F x x 1 3

Nectandra laevis 2 F x x 1 3

Graffenrieda emarginata 1 F x x 1 3

Graffenrieda emarginata 2 F x x 2

Graffenrieda emarginata 3 F x x 2

Cedrela montana 1 N x 1

Cedrela montana 2 A x x 1 3

Cedrela montana 3 A x x 2

Cedrela sp. F x x 2

Guarea kunthiana F x x 2

Guarea cf. kunthiana F x x x 3

Guarea pterorhachis F 2 2

Guarea sp. F x x 1 3

Inga acreana 1 F x 1




Podocarpus oleifolius F x 1 2

Cinchona officinalis N x 1

Heliocarpus americanus 1 N x 1

Heliocarpus americanus 2 A x 1

Heliocarpus americanus 3 A x 1 2

Heliocarpus americanus 4 F x x 2

Occurrence 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 F

F

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.


UNCOThe Sebacinales sequences from pleurothallid orchids

were 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 withGaultheria erecta collected from the road side (Fig. 3,marked by asterisks), Psammisia guianensis shared asequence with Disterigma microphyllum (Fig. 3, marked



by 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 primerGLOM1310rc revealed 33 sequence types of arbuscularmycorrhizal fungi in the 34 trees that belonged to 21species out of 12 families (see Appendix A, Supplemen-tary Table 1). Thirty-two sequence types belonged toGlomus group A, one sequence type belonged to Glomus


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group B as defined by Schußler, Schwarzott, and Walker(2001) (Fig. 4, numbered lines and points). Eighteensequence types occurred at least with two and at mostwith four tree species. Fifteen sequence types were foundin one tree individual only (Fig. 4, points). One–threesequence types were found with one tree individual.Thirty-two tree individuals shared fungal sequence typeswith other trees, two tree individuals (Tabebuia chry-

santha 2 A., Guarea pterorhachis F.) had no commonfungal sequence type with another tree. Differentindividuals of the same tree species showed differentfungal sequence types in most cases (Table 1). In caseswere common fungal sequence types occurred in twotree 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-specific fungal sequence types were found. Foursequence types could be linked with sequences ofidentified morphospecies: sequence type 1 with Glomus

intraradices, type 2 with Glomus vesiculiferum, type 5with Glomus proliferum and type 19 with Glomus

mosseae (Fig. 4).
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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 andTulasnella 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 the



ED PROOF

genus Tulasnella, because these were either the onlyassociated fungal guilds (Aneuraceae; M. Preußingunpublished) or the dominating fungi (trees, Ericaceae,Orchidaceae; Setaro et al., 2006a, b; Suarez et al., 2006,I. Haug unpublished). Secondly, problems resulted fromthe observation that the ribosomal genes can showintraspecific variation especially in case of the Glomer-omycota (Lloyd-Macgilp et al., 1996; Sanders, 2004b;Sanders, Alt, Groppe, Boller, & Wiemken, 1995). Thesefacts pose general, unresolved challenges to a speciesconcept based on meaningful levels of genetic diversity(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 nucSSUhas been 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 basis of a new classification system ofGlomeromycota based on sequences assigned to speciesby spore morphology (Schußler et al., 2001). Ourphylogenetic analysis including these spore-based se-quences from GenBank-detected groups where inter-and intraspecific differences were overlapping(AY635833 Glomus mosseae/U96139 Glomus mosseae

proportion of differing sites 2/1025 ¼ 0.2%; Y17653 G.

caledonium/AJ276085 G. fragilistratum 2/1079 ¼ 0.2%).Nearly every sequenced clonal insert showed at least0.2% differences to other clonal inserts of the same PCRproduct (data not shown). We decided to unitesequences with differences o1% into one sequence typeand evaluated diversity and host preferences of theGlomus mycobionts at this level. Several authorsregarded clustering with a high bootstrap value andsequence differences o2.5% to define sequence types(Helgason et al., 2002; Husband et al., 2002; Vanden-koornhuyse et al., 2002). We chose a lower sequencedifference level in order not to overestimate the numberof fungal species shared by different plants. It cannot beexcluded that even within these sequence types severalspecies were joined together, which would mean that westill overestimated the number of shared fungi. ManyAM fungi that are clearly separated by morphology ande.g. ITS sequences cannot be separated on the SSU base(Schußler pers. communication). We found that 55% ofthe sequence types were shared by different tree speciesof distinct families. Nearly all the investigated treesshared a sequence type with another tree. Seven treespecies were represented by more than one individual,but no species-specific sequence type was recognized.Many sequence types were only isolated once, butfurther investigations may show a wider distribution ofthese sequence types resulting in a broader range ofshared species. Our conclusion that a functional net-


