Sebacinales are common mycorrhizal associates of Blackwell ... · (Taylor et al., 2003), as well as of some photosynthetic orchids related to N. nidus-avis that are partly heterotrophic

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Blackwell Publishing Ltd

Sebacinales are common mycorrhizal associates of

Ericaceae

Marc-André Selosse

1

*, Sabrina Setaro

2

*, Florent Glatard

1

*, Franck Richard

1

, Carlos Urcelay

3

and Michael Weiß

2

1

Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UMR 5175), Equipe coévolution, 1919 Route de Mende, 34293 Montpellier cedex 5, France;

2

Botanisches Institut, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany;

3

Instituto Multidisciplinario de Biologia Vegetal and

FCEFyN, Universidad Nacional de Cordoba, CONICET, CC 495, 5000 Cordoba, Argentina; *These authors contributed equally to this work.

Summary

• Previous reports of sequences of Sebacinales (basal Hymenomycetes) from ericoidmycorrhizas raised the question as to whether Sebacinales are common mycorrhizalassociates of Ericaceae, which are usually considered to associate with ascomycetes.• Here, we sampled 239 mycorrhizas from 36 ericoid mycorrhizal species across theworld (Vaccinioideae and Ericoideae) and 361 mycorrhizas from four species of basalEricaceae lineages (Arbutoideae and Monotropoideae) that do not form ericoidmycorrhizas, but ectendomycorrhizas. Sebacinales were detected using sebacinoid-specific primers for nuclear 28S ribosomal DNA, and some samples were investigatedby transmission electron microscopy (TEM).• Diverging Sebacinales sequences were recovered from 76 ericoid mycorrhizas,all belonging to Sebacinales clade B. Indeed, some intracellular hyphal coils hadultrastructural TEM features expected for Sebacinales, and occurred in living cells.Sebacinales belonging to clade A were found on 13 investigated roots of the basalEricaceae, and TEM revealed typical ectendomycorrhizal structures.• Basal Ericaceae lineages thus form ectendomycorrhizas with clade A Sebacinales,a clade that also harbours ectomycorrhizal fungi. This further supports the propo-sition that Ericaceae ectendomycorrhizas involve ectomycorrhizal fungal taxa.When ericoid mycorrhizas evolved secondarily in Ericaceae, a shift of mycobiontsoccurred to ascomycetes and clade B Sebacinales, hitherto not described as ericoidmycorrhizal fungi.

Key words:

ectendomycorrhizas, Ericaceae evolution, ericoid mycorrhizas, molecularecology, Sebacinales.

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© The Authors (2007). Journal compilation ©

New Phytologist

(2007)

doi

: 10.1111/j.1469-8137.2007.02064.x

Author for correspondence:

Marc-André Selosse Tel:

+

33 467 61 32 31 Fax:

+

33 467 41 21 38 Email: [email protected]

Received:

8 December 2006

Accepted:

11 February 2007

Introduction

Recent works on mycorrhizal communities have stimulatedconsiderable interest in a neglected group of fungi related tothe genus

Sebacina

, recently raised to the order Sebacinales(Weiß

et al

., 2004). This basal order of Hymenomycetes(Basidiomycetes) encompasses fungi with longitudinally septatebasidia and imperforate parenthesomes (i.e. the derivates ofthe endoplasmic reticulum covering septal pores and allowingcommunication between cells). They also lack cystidia andstructures formed during cytokinesis on some basidiomycetous

hyphae, the so-called clamp connections. Cultivable speciesexhibit monilioid hyphae (i.e. hyphal cells that look like pearlsin a chain). This is why sebacinoids with no known sexualstage were placed in the polyphyletic form genus

Rhizoctonia

.Although some sebacinoids are described as valid species,

and some can grow in pure culture, most of our knowledge onSebacinales and their diverse host species comes from molec-ular ecology studies during the last 4 yr, that is, from directamplification of fungal ribosomal DNA (rDNA) of environ-mental samples. Phylogenetic analysis, using sequences fromcultures, fruitbodies or environmental samples, revealed that


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Sebacinales are divided into two clades, A and B, that differ intheir ecology (Weiß

et al

., 2004). The first clade A sequenceswere derived from fruitbodies (Weiß & Oberwinkler, 2001),and subsequently from mycorrhizas of the achlorophyllousorchids

Neottia nidus-avis

(McKendrick

et al

., 2002; Selosse

et al

., 2002a) and

Hexalectris spicata

(Taylor

et al

., 2003), aswell as of some photosynthetic orchids related to

N. nidus-avis

that are partly heterotrophic (Selosse

et al

., 2004; Julou

et al

.,2005). At the same time, many clade A sebacinoids, includingthose from the orchids just mentioned, were demonstrated toform ectomycorrhizas on tree roots (Selosse

et al

., 2002a,b;Urban

et al

., 2003; Walker & Parrent, 2004; Moyersoen,2006). They are among the most common ectomycorrhizalspecies in temperate and Mediterranean forests (Glen

et al

.,2002; Avis

et al

., 2003; Kennedy

et al

., 2003; Walker

et al

.,2004; Richard

et al

., 2005; Tedersoo

et al

., 2006). Up to now,fruitbodies are only known from clade A sebacinoids. CladeB displays a larger array of associations: some species aremycorrhizal on green, autotrophic orchids (Warcup, 1988;Bougoure

et al

., 2005), and others associate with liverwortthalli (Kottke

et al

., 2003). Clade B also contains the non-specific root endophyte

Piriformospora indica

(Verma

et al

.,1998, 2001; Pe

S

kan-Berghöfer

et al

., 2004). Recently, cladeB rDNA sequences were amplified from roots of severalEricaceae (Berch

et al

., 2002; Allen

et al

., 2003; Bougoure &Cairney, 2005; Setaro

et al

., 2006a), raising the intriguing pos-sibility that sebacinoids could be overlooked mycorrhizalfungi of these plants.

Ericaceae form a large, ecologically relevant family of treesand shrubs growing all over the world (Kron

et al

., 2002a),especially at high latitude and high elevation stands. MostEricaceae have a particular mycorrhizal association adaptingthem to the nutrient-poor, acidic soils of such areas (Smith &Read, 1997). In the so-called ericoid mycorrhiza (ERM),fungi colonize fine roots called ‘hair roots’, lacking corticalparenchyma, and form hyphal coils in large epidermal cells.Most cells are independently colonized from the soil (Bergero

et al

., 2000), with some cell-to-cell hyphal connections(Massicotte

et al

., 2005), so that each hair root harbours sev-eral fungi (Perotto

et al

., 1996). All ERM fungi reported so farare ascomycetes (Smith & Read, 1997; McLean

et al

., 1999;Cairney & Ashford, 2002; Midgley

et al

., 2004). However,the cloning of fungal rDNA sequences amplified from ERMroots suggested that sebacinoids colonized roots of

Gaultheriashallon

(Berch

et al

., 2002; Allen

et al

., 2003) and

Epacris pulchella

(Bougoure & Cairney, 2005). These sebacinoids are likely tohave been ignored by classical approaches, as: (i) clamplesshyphae of sebacinoids cannot be distinguished from ascomyc-etous hyphae by light microscopy; and (ii)

in vitro

isolationfailed to reveal them because they have been difficult to getinto culture up until now (Berch

et al

., 2002). However,electron microscope investigations (Bonfante-Fasolo, 1980;Peterson

et al

., 1980; Allen

et al

., 1989; Setaro

et al

., 2006b)have sometimes revealed that basidiomycetes with imperforate

parenthesomes, the type present in Sebacinales, may colonizeliving roots of ERM Ericaceae. Recently, an unusual mycor-rhizal association with clade B sebacinoids was reported fromthe neotropical

Cavendishia nobilis

(Ericaceae), a member ofthe Vaccinioideae (Kron

et al

., 2002a,b), with both intracellularcolonization, as in ERM, and growth between and aroundthe cortical parenchyma cells (cavendishioid mycorrhizas;Setaro

et al

., 2006a). These data raise three questions: (i) aresebacinoid partners common in other ERM species, especiallyfrom the large ERM clade Ericoideae; (ii) if so, do all theseERM sebacinoids belong to clade B, as suggested by availablesequences (Weiß

et al

., 2004; Setaro

et al

., 2006a); and (iii) whatare the ultrastructural features of sebacinoid infection in hairroots, as compared with those of ERM ascomycetes?

