Response of ectomycorrhizal communitystructure to gap opening in natural and managedtemperate beech-dominated forests

Tine Grebenc, Morten Christensen, Ursa Vilhar, Matjaz Cater, Marıa P. Martın,Primoz Simoncic, and Hojka Kraigher

Abstract: Data on the impact of forest management practices on ectomycorrhizal community structure remains fragmen-tary and mainly originates from studies in northern coniferous forests. This study focuses on a comparison of ectomycor-rhizal communities between canopy gaps and closed canopy areas within natural and managed beech-dominated forests atfour locations in Europe. We used high resolution rDNA techniques to identify ectomycorrhiza-forming fungi and at-tempted to extract potential stand-, gap-, soil-, and selected environmentally derived variables by applying multivariateanalysis and ordination for pooling of ecological groups of ectomycorrhiza. A significant reduction of diversity indices, ec-tomycorrhizal and fine root dynamics, in gaps in comparison with closed canopy stands indicates an effect of forest man-agement practice and the high importance of maintaining and protecting natural forest areas for conservation of soilbiodiversity and forest genetic resources. The ordination analysis revealed three groups of ectomycorrhiza correlated withchanging environmental conditions. The litter and soil pH, number of beech seedlings, and presence of a gap had a pro-nounced effect on the ectomycorrhizal community. Combined analysis of ectomycorrhiza and environmental factors usingcorrespondence analysis provided an insight into the ecological preferences of the analysed species and confirmed that en-vironmental factors drive ectomycorrhizal community changes.

Resume : Les donnees concernant l’impact des pratiques d’amenagement forestier sur la structure de la communaute ecto-mycorhizienne demeurent fragmentaires et proviennent principalement d’etudes menees dans les forets nordiques de coni-feres. Cette etude met l’accent sur la comparaison des communautes ectomycorhiziennes entre les zones de trouees dans lacanopee et les zones de canopee fermee, a quatre endroits en Europe, dans des forets naturelles et amenagees domineespar le hetre. Nous avons utilise des techniques de detection d’ADNr a haute resolution pour identifier les champignons quiforment des ectomycorhizes et nous avons essaye d’extraire des variables potentielles selectionnees sur une base environ-nementale et derivees des peuplements, des trouees et du sol, en utilisant l’analyse multivariee et l’ordination pour rassem-bler les groupes ecologiques d’ectomycorhizes. Une reduction significative des indices de diversite ainsi que de ladynamique des ectomycorhizes et des racines fines dans les trouees, comparativement aux peuplements fermes, indiqueque les pratiques d’amenagement ont un impact et illustre l’importance de conserver et de proteger des zones de foret na-turelle pour la conservation de la biodiversite du sol et des ressources genetiques de la foret. L’analyse d’ordination a rev-ele qu’il y avait trois groupes d’ectomycorhizes correles aux conditions environnementales changeantes. La litiere, le pHdu sol, le nombre de semis de hetre et la presence d’une trouee avaient un effet prononce sur la communaute ectomycorhi-zienne. L’analyse combinee des ectomycorhizes et des facteurs environnementaux a l’aide de l’analyse de correspondancea fourni des informations sur les preferences ecologiques des especes etudiees et confirme que les facteurs environnemen-taux regissent les changements dans les communautes ectomycorhiziennes.

[Traduit par la Redaction]

Introduction

Forest management systems, as well as natural successionor disturbances, can cause the opening of canopy gaps inforest stands. Opening due to senescence, wind-throw, pestsand diseases, etc. of individual trees or groups of trees is thedominant process driving the natural forest dynamics(Mlinsek 1985). Partial or complete removal of trees–

canopies, resulting in canopy gaps, can change the environ-ment at the forest floor level by altering the light condi-tions (Jones et al. 2003a), temperature regime andmicroclimate extremes (Ballard 2000), precipitation withsubsequent ground and soil water regimes (Perry et al.1987), nutrient dynamics (Jones et al. 2003a), reduction ofthe number of fine roots (Parsons et al. 1994; Jones et al.2003b), changes in the spatial distribution and frequency

Received 27 November 2008. Accepted 19 May 2009. Published on the NRC Research Press Web site at cjfr.nrc.ca on 15 July 2009.