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work might potentially be formed between the highlydiverse trees in the tropical mountain forest appears sofar well supported. The conclusion is in generalagreement with the conceptual model of AM fungalgrassland communities (Johnson et al., 2005 andliterature therein) which considers the possibility that asmall number of host-specific arbuscular mycorrhizalspecies occur alongside 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,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 Tulasnella sequences were shared among theepiphytic orchids and the liverworts from rotten woodor rocks, but identical Tulasnella sequences were foundamong different orchid species and among the Aneur-aceae. Thus, common guilds of Basidiomycota likelyonly exist among plants of the same family. None of thesequences detected in this study were identical or near-identical to a sequence obtained from terrestrial orchids,however, only sequences obtained from mycobionts ofterrestrial orchids from Australia or the northernhemisphere were available for this study. Further studiesshould investigate the mycobionts of terrestrial orchidsin the study site to examine possible linkages withericads 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.



D PROOF

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.

References

Alexander, I. J., & Lee, S. S. (2005). Mycorrhizas and

ecosystem processes in tropical rain forest: Implications

for diversity. In D. F. R. P. Burslem, M. A. Pinard, & S. E.

Hartley (Eds.), Biotic interactions in the tropics: Their role in

the maintenance of species diversity (pp. 165–203). Cam-

bridge: Cambridge University Press.

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,

Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and

PSI-Blast: A new generation of protein database search

programs. Nucleic Acids Research, 25, 3389–3402.

Beck, A., Kottke, I., & Oberwinkler, F. (2005). Two members

of the Glomeromycota form distinct ectendomycorhizas

with Alzatea verticillata, a prominent tree in the mountain

rain forest of southern Ecuador. Mycological Progress, 4,

11–22.

Berch, S. M., Allen, T. R., & Berbee, M. L. (2002). Molecular

detection, community structure and phylogeny of ericoid

mycorrhizal fungi. Plant and Soil, 244, 55–66.

Bidartondo, M. I., Bruns, T. D., Weiß, M., Sergio, C., & Read,

D. (2003). Specialized cheating of the ectomycorrhizal

symbiosis by an epiparasitic liverwort. Proceedings of the

Royal Society of London, Series B – Biological Sciences,

270, 835–842.

Bidartondo, M. I., Burghardt, B., Gebauer, G., Bruns, T. D.,

& Read, D. J. (2004). Changing partners in the dark:

Isotopic and molecular evidence of ectomycorrhizal liaisons

between forest orchids and trees. Proceedings of the Royal

Society of London, Series B – Biological Sciences, 271,

1799–1806.

Francis, R., & Read, D. J. (1994). The contribution of

mycorrhizal fungi to the determination of plant community

structure. Plant and Soil, 159, 11–25.

Grime, J. P., Mackey, J. M., Hillier, S. H., & Read, D. J.

(1987). Floristic diversity in a model system using experi-

mental microcosms. Nature, 328, 420–422.

Hartnett, D. C., & Wilson, G. W. (1999). Mycorrhizae

influence plant community structure and diversity in

tallgrass prairie. Ecology, 80, 1187–1195.

Haug, I., Lempe, J., Homeier, J., Weiß, M., Setaro, S.,

Oberwinkler, F., et al. (2004). Graffenrieda emarginata

(Melastomateceae) forms mycorrhizas with Glomeromyco-

ta and with a member of the Hymenoscyphus ericae

aggregate in the organic soil of a neotropical mountain

rain forest. Canadian Journal of Botany, 82, 340–356.