ERM association is thought to have arisen once duringEricaceae evolution (Cullings, 1996; Kron

et al

., 2002a), andbasal subfamilies (Pyroleae and Arbutoideae) display totallydifferent mycorrhizas. They form ectendomycorrhizas (EEM),where fungi colonize both the cortical cells and the root surface,forming a sheath, an intercellular network between corticalcells, and intracellular structures that depend on the host taxon(Smith & Read, 1997). Here, a single fungal individual colonizeseach root tip in most cases. Ascomycetes and/or basidiomycetesare present on EEM roots in Pyroleae (Robertson & Robertson,1985; Bidartondo, 2005; Tedersoo

et al

., 2007) and Arbutoideae(Giovannetti & Lioi, 1990; Münzenberger

et al

., 1992;Richard

et al

., 2005). These fungi belong to species thatusually form ectomycorrhizas on tree roots. Sebacinoids werereported to occur on EEM roots of

Arbutus unedo

(Arbutoideae;Richard

et al.

, 2005) and

Orthilia secunda

(Pyroleae; Tedersoo

et al.

, 2007), again raising three questions: (i) do sebacinoidsoften colonize EEM Ericaceae; (ii) which Sebacinalesclade(s) are EEM plants associated with; and (iii) what are theultrastructural features of their infection?

To answer these questions on identity, diversity and struc-tural interactions of sebacinoids associated with Ericaceae, wecollected ERM and EEM roots, using worldwide sampling forERM roots. First, detection of sebacinoids was achieved usingspecific PCR primers. Second, the sequences obtained wereused to find their phylogenetic position. Third, microscopeinvestigations allowed characterization of the ultrastructuralfeatures, and thus the mycorrhizal status, of these sebacinoids.

Materials and Methods

Root sampling

Ericoid mycorrhiza roots were sampled from various sites inEurope, La Réunion Island (Indian Ocean), and North andSouth America between 2002 and 2004 (Table 1; Supple-mentary Material, Table S1). Freshly harvested ERM rootswere checked for connection to aerial plant parts to ensurehost identity. They were washed carefully under a dissectionmicroscope in order to remove soil and organic matter particles

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Table 1 A summary of ericoid mycorrhizal roots of Ericaceae (Vaccinioideae and Ericoideae) investigated and successful PCR amplification and sequencing of a sebacinoid ribosomal DNA sequence, using primers ITS3Seb and TW13 (for more details and GenBank accession numbers, see Supplementary Material)

TaxaRegion of origina

No. of samples investigated

No. of successful amplificationsb

No. of sequences recoveredb

Vaccinioideae 115 64 (55.6%) 45 (39.1%)Agauria buxi Re (1) 3 0 0Agauria salicifolia Re (1) 5 4 3 (all identical)Andromeda glauca Ca (1) 2 1 1Andromeda polifolia Eu (2) 8 7 5c

Chamaedaphne calyculata Ca (2) 4 0 0Chiogenes hispidula Ca (2) 7 4 3Gaultheria poeppigii Ar (3) 22 13 8 (5 identical)Gaultheria procumbens Ca (3) 6 1 1Gaultheria sp. Ar (1) 2 1 0Vaccinium angustifolia Ca (3) 5 1 1Vaccinium macrocarpum Ca (1) 2 2 2Vaccinium myrtilloides Ca (2) 4 0 0Vaccinium myrtillus Eu (12) 20 10 7Vaccinium oxycoccos Ca (2), Eu (1) 6 4 2Vaccinium uliginosum Eu (7) 11 10 7Vaccinium vitis-idaea Eu (2) 8 6 5c

Ericoideae 124 55 (44.3%) 31 (25.0%)Calluna vulgaris Eu (19) 34 20 14Empetrum nigrum Eu (3) 6 3 3Erica arborea Eu (3) 8 2 2Erica ciliaris Eu (1) 1 1 1c

Erica cinerea Eu (9) 15 7 4Erica gallioides Re (3) 7 1 0Erica multiflora Eu (2) 8 6 2Erica reunionensis Re (4) 7 3 1Erica vagans Eu (2) 3 1 1Kalmia angustifolia Ca (2) 5 1 0Kalmia polifolia Ca (2) 5 0 0Ledum palustre Ca (1) 3 3 1Loiseleuria procumbens Eu (1) 2 0 0Rhododendron canadensis Ca (2) 3 1 0Rhododendron conicum Eu (1) 2 0 0Rhododendron decorum Eu (1) 2 1 0Rhododendron ferrugineum Eu (1) 4 3 1c

Rhododendron fortunei Eu (1) 2 1 0Rhododendron groenlandicum Ca (3) 5 1 1Rhododendron japonicum Eu (1) 2 0 0

Subtotal for each global regiond

Europe 140 81 (57.8%)a 54Canada 53 16 (30.2%)b 10Argentina 24 14 (58.3%)a 8Réunion Island 22 8 (40.9%)a b 4Total 239 119 (49.8%) 76c,e (31.8%)

aAr, Argentina; Ca, Canada; Eu, Europe; Re, La Réunion. Brackets contain the number of independent sampled sites (i.e. areas of < 10 m2 separated by > 1 km from the other sites).bPercentages in brackets are related to the total number of samples investigated.cFive sequences were obtained as a consensus of at least seven clones obtained by cloning the PCR product (namely, EF030919 and EF030867 on Andromeda polifolia, EF030944 on Erica ciliaris, EF030936 on Rhododendron ferrugineum and EF030875 on Vaccinium vitis-idaea).dValues followed by different letters differ according to a chi-squared test (P < 0.001).eGiven that some sequences were found on different plants (for Agauria salicifolia and Gaultheria poeppigii), this means a total of 70 different sequences.

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as well as old and nonmycorrhizal roots. Then a 0.1–0.5 g(fresh weight) subsample, containing several hair roots, wasfrozen at −80°C, either directly or after storage in 60%ethanol in water (v/v) for < 5 d before transport. The samplesfrom Argentina were oven-dried at 60°C for 72 h beforetransport, and stored in silicagel until DNA extraction. Samplesfrom Calluna vulgaris and Erica cinerea from Lanno in Belle-Isleen Mer (France, one sampling of a cluster of co-occurringroots for each species) and Gaultheria poeppigii from Pampade Achala (Argentina, two samplings of a root cluster; seeTable S1 for locations) were prepared for transmission electronmicroscopy (TEM) in a fixating buffer (2% glutaraldehyde in0.1 M phosphate buffer (0.2 M KH2PO4/Na2HPO4), pH = 7.2)at 4°C.