T. Grebenc, U. Vilhar, M. Cater, P. Simoncic, and H. Kraigher.1 Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljubljana,Slovenia.M. Christensen. University of Copenhagen, Faculty of Life Sciences, Forest and Landscape, Rolighedsvej 23, Dk-1958 Frederiksberg C,Denmark.M.P. Martın. Department of Mycology, Real Jardın Botanico, Plaza de Murillo 2, 28014 Madrid, Spain.

1Corresponding author (e-mail: hojka.kraigher@gozdis.si).

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Can. J. For. Res. 39: 1375–1386 (2009) doi:10.1139/X09-072 Published by NRC Research Press

(quantity) of coarse woody debris (Kraigher et al. 2002),and other parameters. It can cause the forest understoreyplant community to change into fast-growing perennials(Schroeer et al. 1999). Relatively few studies have ad-dressed the below-ground effects of canopy gap formation,such as soil-related aspects, ephemeral root gaps (Jones etal. 2003b), or the ectomycorrhizal community in sub-Mediterranean oak forests (Mosca et al. 2007), and notmuch is known about the response of ectomycorrhizalroots and ectomycorrhizal fungi sporocarp formation togap opening and its succession in European temperate for-est ecosystems (Diaci et al. 2000; Jones et al. 2003b).

Forests dominated by common beech (Fagus sylvatica L.)and silver fir (Abies alba Mill.), which were the main focusof the study, are among the best preserved, large forest com-plexes and contain the highest biodiversity in Slovenia —they form part of the ‘‘biodiversity hot belt’’ around theMediterranean region (Odor et al. 2006). Beech and silverfir are known to form ectomycorrhiza with a variety of basi-diomycetes and ascomycetes. About 40 different ectomycor-rhizal types have been identified and comprehensivelydescribed on beech (Agerer 1987), while only very limiteddata exist for ectomycorrhiza on silver fir, the second mostabundant species in the studied area, except for a few shortdescriptions of ectomycorrhiza with limited ecological com-ments (Pacioni et al. 2001).

Data on fungal population shifts under changing environ-mental and other anthropogenic impacts are known fromsporocarp mapping (Arnolds 1991; Lilleskov et al. 2002).However, this only poorly reflects the composition of thebelow-ground ectomycorrhizal community (Gardes andBruns 1996; Kraigher et al. 1996), mycelia in the soil, andtheir spatial distribution (Hirose et al. 2004). Sufficient datanow exist on the influence on ectomycorrhiza directly. A de-crease of ectomycorrhiza species richness and majorchanges in community composition were observed in clear-cut sites in comparison with undisturbed lodgepole pine(Pinus contorta Doug. ex Loud.) sites (Byrd et al. 2000),and the quality and diversity of ectomycorrhizal fungal inoc-ulum in soil also changed in Douglas-fir (Pseudotsuga men-ziesii (Mirb.) Franco) forests (Hagerman et al. 2001).Thinning activities in beech stands also caused drift in theectomycorrhiza community structure and activity (Buee etal. 2005). Since a single root tip was estimated to remainfunctional for 31–85 days for most mycorrhiza (Downes etal. 1992), the relatively fast turnover and constant presenceof ectomycorrhizal roots in the soil (Majdi et al. 2001) com-pared with sporocarp production makes ectomycorrhiza abetter and more accurate indicator of environmentalchanges. Some of the described species were clearly corre-lated with different stress factors, such as the drought re-sponse of Xerocomus (Boletus) chrysenteron (Shi et al.2002), and several types of ectomycorrhizae from otherplant partners (Norway spruce, Picea abies (L.) Karst.) havebeen reported to be applicable in mycobioindication of stress(Kraigher et al. 1996), as reviewed recently (Kraigher et al.2007; Cudlin et al. 2007).

We aimed to describe differences in the ectomycorrhizalcommunity and the preferential occurrence of ectomycorrhi-zal fungal species in four sites in the southern part of centralEurope (Slovenia) and in northern Europe (Denmark), se-

lected in undisturbed areas of a closed canopy stand or insmall canopy gaps in the same forest. The sites were locatedeither in managed or unmanaged beech-dominated foreststands. Based on knowledge of the relatively fast adaptationof ectomycorrhiza to spatial and temporal changes and de-tected changes in environmental conditions and gap open-ings, a change was expected in the ectomycorrhizalcommunity. We have analysed the ectomycorrhizal com-munity structure in a closed canopy and in acanopy gapopening and the change in measured environmental parame-ters, both as a potential cause for the expected drift of theectomycorrhizal community after gap formation.