Haug, I., Weiß, M., Homeier, J., Oberwinkler, F., & Kottke, I.

(2005). Russulaceae and Thelephoraceae form ectomycor-

rhizas with members of the Nyctaginaceae (Caryophyllales)

in the tropical mountain rain forest of southern Ecuador.

New Phytologist, 165, 923–936.

Helgason, T., Daniell, T. J., Husband, R., Fitter, A. H., &

Young, J. P. (1998). Ploughing up the wood-wide web?

Nature, 394, 431.


.2007.03.007

dx.doi.org/10.1016/j.baae.2007.03.007

dx.doi.org/10.1016/j.baae.2007.03.007

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3

5

7

9

11

13

15

17

19

21

23

25

27

29

31

33

35

37

39

41

43

45

47

49

51

53

55

57

59

61

63

65

67

69

71

73

75

77

79

81

83

85

87

89

91

93

95

97

99

101

103

105

107

ARTICLE IN PRESS

BAAE : 50160


UNCORRECT

Helgason, T., Merryweather, J. W., Denison, J., Wilson, P.,

Young, J. P., & Fitter, A. H. (2002). Selectivity and

functional diversity in arbuscular mycorrhizas of co-

occurring fungi and plants from a temperate deciduous

woodland. Journal of Ecology, 90, 371–384.

Homeier, J., Dalitz, H., & Breckle, S. W. (2002). Waldstruktur

und Baumdiversitat im montanen Regenwald der Estacion

Cientıfica San Francisco in Sudecuador. Berichte der

Reinhessischen Tuxen-Gesellschaft, 14, 109–118.

Husband, R., Herre, E. A., Turner, S. L., Gallery, R., &

Young, J. P. (2002). Molecular diversity of arbuscular

mycorrhizal fungi and patterns of host association over

time and space in a tropical forest. Molecular Ecology, 11,

2669–2678.

Johnson, D., lJdo, M., Genney, D. R., Anderson, I. C., &

Alexander, I. J. (2005). How do plants regulate the

function, community structure, and diversity of mycor-

rhizal fungi? Journal of Experimental Botany, 56,

1751–1760.

Kottke, I. (2002). Mycorrhizae–Rhizosphere determinants of

plant communities. In Y. Waisel, A. Eshel, & U. Kafkafi

(Eds.), Plant Roots: The Hidden Half (3rd ed., pp. 919–932).

New York, Basel: Marcel Dekker, Inc.

Kottke, I., Beck, A., Oberwinkler, F., Homeier, J., & Neill, D.

(2004). Arbuscular endomycorrhizas are dominant in the

organic soil of a neotropical montane cloud forest. Journal

of Tropical Ecology, 20, 125–129.

Lloyd-Macgilp, S. A., Chambers, S. M., Dodd, J. C., Fitter, A.

H., Walker, C., & Young, J. P. (1996). Diversity of the

ribosomal internal transcribed spacer within and among

isolates of Glomus mosseae and related mycorrhizal fungi.

New Phytologist, 133, 103–111.

Parolly, G., Kurschner, H., Schafer-Verwimp, A., & Grad-

stein, S. R. (2004). Cryptogams of the Reserva Biologica

San Francisco (Province Zamora-Chinchipe, Southern

Ecuador) III. Bryophytes – Additions and new species.

Cryptogamie, Bryologie, 25, 271–289.

Read, D. (1998). Plants on the web. Nature, 396, 22–23.

Russel, J., & Bulman, S. (2004). The liverwort Marchantia

foliaceae forms a specialized symbiosis with arbuscular

fungi in the genus Glomus. New Phytologist, 165, 567–579.

Saito, K., Suyama, Y., Sato, S., & Sugawara, K. (2004).

Defoliation effects on the community structure of arbus-

cular mycorrhizal fungi based on 18S rDNA sequences.

Mycorrhiza, 14, 363–373.

Sanders, I. R. (2004a). Plant and arbuscular mycorrhizal

fungal diversity – Are we looking at the relevant levels of

diversity and are we using the right techniques? New

Phytologist, 164, 415–418.