For EEM roots, each sample was stored separately at −80°C.For Arbutus unedo, mycorrhizal root tips were harvested in theFango forest (Corsica, France, Table 2). From two soil coressituated 10 m apart, four and three tips, respectively, wereselected that fitted the sebacinoid morphotype described fromthe same site by Richard et al. (2005). This is a trichotomicmycorrhiza found in large aggregates, exclusively in theorganic soil layer. These mycorrhizas are highly clavate, haveno emanating hyphae or rhizomorphs, and are orange incolour and almost yellow at the tips. The seven selected tipswere cut in two parts: one was placed in TEM fixating bufferand the other was used for DNA extraction. Four additionalsequences from the Fango forest were obtained (GenBankaccession numbers EF030880, EF030929, EF030911 andEF030912) by further sequencing A. unedo EEM DNAsamples that produced sebacinoid internal transcribed spacer(ITS) sequences in a previous study (sebacinoid #1 to #4 inRichard et al., 2005). For the Pyroleae, EEM tips or root sec-tions that showed external hyphae were recovered from a 6 m2

patch of Orthilia secunda and a 4 m2 patch of Pyrola chlorantha

(100 EEM per species) at the Chauriat forest (Puy de Dôme,France, Table 2). A set of 49 mycorrhizal roots of Orthiliasecunda were collected from a 4 m2 patch at Abrahams Lake(Nova Scotia, Canada, Table 2). For Arctostaphylos uva-ursi,70 mycorrhizal roots were harvested from a 6 m2 patch atKukka bog reserve (Hiiumaa island, Estonia, Table 2) and 35other roots from several plants at Crêt de l’Oeillon (Monts duPilat, France, Table 2). The last four species were not sampledfor TEM analysis, as no description of sebacinoid morpho-types was available.

DNA extraction, PCR and sequencing

All roots were submitted to DNA extraction using theDNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France),according to the manufacturer’s instructions, and DNA wasrecovered in 40 µl of distilled water. Quality of DNA extractand fungal colonization were tested by PCR amplification ofthe fungal ITS, using a set of primers universal for fungi(ITS1F + ITS4) as in Selosse et al. (2002a). They were thensubmitted to amplification of a fragment of partial ITS plusthe 5′ part of the 28S rDNA (D1/D2 region), using theprimers ITS3Seb (5′-TGAGTGTCATTGTAATCTCAC-3′,internal to ITS and specific for Sebacinales, kindly providedby M. Berbee) and TW13 (5′-GGTCCGTGTTTCAAGACG-3′, universal for fungi and internal to 28S rDNA; White et al.,1990), using DNA from a sebacinoid fruitbody as a positivecontrol. PCR was carried out in 50 µl, with final con-centrations of 66 µM for each dNTP, 0.6 µM for each of theprimers (Laboratoires Eurobio, Les Ulis, France), 10 mM Tris-HCl,50 mM KCl, 1.5 mM MgCl2, 0.2 mg ml−1 gelatin, 0.1% (v/v)Triton X100, 5% (v/v) dimethyl sulphoxide and 1.5 units ofTaq DNA polymerase (Quantum Appligène, Illkirch, France).We used 1 µl of the extracted DNA solution, but higher

Table 2 A summary of sebacinoid sequences amplified from ectendomycorrhizal (EEM) Ericaceae using primers ITS3Seb and TW13

Species SiteNo. of EEM samples

No. of successful PCRs

Sequences (GB accession numbers)a

Arbutus unedob Fango forest, France 7 7b EF030913 (n = 7)(42°20′N; 8°49′E)

Arctoctaphylos uva-ursi Kukka bog, Estonia 70 0 –(58°14′N; 22°00′E)Crêt de l’Oeillon, France 35 0 –(45°24′N; 4°37′E)

Pyrola chlorantha Chauriat forest, France 100 1 EF030896c (n = 1)(45°46′N; 3°17′E)

Orthilia secunda Chauriat forest, France 100 4 EF030895c (n = 3)(45°46′N; 3°17′E) EF030946 (n = 1)Abrahams Lake, Canada 49 1 EF030894 (n = 1)(45°09′N; 62°36′W)

aWith number (n) of samples producing the sequence.bOnly sebacinoid morphotypes after Richard et al. (2005) were harvested and sequenced for A. unedo.cOne of the sequences obtained from O. secunda at the Chauriat forest (EF030895) was 100% identical to that retrieved from P. chlorantha (EF030896) at the same site.

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volumes (up to 10 µl) or dilutes (up to 10×) were tested whenamplification failed. Reactions were performed in a TRIO-Thermoblock (Biometra, Göttingen, Germany) under thefollowing thermoprofile: initial denaturation at 94°C for4 min, followed by 35 cycles of denaturation at 94°C for 30 s,annealing at 53°C for 30 s, and extension at 72°C for 30 s.After the 35th cycle, a terminal extension of 10 min at 72°Cwas carried out. PCR products were sequenced as in Selosseet al. (2002a). Five randomly chosen PCR products for whichno direct sequencing was possible were submitted to cloning(Table S1), as in Julou et al. (2005), and at least seven clones weresequenced for each of them. Sequences were edited, aligned usingSequencher 4.5 from Genes Codes (Ann Arbor, MI, USA), andconfirmed as sebacinoid sequences by BLAST analysis (Altschulet al., 1997) against GenBank (NCBI; www.ncbi.nlm.nih.gov).All sequences (including consensus of cloned sequences) weredeposited in GenBank (EF030867 to EF030946).

Transmission electron microscopy

For ERM, only the narrowest roots (hair roots) were used forinvestigation. About 20 hair roots were selected perglutaraldehyde-fixed sample and processed as in Setaro et al.(2006b) to obtain semithin sections (1 µm) and, when appro-priate, ultrathin sections (0.05 µm). For EEM tips, ultrathinsections were obtained directly, by the same procedure as inSelosse et al. (2002b).

Phylogenetic reconstruction

The nucLSU portions of the retrieved DNA sequences werealigned with representative reference sequences taken fromGenBank using MAFFT, version 5.850 (Katoh et al., 2002).To estimate phylogenetic relationships, the alignmentwas analysed using heuristic maximum likelihood (ML) asimplemented in the PHYML software, version 2.4.4 (Guindon& Gascuel, 2003), starting from a BIONJ tree (Gascuel,1997), with a general time-reversible model of nucleotidesubstitution and additionally assuming a percentage ofinvariant sites and gamma-distributed substitution rates atthe remaining sites (GTR + I + G). The gamma distributionwas approximated with four discrete rate categories. All modelparameters were estimated using ML. Branch support wasinferred from 1000 replicates of nonparametric bootstrapping(Felsenstein, 1985), with model parameters estimated via MLindividually for each bootstrapped alignment. Additionally,we performed a Bayesian Markov chain Monte Carlo (MCMC)analysis using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003).We ran two independent MCMC analyses, each involvingfour incrementally heated chains over five million generations,using the GTR + I + G model of nucleotide substitution andstarting from random trees. Model parameters were not fixedbut sampled during MCMC. Trees were sampled every 100generations, resulting in an overall sampling of 50 000 trees

per run, from which the first 20 000 trees of each run werediscarded (burn in). The remaining 30 000 trees sampledfrom each run were pooled and used to compute a majority ruleconsensus tree to get estimates for the posterior probabilities.Stationarity of the process was assessed using the Tracersoftware (Rambaut & Drummond, 2003).