Materials and methods

Experimental sitesFour research sites were selected in Slovenia and Den-

mark, representing the southern and the northern optimalarea for beech growth (Fig. 1). General data about the foursites is given in Table 1.

Rajhenavski Rog forest reserve is regarded as virgin for-est (after Peterken 1996). Such natural forests (virgin forestremnants), with little or no human influence, are consideredan important source of information about the dynamics andnatural processes in forest ecosystems, because of the highpercentage of coarse woody debris, continuous fluctuationof species composition, and a mosaic structure of develop-mental stages (Mlinsek 1985; Hocevar et al. 1995; Kraigheret al. 2002). Because of the high importance of virgin forestremnants in the region and their conservation, work at thesites was as noninvasive as possible to keep the area, andespecially the forest floor, undisturbed and in close-to-natureconditions throughout the work. The Snezna jama site ischaracterized by sustainable and multifunctional forest man-agement, which involves small-scale flexible forest manage-ment adapted to natural development and the preservation ofdiversity. The two selected sites in Slovenia shared similarecological conditions on a local scale.

The two research areas located in northern Europe wereRude Skov and Ravnsholte Skov, in Zealand in Denmark,both selected on the basis of similarity of management ap-proaches and forest characteristics to the southern sites.Rude Skov is part of a larger forest area dominated by beechin northern Zealand, with stands originating from natural re-generation dating back to 1755. The area is designated asunmanaged, with no forestry activities. The Ravnsholte site,which forms a part of the Skjoldenæsholm forest area, is lo-cated in central Zealand. The site occupies an area of 3.7 ha,consisting of 75-year-old beech trees with an average can-opy height of about 27 m. In contrast with the Sloveniansites, at both Danish sites, the maximum difference in heightabove sea level across the area of the site was less than 2 m.

All four studied gaps were approximately 20 m in diame-ter (Kraigher et al. 2002) and classified as small gaps basedon a ratio of gap diameter to mean height of the surroundingstand (d/h) of less than 1.0 (Runkle 1982).

Sampling strategies and light conditions measurementSporocarps of ectomycorrhizal species were intensively

mapped on a grid that covered the whole gap and a compa-rable surface of the closed canopy area. Sporocarps were

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mapped twice per month for two seasons (2001 and 2002)throughout the vegetation period. Each sporocarp was ex-actly positioned on the grid. At least one sporocarp of eachmapped species per year was deposited in the Herbariumand Mycotheca of the Slovenian Forestry Institute or in theHerbarium of the University of Copenhagen (Botanical Mu-seum, herb. C), subsequently used as a reference materialfor restriction fragment length polymorphism of amplifiedribosomal DNA internal transcribed spacers (PCR–ITS–RFLP) or sequenced and deposited in the UNITE database(unite.ut.ee/) (accession numbers: UDB000001,UDB000002, UDB000004–UDB0000011, UDB0000017–UDB0000019, UDB0000029–UDB0000033, UDB0000036,and UDB0000037) as described below.

Soil cores for analysis of ectomycorrhizae were collectedonce per season, immediately after leaf fall, at the beginningof November in 2001 and 2002. Six samples per site wererandomly taken in the canopy gap and four in the closedstand surrounding the gap. A standardized sampling methodwas applied, taking 274 mL of soil, including the litter layerand organic and mineral levels down to 20 cm (Kraigher1999). From each soil sample, all roots were carefullywashed and analysed under a stereomicroscope.

Light conditions were measured at sampling points usingWinScanopy Pro 2003d analysed hemispherical site photos

to ensure clear and uniform gap or closed canopy condi-tions. The criteria for light conditions were indirect site fac-tor, relative proportion of diffuse light intensity above thedefined area compared with the area without shading (in%), and total site factor.

Identification of ectomycorrhizaDifferent types of ectomycorrhiza were differentiated and

separated based on morphological and anatomical character-istics observed under an Olympus SZX12 stereomicroscope(magnification 3.5� – 45� ) and an Olympus BX51 micro-scope (magnification 100� – 2000� ). Morphotypes werebriefly described and identified (Grebenc and Kraigher2007) following the procedure and identification keys inAgerer (1987) and Pacioni et al. (2001).

Total DNA was extracted from a single fresh and vital ec-tomycorrhizal root tip in up to three repetitions for each un-identified anatomotype and soil sample, homogenized with amicropestle in 2% CTAB (cetyltrimethylammonium bro-mide) buffer or an EZNA Fungi DNA miniprep kit (OmegaBiotek).