Sanders, I. R. (2004b). Intraspecific genetic variation in

arbuscular mycorrhizal fungi and its consequences for

molecular biology, ecology, and development of inoculum.

Canadian Journal of Botany, 82, 1057–1062.

Sanders, I. R., Alt, M., Groppe, K., Boller, T., & Wiemken, A.

(1995). Identification of ribosomal DNA polymorphisms

among and within spores of the Glomales: Application to

studies on the genetic diversity of arbuscular mycorrhizal

fungal communities. New Phytologist, 130, 419–427.



ED PROOF

Scheublin, T. R., Ridgway, K. P., Young, J. P. W., & van der

Heijden, M. G. A. (2004). Nonlegumes, legumes, and root

nodules harbor different arbuscular mycorrhizal fungal

communities. Applied and Environmental Microbiology, 70,

6240–6246.

Schußler, A., Schwarzott, D., & Walker, C. (2001). A new

fungal phylum, the Glomeromycota: Phylogeny and evolu-

tion. Mycological Research, 105, 1413–1421.

Setaro, S., Oberwinkler, F., & Kottke, I. (2006a) Anatomy and

ultrastructure of mycorrhizal associations of neotropical

Ericaceae. Mycological Progress DOI:10.1007/s11557-006-

0516-7.

Setaro, S., Weiß, M., Oberwinkler, F., & Kottke, I. (2006b).

Sebacinales form ectendomycorrhizas with Cavendishia

nobilis, a member of the Andean clade of Ericaceae, in

the mountain rain forest of southern Ecuador. New


Simard, S. W., & Durall, D. M. (2004). Mycorrhizal networks:

A review of their extent, function, and importance.

Canadian Journal of Botany, 82, 1140–1165.

Smith, S. E., & Read, D. J. (1997). Mycorrhizal Symbiosis (2nd

ed.). San Diego, London: Academic Press.

Suarez, J. P., Weiß, M., Abele, A., Garnica, S., Oberwinkler,

F., & Kottke, I. (2006). Diverse tulasnelloid fungi form

mycorrhizas with epiphytic orchids in an Andean cloud

forest. Mycological Research, 110, 1257–1270.

Van der Hammen, T. (1989). History of the montane forests of

the northern Andes. Plant Systematics and Evolution, 162,

109–114.

Van der Heijden, M. G., Klironomos, J. N., Ursic, M.,

Moutoglis, P., Streitwolf-Engel, R., Boller, T., et al. (1998).

Mycorrhizal fungal diversity determines plant biodiversity,

ecosystem variability and productivity. Nature, 396, 69–72.

Van der Heijden, M. G., Wiemken, A., & Sanders, I. R. (2003).

Different arbuscular mycorrhizal fungi alter coexistence

and resource distribution between co-occurring plants. New


Vandenkoornhuyse, P., Husband, R., Daniell, T. J., Watson, I.

J., Duck, J. M., Fitter, A. F., et al. (2002). Arbuscular

mycorrhizal community composition associated with two

plant species in a grassland ecosystem. Molecular Ecology,

11, 1555–1564.

Warcup, J. H. (1988). Mycorrhizal associations of isolates of

Sebacina vermifera. New Phytologist, 110, 227–231.

Warcup, J. H., & Talbot, P. H. (1967). Perfect states of

Rhizoctonias associated with orchids. New Phytologist, 66,

631–641.

Weiß, M., Selosse, M.-A., Rexer, K.-H., Urban, A., &

Oberwinkler, F. (2004). Sebacinales: A hitherto overlooked

cosm of heterobasidiomycetes with a broad mycorrhizal

potential. Mycological Research, 108, 1003–1010.

Wubet, T., Weiß, M., Kottke, I., Teketay, D., & Oberwinkler,

F. (2006). Phylogenetic analysis of nuclear small subunit

rDNA sequences suggests that the endangered African

Pencil Cedar, Juniperus procera Hochst. ex Endl., is

associated with distinct members of Glomeraceae. Mycolo-

gical Research, 110, 1059–1069.

109


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Education

Guilds Of Mycorrhizal Fungi And Their Relation To Trees Ericads Orchids And Liverworts In A Neotropical Mountain Rain Forest