Results

Sebacinoids from ERM samples

In all, 239 ERM root samples were recovered from 36 Ericaceaespecies (Table 1), that is, from 50 sites worldwide (Table S1).ITS amplification using the universal fungal primers ITS1Fand ITS4 produced multiple PCR fragments for all samples,demonstrating that several fungal species were present onthese roots (data not shown). The sebacinoid-specific primerset ITS3Seb and TW13 produced either a single 900 bpfragment (in 49.8% of the samples, i.e. in 29 out of the 36investigated species, Table 1), or no PCR product. Althoughvariable percentages of successful PCR amplification wereobtained, none of these differences was significant (usingpairwise chi-squared tests; P > 0.05, not shown) amongVaccinioideae and Ericoideae, nor among the most extensivelysampled species (V. myrtillus, C. vulgaris and E. cinerea), noreven among the most extensively sampled genera (Gaultheria,Vaccinium, Erica and Rhododendron). Only samples fromCanada yielded significantly fewer PCR products than thosefrom Europe and Argentina according to chi-squared tests(P < 0.001, Table 1).

Sequences were obtained by direct sequencing of the 900 bpfragment from 71 samples (i.e. 59.6% of samples producinga PCR product – Table S1). BLAST analysis confirmed that theywere closely related to sebacinoid sequences from GenBank.In some cases (e.g. EF030869, EF030888 or EF030899), twobases were present at some positions, suggesting that two closesequences were sequenced as a result of heterozygosities or thepresence of two closely related sebacinoids. All other PCRproducts yielded a mix of different sebacinoid sequences, sothat the conserved areas were the same but many polymorphicsites were detected in the variable regions (not shown). Five ofthese PCR products were cloned and sequenced (Table 1). Inall cases, a unique sequence (with the exception of a few PCRand cloning errors, not shown) was found among clones,suggesting that at least one sebacinoid was present in thesesamples. (Perhaps other minor sequences were present that werenot seen because only seven clones were sequenced). In all,sebacinoid sequences were obtained in 31.8% of the samples(Table 1) and no nonsebacinoid sequences were recovered.

All sequences differed by at least some base pairs, even forplants growing on the same site: as the only exceptions, iden-tical sequences were found in five co-occurring Gaultheriapoeppigii samples from Argentina (EF030889) and threeco-occurring Agauria salicifolia samples from La Réunion


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(EF030898, Table S1). As a result, 70 different sequences werefound. Up to five different sequences were found from the samesite, that is from < 10 m2 (Table S1), and different sequenceswere recovered from the same host at the same site (e.g. Vacciniummyrtillus, V. uliginosum or Calluna vulgaris produced two dif-ferent sebacinoid sequences at some sites; Table S1).

Light and transmission electron microscopy of fungal colonization in ERMs

Semithin sections of clusters of hair roots from C. vulgaris(n = 7), Erica cinerea (n = 2) and Gaultheria poeppigii (n = 8)displayed typical ERM colonization, with intracellular hyphalcolonization (Fig. 1). TEM revealed both ascomycetes andhymenomycetous basidiomycetes as associated fungi (Fig. 2),with ultrastructure clearly distinguishing them. Ascomyceteshad simple septal pores with Woronin bodies (Fig. 2b,c) andelectron-light cell walls (Fig. 2b,c,e), whereas basidiomyceteshad imperforate parenthesomes (i.e. the parenthesomal typealso present in Sebacinales; Fig. 2d,e) and electron-densehyphal cell walls (Fig. 2a,b,d,e). Sebacinales-like basidiomyceteswere found on E. cinerea (in n = 1 ultrathin section) and G.poeppigii (n = 2). Both asco- and basidiomycetes formedintracellular coils in living cortical cells of hair roots. Thevitality of the host cells was indicated by the mitochondria-rich cytoplasm (Fig. 2a,b,f ). Basidiomycetous colonization ofcortical cells was found to be exclusive (Fig. 2a) or dual, thatis, with both Sebacinales-like basidiomycetes and ascomycetesforming coils within a single host cell (in G. poeppigii, Fig. 2b,e).

Sebacinoids from EEM samples

The seven EEM root tips of A. unedo from the Fango forestshowing a sebacinoid morphotype harboured a unique

sebacinoid sequence (EF030913, Table 2). It differed fromthe four sequences already obtained by Richard et al. (2005)at this site, suggesting that sebacinoids are diverse in this forest.Two Pyroleae (O. secunda and P. chlorantha) from the Chauriatforest produced the same sequence (EF030895 = EF030896,Table 2), so that the related sebacinoid was probably nothost-specific. A second sebacinoid occurred in O. secunda atthe Chauriat. A single EEM O. secunda root from AbrahamsLake harboured a sebacinoid sequence that differed from theprevious ones (Table 2). No sequences were obtained fromA. uva-ursi at the two sites investigated (Table 2). Because ofthe low frequency of sebacinoid colonization on other hosts,this does not imply that A. uva-ursi is not a host (pairwisechi-squared tests not significant (P > 0.05), not shown).

Ultrastructure of fungal colonization in Arbutus unedo EEMs

Transmission electron microscopy performed on threerandomly selected EEM tips of Arbutus unedo (n = 3) fromthe Fango forest revealed typical arbutoid ectendomycorrhizaswith hyphal sheath, Hartig net and intracellular colonization(Fig. 3a,b). Although vitality of the plant cell is not visible,these structures indicate an ectomycorrhizal association.Peripheral hyphae showed thick cell walls (Fig. 3d), as describedfor ectomycorrhizal sebacinoids (Selosse et al., 2002b). Theassociated fungi have dolipores and imperforate parenthesomes,as is typical for Sebacinales (Fig. 3b,c).

Phylogeny of sebacinoids from Ericaceae

The results of our molecular phylogenetic analysis involving thenucLSU sequences from the present study and a comprehensiveset of reference sequences published in GenBank are shown in

Fig. 1 Semithin sections of ericoid mycorrhiza of Gaultheria poeppiggi. The cortical cells are well colonized by fungal hyphae. Bar, 10 µm.


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Fig. 2 Transmission electron micrographs of ericoid mycorrhiza (ERM) colonized by sebacinoids. (a) Cortical cell of an Erica cinerea hair root colonized by sebacinoid hyphae (s). Black arrowhead, a dolipore of an intracellular hypha; white arrowheads, mitochondria of the plant cell. Bar, 2 µm. (b) Cortical cell of an ERM of Gaultheria poeppigii with dual colonization of ascomycetes (a) and Sebacinales (s). The plant cell cytoplasm is alive, indicated by the nucleus (NU) and mitochondria (white arrowheads); the black arrowhead points to an ascomycetous septum with Woronin bodies. Bar, 2 µm. (c) Detail of the ascomycetous hyphae with septum and Woronin body (arrowhead) of panel (b); M, mitochondria; *, the electron-light hyphal wall. Bar, 1 µm. (d) Dolipore of a sebacinoid hypha forming ERM with G. poeppigii. Arrowheads point to imperforate parenthesomes; *, the electron-dense hyphal wall. Bar, 0.1 µm. (e) Sebacinoid hyphae (s) with dolipore (arrowhead) and imperforate parenthesomes forming dual colonization with ascomycetes (a) in a cortical cell of G. poeppigii. White and black asterisks mark the electron-light cell walls of the ascomycetes and the electron-dense cell walls of the sebacinoids, respectively. Bar, 2 µm. (f) Detail of mitochondria (M) in the cytoplasm of the plant cell dually colonized by Sebacinales and ascomycetes. Bar, 0.1 µm.


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Fig. 4. The outcome of the heuristic ML analysis and the twoindependent MCMC runs was widely consistent, with fewexceptions, the most prominent of which is the differentplacement of a sebacinoid detected in Vaccinium vitis-idaea(EF030882). In the ML tree, this sequence clustered, althoughwithout significant support, together with a similar sequencefrom Vaccinium vitis-idaea (EF030883) at the base of Sebacinalesgroup A (Fig. 4). In MCMC analysis, however, the sequenceEF030882 was placed within Sebacinales clade B with aposterior probability of 100%, while EF030883 was stillplaced at the base of clade A with a posterior probability of97% (not shown). Apart from these exceptions, both ML andMCMC analyses separated the Sebacinales into the knownclades A and B.