DNA was amplified with 1 mL of template DNA in a totalvolume of 40 mL. PCR was performed according to Kraigheret al. (1995), with an annealing temperature of 53 8C or us-

Fig. 1. Beech (Fagus sylvatica) distribution in Europe (with the permission of European Forest Genetic Resources Programme (EU-FORGEN); modified from Von Wuelisch 2008) with indicated approximate locations of northern and southern study sites RajhenavskiRog (1), Snezna jama (2), Rude Skov (3), and Ravnsholte Skov (4).

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ing Ready-to-Go PCR Beads (GE Healthcare, UK). Forwardprimer ITS1F (Gardes and Bruns 1993) and reverse primersITS4 (White et al. 1990) and ITS4B (Gardes and Bruns1993) were used to amplify both internal transcribed spacer(ITS) regions, including the 5.8S of the ribosomal RNAgene cluster and small flanking parts of the small subunit(SSU) and large subunit (LSU) genes of Ascomycotina(ITS1F/ITS4) or Basidiomycotina (ITS1F/ITS4B). All PCRproducts were dedicated for RFLP analysis and werecleaved in a single enzyme digest, with Hinf I, Mbo I, andTaq I (Karen et al. 1997), following the manufacturer’s in-structions. Gels were read and fragment size data from Po-laroid photos was processed using Adobe Photoshopsoftware and analysed in the Taxotron software system (In-stitute Pasteur), especially designed for RFLP data process-ing as described in Grebenc et al. (2000) and subsequentlycompared with the PCR–RFLP database at the SlovenianForestry Institute (www.digitalna-knjiznica.bf.uni-lj.si/dd_grebenc_tine.pdf) (Grebenc et al. 2000; Grebenc andKraigher 2007).

PCR products of unidentified ectomycorrhiza or those re-quiring confirmation of identification based on anatomicalcharacteristics and RFLP were purified using Promega Wiz-ard SV Gel and PCR Clean-up System, and both strandswere sequenced separately using primers ITS1F and ITS4 atthe Automatic Sequencing Service (CIB-CSIC, Madrid). Se-quence fragments were assembled with Sequence NavigatorSequence Comparison software (Applied Biosystems). Nu-cleotide BLAST searches were used to compare unidentifiedectomycorrhizae sequences with DNA sequences in theNCBI (www.ncbi.nlm.nih.gov/) and UNITE database (unite.ut.ee/) using attached search tools. Anatomotypes with lessthan 98% sequence similarity were assigned to the genuslevel, and those with less than 95% were assigned as un-known types of ectomycorrhiza. The new obtained sequen-

ces were entered in the European Molecular BiologyLaboratory (EMBL, www.embl.org/) database with the ac-cession numbers listed in Table 2.

All vital ectomycorrhiza roots were counted and percen-tages were calculated in terms of the position in the site(gap or closed canopy area) and among sites.

Ecological dataPrimary data describing environmental, pedological, and

biochemical variables were assessed or modelled (Table 3)and correlated to abundance of ectomycorrhiza only for Raj-henavski Rog and Snezna jama sites. Soil, vegetation, nu-trient, and water leaching parameters were measured ormodelled for each 1 m2 subplot surrounding the point wheresoil cores for ectomycorrhizal analysis were taken. For a de-tailed description of site environmental factors, a full set ofprimary environmental data, including modelling of waterbalance and nutrients flux, see Vilhar et al. (2005).

Statistical analysisThe results of mapping sporocarps were analysed by one-

way ANOVA to analyse gap–canopy differences for eachsite separately and the multiway ANOVA test to compareall sites and replicates (StatGraphic Plus 5.1, StatisticalGraphics Corp.). All analyses were based on 36 randomlytaken samples from the pool of data for each gap and acomparable number of samples of the same size from theclosed canopy area. The total studied areas in gap andclosed canopy were of the same size at the two sites. Analy-ses focused on the influence of gap and site management onthe occurrence and number of the mapped species. For fac-tors with statistically significant difference among standarddeviations at a 95% confidence level, the Kruskal–Wallismedian test was applied instead of ANOVA.

The number of vital ectomycorrhizal roots, number of old

Table 1. General characteristics of studied sites.