Consistent with earlier studies, group A also includes allsequences retrieved from fruitbodies, from ectomycorrhizal

samples and from heterotrophic orchids (Fig. 4). Sebacinoidsequences from EEMs (from Arbutus unedo, Orthilia secunda,Pyrola chlorantha) also appear in this group. All the sequencesobtained from ERMs, on the other hand, clustered withinSebacinales clade B, the clade that also includes all sequencesfrom cavendishioid mycorrhizas and liverwort thalli (Fig. 4).Clade B also contains the sequences of Sebacina vermiferaisolates obtained mostly from Australian green, autotrophicorchids (Warcup, 1988), as well as of Piriformospora indica.

Within clades A and B, the sequences were not strictlygrouped according to the mycorrhizal types from which theywere obtained. Furthermore, no larger biogeographical sub-group was resolved. For example, a well-supported subgroup(97% in ML bootstrap, 100% MCMC support) containssequences from Australia, Argentina, Ecuador, Canada, France,Germany, and Estonia.

Fig. 3 Transmission electron micrographs (TEMs) of ectendomycorrhiza (EEM) from Arbutus unedo and a sebacinoid (corresponding to sequence EF030880). (a) Mycorrhiza with formations of hyphal sheath (hs) and Hartig net (arrowheads) as well as hyphal growth within cortical cells (CC+). CC–, cortical cells without hyphal colonization. Bar, 4 µm. (b) Cortical cell (CC) with intracellular hyphae (ih) and Hartig net (Hn) by a sebacinoid; arrowhead, a dolipore. Bar, 1 µm. (c) Detail of dolipore with imperforate parenthesomes (arrowheads) of a sebacinoid hypha. (d) External sheath hyphae with thick walls (on the left; inner mantle on the right). Bar, 1 µm. (a–c, pictures from Robert Bauer; d, picture from Houria Horcine).


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Discussion

Sebacinoids as common mycorrhizal fungi in Ericaceae roots

Out of 600 samples of Ericaceae roots, a total of 89 produceda sebacinoid sequence (Tables 1, 2), representing 74 differentsequences (because of some identities). Among them, fivewere obtained after cloning, suggesting that the 48 PCRproducts from ERM roots that we were not able to sequencedirectly contain sebacinoid sequences (Table S1). Cross-contamination of samples seems unlikely, as most sequencesare different. Moreover, we provide evidence that hyphae withsebacinoid dolipores and parenthesomes can be detected byTEM analysis and that they occur in (or around) living cells,forming typical ERM (Fig. 2) or EEM (Fig. 3) on the roots ofat least three host species. This underlines the utility of com-bining molecular and TEM approaches, if possible on thesame sample, as already used to demonstrate that sebacinoidsform ectomycorrhizas (Selosse et al., 2002b) and cavendis-hioid mycorrhizas (Setaro et al., 2006a). In this study, the useof specific primers probably enhanced molecular detection.

Sebacinales form ERM-like interactions in hair roots ofEricaceae, whereas only ascomycetes were reported to beinvolved so far (Smith & Read, 1997). Although more speciesshould be investigated at the ultrastructural level, this is inaccordance with the molecular diversity of Sebacinales previ-ously found on hair roots of Vaccinioideae and Styphelioideaespecies from Canada (Gaultheria shallon, Berch et al., 2002; Allenet al., 2003), Australia (Epacris pulchella, Bougoure & Cairney,2005), and the Neotropics (Cavendishia nobilis, Setaro et al.,2006a). It extends this diversity to the Ericoideae tribe. Withthe exception of C. nobilis (Setaro et al., 2006a), and probablyCalluna vulgaris (Bonfante-Fasolo, 1980), we are not aware ofdirect observation of sebacinoids in ERM. Some speciesinvestigated in the present study failed to produce positivePCRs (Table 1). This could be the result of undersampling,as Sebacinales abundance might vary among species. OnG. shallon they represented more than half of cloned ITSsequences (Allen et al., 2003; accordingly 75% of roots didnot yield fungal isolates) compared with less than a quarter onE. pulchella (Bougoure & Cairney, 2005; 8% of roots did notyield fungal isolation). This suggested that Sebacinales colo-nized larger root portions on G. shallon than on E. pulchella.However, these percentages are not directly comparable, sinceisolation procedures were not identical.

Can one say that Sebacinales are ERM mycorrhizal? Ifmycorrhizal interaction is defined as a morphogenetic processuniting roots and soil fungi, a definition we favour and usehere, then we observed ERM mycorrhizas involving sebaci-noids. If the definition additionally implies a mutualisticrelationship, a feature sometimes violated in associations con-sidered as mycorrhizal (Kiers & van der Heijden, 2006) anddifficult to establish, then the question requires further inves-

tigation. Mutualism has been experimentally shown for rela-tives of the ERM sebacinoids in clade B. Piriformospora indicais beneficial for the growth of several plants (Rai et al., 2001;Varma et al., 2001; PeSkan-Berghöfer et al., 2004; Waller et al.,2005) and even induces systemic resistance to fungal diseasesand tolerance to salt stress in barley (Waller et al., 2005).Together with another clade B sebacinoid, it enhanced growthof Nicotiana tabacum, but at the expense of herbivore resistance(Barazani et al., 2005). Similar positive effects on plant growthhave also been shown for several isolates of the Sebacina vermiferaspecies complex (Deshmukh et al., 2006). Such beneficialeffects might thus be expected in the interaction betweengroup B sebacinoids and Ericaceae, but await further studies,for example using in vitro synthetic associations.

Evolution of mycorrhizal structures in the Sebacinales

Mycorrhizal types are not evenly distributed over thephylogeny of Sebacinales (Fig. 4), in agreement with previousinvestigations (Weiß et al., 2004). Clade B encompasses speciesthat grow intracellularly, in hepatics (Kottke et al., 2003),ERM roots (our data), orchid roots (Warcup, 1988), andmany other hosts for P. indica (the model species Arabidopsisthaliana (PeSkan-Berghöfer et al., 2004) and Nicotianatabacum (Barazani et al., 2005), but also species in Fabaceaeand Rhamnaceae (Varma et al., 2001), Asteraceae and Solanaceae(Rai et al., 2001) as well as Poaceae (Waller et al., 2005)).However, the interaction is somewhat different at the cellularlevel for P. indica, which was shown to enhance apoptosis ofplant cells, and colonize dead cells instead of living cells asobserved here (Deshmukh et al., 2006). Several types ofinteraction can therefore be expected at the structural level inclade B. Some well-supported terminal clades within group Bdo not at present contain sequences from different host types(Fig. 4), but this might well be the result of an undersamplingof Sebacinales diversity. Within clade B, species involved incavendishioid mycorrhizas (Setaro et al., 2006a) form abundantextracellular mycelium, in addition to the usual intracellularcolonization. This results in an EEM-like association thatprobably evolved secondarily among ERM plants (Setaroet al., 2006b).

By contrast, clade A mycorrhizal association usually presentsa hyphal sheath (Selosse et al., 2002b; Urban et al., 2003), andsometimes intracellular colonization, as in some ectomycor-rhizas (Selosse et al., 2002b) and in some heterotrophicorchids (Selosse et al., 2002a; Taylor et al., 2003). All EEMsebacinoids from Ericaceae belong to clade A, as observed inindependent studies of Monotropoideae mycorrhizal associates(Pyrola chlorantha and Orthilia secunda, Tedersoo et al., 2007;P. rotundifolia, M.-A. Selosse, unpublished; discussed later).This corroborates the observation that EEM fungi usuallybelong to clades with ectomycorrhizal abilities, in bothArbutoideae (Richard et al., 2005) and Monotropoideae(Bidartondo, 2005; Tedersoo et al., 2007).


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Fig. 4 Phylogenetic relationships within the Sebacinales, with part of the tree relating to clade A and clade B. Phylogram derived by heuristic maximum-likelihood (ML) analysis from an alignment of nucLSU sequences, using a time-reversible model of nucleotide substitution, additionally assuming a portion of invariable sites and gamma-distributed substitution rates at the remaining sites (GTR + I + G). Branch support values were calculated from 1000 replicates of nonparametric ML bootstrap analysis (first numbers), and from Bayesian Markov chain Monte Carlo analysis, also using the GTR + I + G substitution model (second numbers). Values below 50% are indicated with asterisks or omitted. Branch lengths are scaled in terms of expected numbers of nucleotide substitutions per site. The tree was rooted with Auricularia auricula-judae. Mycorrhizal types: ECM, ectomycorrhiza; CVM, cavendishioid mycorrhiza; EEM, ectendomycorrhiza; ERM, ericoid mycorrhiza; JMM, jungermannialean mycorrhiza-like thalli; ORM, orchid mycorrhiza. Sample origin: A, Austria; AUS, Australia; ARG, Argentina; CAN; Canada; CHL, Chile; CHN, P.R. China; ECU, Ecuador; EST, Estonia; FRA, France; GER, Germany; GUY, Guyana; IND, India; NOR, Norway; REU, la Réunion; SPA, Spain.


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Fig. 4 continued


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Clades A and B therefore differ in mycorrhizal associations.The occurrence of ERM sequences basal to group A in Fig. 4is not strongly supported and might have two alternativeexplanations: either future works will support this (in whichcase features specific to clade A are derived in Sebacinalesevolution, such as mycorrhizal associations with sheath), orthis is an artifactual grouping. Indeed, these sequencesappeared in various positions within group B in preliminaryanalyses, and there is a discrepancy concerning this pair ofsequences between ML and MCMC analysis (as detailedin the Results section). For the moment, we regard the place-ment of these sequences in the phylogenetic tree as unstable.Addition of more sequences might resolve this problem infuture analyses.

Evolution of mycorrhizal structures in the Ericaceae

Available Ericaceae phylogenies (Freudenstein, 1999; Kronet al., 2002a) suggest a single most parsimonious scenario forevolution of mycorrhizal association (Fig. 5). Starting from aplesiomorphic association with arbuscular mycorrhizal fungi,still observed in Enkianthoideae (Abe, 2005), a shift occurredto EEM association involving ectomycorrhizal fungi, ascurrently retained in Monotropoideae + Arbutoideae. Then,ERM association arose once in the Ericoideae + Styphelioideae+ Harrimanelloideae + Cassiopoideae + Vaccinioideae clade(Fig. 5; Cullings, 1996). Among these, more derived conditionsevolved, such as the cavendishioid mycorrhizas in the Andeanclade of Vaccinioideae (Setaro et al., 2006a,b), or perhapsthe additional presence of arbuscular mycorrhizal fungi inGaultheria poeppigii (Urcelay, 2002), in Vaccinium spp. andStyphelia tameiameiae from Hawaii (Koske et al., 1990).Significantly, although data on the smaller subfamiliesHarrimanelloideae and Cassiopoideae are lacking so far, the

EEM to ERM shift was associated with a shift from clade Ato clade B sebacinoids (Fig. 5).

Interestingly, two sequences of clade B sebacinoids were foundin cloning of fungal ITS in the Ericaceae Pyrola chlorantha, inaddition to EEM mycorrhizal fungi (Tedersoo et al., in press):cloning allows recovery of various endophytic fungi, and thismay mean that clade B sebacinoids even occur in EEM Eri-caceae as endophytes, together with the main mycorrhizalfungus. Strikingly, Helotiales, the ERM ascomycetous associ-ates of Ericaceae, also encompasses root endophytic species(Vrålstad et al., 2002), raising an intriguing hypothesis. At theemergence of ERM association in Ericaceae, some endophyticfungal clades (namely the Helotiales and the Sebacinales)would have been recruited as mycorrhizal partners and/orexcluded the former EEM symbionts in the ancestor of ERMEricaceae.

We did not detect any significant difference in frequency ofcolonization, or specialization for any sebacinoid subclade inEricoideae, Vaccinioideae, or in any frequently sampled plantspecies (Table 1). Mycobionts from the same ericaceousspecies are distributed over group B in several cases (Fig. 4).However, Sebacinales obtained from some host species fromdifferent localities are very similar, if not identical (e.g. Vacciniummyrtillus mycobionts from France and Germany), and there isa cluster (near the top of the clade B tree) uniting mycobiontsfrom several Vaccinium species. Our sampling design andeffort are nevertheless not suitable for reliable conclusions tobe drawn regarding specificity.

Perspectives on Sebacinales

Our data add to the amazing diversity and ecological abundanceof sebacinoids. So far, no biogeographical subgroup is detectablein Sebacinales. One well-supported subgroup (97%, with a

Fig. 5 Phylogeny of the Ericaceae subfamilies following Freudenstein (1999) and Kron et al. (2002a), with most parsimonious scenario for evolution of the mycorrhizal types. AM, the ancestral arbuscular mycorrhizal condition; CVM, some species form cavendishioid mycorrhizas. Note that emergence of ericoid mycorrhizas (ERMs) corresponds to the rise of the ‘early anther inversion clade’ (Kron et al., 2002a). EEM, ectomycorrhiza.


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long branch; Fig. 4) contains Sebacinales from Europe, SouthAmerica and Australia. Some well-supported terminal cladesat present do not include sequences from different continents:however, ensuring that they represent regional clades wouldrequire much more additional sampling. Finding sebacinoidsamong ascomycetes in ERM roots (sometimes in the samecell, Fig. 2b) again underlines their frequent coexistence withascomycetes (Warcup, 1988; Selosse et al., 2002a; Urbanet al., 2003; Setaro et al., in press), an intriguing and hithertounexplained feature.

Interestingly, in > 60% of the ERM samples successfullyamplified, a single or, at most, two nearly identical sequences(when two bases are present at certain positions) wereobtained. In Andean Ericaceae investigated by Setaro et al.(2006), different sebacinoids co-occurred in a given sampledroot. The sequence homogeneity observed on our samplesencompassing many roots from a given root system, on is ofunclear significance, since the meaning of the differencesbetween ITS + 28S rDNA sequences is unknown. If thesedifferences represent interspecific variations, we observe herepatchiness in species distribution that often leads to thecolonization of a root system by a single species (includingpossibly several individuals), and a huge specific diversityexists among Sebacinales. If differences between sequencesrepresent intraspecific polymorphism, then a single individual(or several closely related ones) often covers each root system.Although there is evidence from other basidiomycetes that thevariability of 28S rDNA sequences is low at the intraspecificlevel (Fell et al., 2000), no rigorous conclusion can be drawn.This frustrating problem arises for sebacinoids because theabsence of meaningful morphological characters makesdistinction of morphological species impossible. Similarly, theabsence of haploid cultures for most ERM and EEM speciesdoes not allow distinction of biological species using inter-fertility tests. If other loci were available, the phylogeneticspecies concept (Taylor et al., 2000) would be interesting to apply:the fingerprint of recombination in a taxon is that phylogeniesof individuals of the same biological species are not congruentfor different loci. Conversely, in a comparison between phylo-genetic trees of different loci, the level at which congruencebetween loci arises delineates biological species (Taylor et al.,2000). This potentially applies to taxa known only fromenvironmental DNA, if at least two loci are available. Forsebacinoids, specific primers should be designed, sinceenvironmental DNA extracts harbour DNA from manyfungal species. This is a common problem with mycorrhizaltaxa mostly reported from molecular studies, such as tullasnel-loids (Bidartondo et al., 2004) or thelephoroids (Koljalg et al.,2001).

More diversity and biological interactions may still befound in Sebacinales. They may colonize many other hosts, assuggested by their isolation from roots that were first sup-posed to be arbuscular mycorrhizal (Williams, 1985; Milligan& Williams, 1987). In particular, clade B sebacinoids may

grow in roots of many plant taxa: some were recoveredfrom Phragmites australis (Neubert et al., 2006) and hepatics(Kottke et al., 2003), and P. indica showed compatibility withtremendously diverse hosts in glasshouse experiments, asmentioned earlier. Additionally, sebacinoid mycelia may beshared by different root systems. Thus, we might expect a majorrole in interplant interactions, or even in carbon transfersbetween plants, as already demonstrated for clade A sebaci-noids associated with achlorophyllous orchids (McKendricket al., 2002; Selosse et al., 2002a,b; Taylor et al., 2003). A rolefor sebacinoids in shaping plant communities is an intriguingpossibility raised by their frequent detection in molecularmicrobial ecology.

Acknowledgements

The authors wish to thank Sigisfredo Garnica, Thomas Julou,Anne-Marie Mollet, Carine Saison and Claude Selosse forhelp in root sampling, as well as Robert Bauer and HouriaHorcine for TEM analysis of Fig. 3. We also thank twoanonymous referees for helpful comments. Marc-André Selosseis funded by the Centre National de la Recherche Scientifiqueand the Société Française d’Orchidophilie.

ReferencesAbe J. 2005. An arbuscular mycorrhizal genus in the Ericaceae. Abstracts of

the MSA Symposium 6.Allen WK, Allaway WG, Cox GC, Valder PG. 1989. Ultrastructure of

mycorrhizas of Dracophyllum secundum R. Br. (Ericales, Epacridaceae). Australian Journal of Plant Physiology 16: 147–535.

Allen TR, Millar T, Berch SM, Berbee ML. 2003. Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytologist 160: 255–272.

Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Research 25: 3389–3402.

Avis PG, McLaughlin DJ, Dentinger BC, Reich PB. 2003. Long-term increase in nitrogen supply alters above- and below-ground ectomycorrhizal communities and increases the dominance of Russula spp. in a temperate oak savanna. New Phytologist 160: 239–253.

Barazani O, Benderoth M, Groten K, Kuhlemeier C, Baldwin IT. 2005. Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata. Oecologia 146: 234–243.

Berch SM, Allen TR, Berbee ML. 2002. Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant and Soil 244: 55–66.

Bergero R, Perotto S, Girlanda M, Vidano G, Luppi AM. 2000. Ericoid mycorrhizal fungi are common root associates of Mediterranean ectomycorrhizal plants (Quercus ilex). Molecular Ecology 9: 1639–1649.

Bidartondo MI. 2005. The evolutionary ecology of myco-heterotrophy. New Phytologist 167: 335–352.

Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ. 2004. Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proceedings of the Royal Society London Series B 271: 1799–1806.

Bonfante-Fasolo P. 1980. Occurrence of a basidiomycete in living cells of mycorrhizal hair roots of Calluna vulgaris. Transactions of the British Mycological Society 75: 320–325.


Research 877

Bougoure DS, Cairney JWG. 2005. Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella. Environmental Microbiology 7: 819–827.

Bougoure JJ, Bougoure DS, Cairney JW, Dearnaley JD. 2005. ITS-RFLP and sequence analysis of endophytes from Acianthus, Caladenia and Pterostylis (Orchidaceae) in southeastern Queensland. Mycological Research 109: 452–460.

Cairney JWG, Ashford AE. 2002. Biology of mycorrhizal associations of epacrids (Ericaceae). New Phytologist 154: 305–326.

Cullings KW. 1996. Single phylogenetic origin of the world’s heath plants indicated by 28S ribosomal RNA gene sequences and the ericoid mycorrhizal symbiosis. Canadian Journal of Botany 74: 1896–1909.

Deshmukh S, Hückelhoven R, Schäfer P, Imani J, Sharma M, Weiß M, Waller F, Kogel KH. 2006. The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley. Proceedings of the National Academy of Sciences, USA 103: 18450–18457.

Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. 2000. Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. International Journal of Systematic and Evolutionary Microbiology 50: 1351–1371.

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.

Freudenstein JV. 1999. Relationships and character transformation in Pyroloideae (Ericaceae) based on ITS sequences, morphology, and development. Systematic Botany 24: 398–408.

Gascuel O. 1997. BIONJ: An improved version of the NJ algorithm based on a simple model of sequence data. Molecular Biology and Evolution 14: 685–695.

Giovannetti M, Lioi L. 1990. The mycorrhizal status of Arbutus unedo in relation to compatible and incompatible fungi. Canadian Journal of Botany 68: 1239–1244.

Glen M, Tommerup IC, Bougher NL, O’Brien PA. 2002. Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests? Mycorrhiza 12: 243–247.

Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696–704.

Julou T, Burghardt B, Gebauer G, Berveiller D, Damesin C, Selosse M-A. 2005. Evolution of mixotrophy in orchids: insight from a comparative study of green and achlorophyllous Cephalanthera damasonium. New Phytologist 166: 639–653.

Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transformation. Nucleic Acids Research 30: 3059–3066.

Kennedy PG, Izzo AD, Bruns TD. 2003. There is high potential for the formation of common mycorrhizal networks between understorey and canopy trees in a mixed evergreen forest. Journal of Ecology 91: 1071–1080.

Kiers ET, van der Heijden GA. 2006. Mutualistic stability in the arbuscular mycorrhizal symbiosis: exploring hypotheses of evolutionary cooperation. Ecology 87: 1627–1636.

Koljalg U, Dahlberg A, Taylor A, Larsson E, Hallenberg N, Stenlid J, Larsson KH, Fransson P, Karen O, Jonsson L. 2001. Diversity and abundance of tomentelloid fungi in Swedish boreal forests. Molecular Ecology 9: 1985–1996.

Koske RE, Gemma JN, Englander L. 1990. Vesicular-arbuscular mycorrhizae in Hawaiian Ericales. American Journal of Botany 77: 64–68.

Kottke I, Beiter A, Weiß M, Haug I, Oberwinkler F, Nebel M. 2003. Heterobasidiomycetes form symbiotic associations with hepatics: Jungermanniales have sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species. Mycological Research 107: 957–968.

Kron KA, Judd WS, Stevens PF, Crayn DM, Anderberg AA, Gadek PA, Quinn CJ, Luteyn JL. 2002a. Phylogenic classification of Ericaceae: molecular and morphological evidence. Botanical Review 68: 335–423.

Kron KA, Powell EA, Luteyn JL. 2002b. Phylogenetic relationships within the blueberry tribe (Vaccinieae, Ericaceae) based on sequence data from matK and nuclear ribosomal ITS regions, with comments on the placement of Satyria. American Journal of Botany 89: 327–336.

Massicotte HB, Melville LH, Peterson RL. 2005. Structural characteristics of root–fungal interactions for five ericaceous species in eastern Canada. Canadian Journal of Botany 83: 1057–1064.

McKendrick SL, Leake JR, Taylor DL, Read DJ. 2002. Symbiotic germination and development of the myco-heterotrophic orchid Neottia nidus-avis in nature and its requirement for locally distributed Sebacina spp. New Phytologist 154: 233–247.

McLean CB, Cunnington JH, Lawrie AC. 1999. Molecular diversity within and between ericoid endophytes from the Ericaceae and Epacridaceae. New Phytologist 144: 351–358.

Midgley DJ, Chambers SM, Cairney JWG. 2004. Utilisation of carbon substrates by multiple genotypes of ericoid mycorrhizal fungal endphytes from eastern Australian Ericaceae. Mycorrhiza 14: 245–251.

Milligan MJ, Williams PG. 1987. Orchidaceous rhizoctonias from roots of nonorchids: mycelial and cultural characteristics of field and pot culture isolates. Canadian Journal of Botany 65: 598–606.

Moyersoen B. 2006. Pakaraimaea dipterocarpacea is ectomycorrhizal, indicating an ancient Gondwanaland origin for the ectomycorrhizal habit in Dipterocarpaceae New Phytologist 172: 753–762.

Münzenberger B, Kottke I, Oberwinkler F. 1992. Ultrastructural investigations of Arbutus unedo – Laccaria amethystea mycorrhiza synthesized in vitro. Trees 7: 40–47.

Neubert K, Mendgen K, Brinkmann H, Wirsel SGR. 2006. Only a few fungal species dominate highly diverse mycofloras associated with the common reed. Applied and Environmental Microbiology 72: 1118–1128.

Perotto S, Actis-Perino E, Perugini J, Bonfante P. 1996. Molecular diversity of fungi from ericoid mycorrhizal roots. Molecular Ecology 5: 123–131.

Peskan-Berghöfer T, Shahollari B, Giong PH, Hehl S, Markert C, Blanke V, Kost G, Varma A, Oelmüller R. 2004. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiologia Plantarum 122: 465–477.

Peterson TA, Mueller WC, Englander L. 1980. Anatomy and ultrastructure of a Rhododendron root–fungus association. Canadian Journal of Botany 58: 2421–3325.

Rai M, Acharya D, Singh A, Varma A. 2001. Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 11: 123–128.

Rambaut A, Drummond A. 2003. Tracer MCMC trace analysis tool, version 1.2.1. Oxford, UK: University of Oxford. [http://evolve.zoo.ox.ac.uk/software.html.]

Richard F, Millot S, Gardes M, Selosse MA. 2005. Diversity and structuration by hosts of the below-ground mycorrhizal community in an old-growth Mediterranean forest dominated by Quercus ilex L. New Phytologist 166: 1011–1023.

Robertson DC, Robertson JA. 1985. Ultrastructural aspects of Pyrola mycorrhizae. Canadian Journal of Botany 63: 1089–1098.

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.

Selosse MA, Bauer R, Moyersoen B. 2002b. Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees in silva: microscopic and molecular evidence. New Phytologist 155: 183–195.

Selosse MA, Faccio A, Scappaticci G, Bonfante P. 2004. Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including truffles. Microbial Ecology 47: 416–426.


Research878

Selosse MA, Weiß M, Jany JL, Tillier A. 2002a. Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M. Rich. & neighbouring tree ectomycorrhizae. Molecular Ecology 11: 1831–1844.

Setaro S, Kottke I, Oberwinkler F. 2006b. Anatomy and ultrastructure of mycorrhizal associations of neotropical Ericaceae. Mycological Progress 5: 243–254.

Setaro S, Weiß M, Oberwinkler F, Kottke I. 2006a. Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador. New Phytologist 169: 355–365.

Smith SE, Read DJ. 1997. Mycorrhizal symbiosis, 2nd edn. London, UK: Academic Press.

Taylor DL, Bruns TD, Szaro TS, Hodges SA. 2003. Divergence in mycorrhizal specialization within Hexalectris spicata (Orchidaceae), a non-photosynthetic desert orchid. American Journal of Botany 90: 1168–1179.

Taylor JW, Jacobson DJ, Kroken S. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21–32.

Tedersoo L, Pellet P, Kõljalg U, Selosse MA. 2007. Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae. Oecologia 151: 206–217.

Tedersoo L, Suvi T, Larsson E, Kõljalg U. 2006. Diversity and community structure of ectomycorrhizal fungi an a wooded meadow. Mycological Research 110: 734–748.

Urban A, Weiß M, Bauer R. 2003. Ectomycorrhizae involving sebacinoid mycobionts. Mycological Research 107: 3–14.

Urcelay C. 2002. Co-occurrence of three fungal root symbionts in Gaultheria poeppigii DC in Central Argentina. Mycorrhiza 12: 89–92.

Varma A, Singh A, Sudha S, Sharma J, Roy A, Kumari M, Rana D, Thakran S, Deka D, Bharti K, Hurek T, Blechert O, Rexer KH, Kost G, Hahn A, Maier W, Walter M, Strack D, Kranner I. 2001. Piriformospora indica – an axenically culturable mycorrhiza-like endosymbiotic fungus. In: Hock B, ed. Mycota IX. Berlin, Germany: Springer, 123–150.

Verma S, Varma A, Rexer KH, Hassel A, Kost G, Sarbhoy A, Bisen P, Bütehorn B, Franken P. 1998. Piriformospora indica, gen. et sp. nov., a new root-colonizing fungus. Mycologia 90: 896–903.

Vrålstad T, Myhre E, Schumacher T. 2002. Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytologist 155: 131–148.

Walker JF, Miller OK, Horton JL. 2004. Hyperdiversity of ectomycorrhizal fungus assemblages on oak seedlings in mixed forests in the southern Appalachian Mountains. Molecular Ecology 14: 829–838.

Walker JF, Parrent JL. 2004. Molecular phylogenetic evidence for the mycorrhizal status of Tremellodendron (Sebacinaceae). Memoirs of the New York Botanical Garden 89: 291–296.

Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Hückelhoven R, Neumann C, von Wettstein D, Franken P, Kogel KH. 2005. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proceedings of the National Academy of Sciences, USA 102: 13386–13391.

Warcup JH. 1988. Mycorrhizal associations of isolates of Sebacina vermifera. New Phytologist 110: 227–231.

Weiß M, Oberwinkler F. 2001. Phylogenetic relationships in Auriculariales and related groups – hypotheses derived from nuclear ribosomal DNA sequences. Mycological Research 105: 403–415.

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.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand H, Sninsky JS, White TJ, eds. PCR protocols: a guide to methods and applications. New York, NY, USA: Academic Press, 315–322.

Williams PG. 1985. Orchidaceous rhizoctonias in pot cultures of vesicular-arbuscular mycorrhizal fungi. Canadian Journal of Botany 63: 1329–1333.

Supplementary Material

The following supplementary material is available for thisarticle online:

Table S1 List of the 239 samples of ericoid mycorrhizal(ERM) roots investigated in this work, in alphabetic order ofEricaceae host name, with geographic origin and GenBankaccession numbers of the recovered sequences of the ITS +28SrDNA

This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-8137.2007.02064.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsible for thecontent or functionality of any supplementary materials suppliedby the authors. Any queries (other than missing material)should be directed to New Phytologist Central Office.

http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-8137.2007.02064.x

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Sebacinales are common mycorrhizal associates of Blackwell ... · (Taylor et al., 2003), as well as of some photosynthetic orchids related to N. nidus-avis that are partly heterotrophic