Rajhenavski Rog Snezna jama Rude Skov Ravensholte SkovCountry Slovenia Slovenia Denmark DenmarkLocation Dinaric Alps,

SW SloveniaDinaric Alps,

SW SloveniaNorthern Zealand Central Zealand,

Skjoldenæsholmforest area

Coordinates 45839’36@N,1580’36@E

45839’15@N,1581’40@E

55851’N,12828’E

55831’N,11854’E

Altitude (m) 850–900 850–900 80–90 80–90Management applied Virgin forest Managed forest

(selective thinning,equal thinning pres-sure on all species)

Unmanaged,planted in 1755

Managed forest

Exposition SW, hilly SW, hilly Flat to gently rolling Flat to gently rollingDominant tree species Beech (60%),

silver fir (40%)Beech (60%),

silver fir (40%)Beech (dominant),

sycamore maple(solitary)

Beech (dominant),sycamore maple(solitary)

Understory vegetation Patches of beechseedlings

No regeneration Beech and sycamoremaple seedlings

Scattered regenera-tion of sycamoremaple

Soil type Rendzic Leptosol,Eutric Cambisol

Rendzic Leptosol,Eutric Cambisol

Haplic Luvisol Eutric Cambisol

Gap formation Windfall Clear-cut Windfall Clear-cutGap age (at the year of

the first sampling)6 2 5 2

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Table 2. Types of ectomycorrhizae recorded in soil samples from four research sites, with identification method of ectomycorrhizae andthe percentage of each ectomycorrhizal anatomotype in canopy or gap per site.

RajhenavskiRog Snezna jama Rude Skov

RavnsholteSkov

Type of ectomycorrhiza Method of identification Canopy Gap Canopy Gap Canopy Gap Canopy GapByssocorticum atrovirens (Fr.) Bond.

& Sing. ex Sing.M — — 1.1 — — — — —

Cenococcum geophilum Fr. M, S 3.8 14.9 7.5 34.0 12.8 12.1 26.7 12.1Clavulina cinerea (Bull.) J. Schrot S; AJ937997 (98%) 2.5 — 0.3 — — — — —Entoloma rhodopolium

Berk. & BroomeM, S; AJ938003 (100%) 4.4 1.2 — — — — — —

Genea hispidula Berk. exTul. & C. Tul.

M — — — — 0.7 0.3 — 2.1

Hebeloma sinapizans (Fr.) Sacc. S; AJ938001 (99%) 9.9 — — — — — — —Laccaria amethystina Cooke M, R, S; AJ937999 (99%) 3.0 0.6 9.2 — — — 2.6 2.1Lactarius acris (Bolton) Gray M, R, S; AJ937991 (99%) — 9.0 — — — — — —Lactarius blennius (Fr.) Fr. M — — 0.1 — 0.4 9.9 4.1 2.6Lactarius camphorates (Bull.) Fr. M, R — — — — 0.4 — — 11.4Lactarius pallidus Pers. M 3.1 9.1 23.1 14.0 8.0 1.4 — 1.5Lactarius rubrocinctus Fr. M — — — — — — — 0.3Lactarius salmonicolor

R. Heim & LeclairM 9.0 — — — — — — —

Lactarius subdulcis (Bull.) Gray M, R, S; AJ937986 (99%) 3.2 12.6 2.4 — 7.0 16.2 11.6 3.1Lactarius subsericatus

Kuhner & Romagn.S; AJ937989 (99%) — — 2.5 — — — — —

Russula cyanoxantha (Schaeff.) Fr. M, R, S; AJ937993 (98%) 1.9 12.5 1.5 — — — — —Russula fellea (Fr.) Fr. M, R — — — — 2.1 2.5 0.6 —Russula illota Romagn. M, R, S; AJ937998 (98%) 1.2 2.4 — — — — — —Russula lepida Fr. M, R — — — — 5.8 — — —Russula mairei Sing. M, R 2.1 8.1 — — 20.6 — — —Russula ochroleuca (Pers.) Fr. M — 0.4 — 2.9 33.4 47.3 29.6 21.7Tomentella terrestris

(Berk. & Broome) M.J. LarsonS; AJ938002 (98%) 6.2 8.7 0.0 — — — — —

Tricholoma scioides (Pers.) C. Martın M 4.1 6.6 — — 2.5 — — 1.3Xerocomus chrysenteron

(Bull.) QueletM, R — — — — 0.2 10.3 8.0 6.2

Fagirhiza fusca M 2.6 0.6 — — — — — —Fagirhiza pallida M — — — — 0.9 — — —Fagirhiza setifera M — — — — 0.3 — — —Fagirhiza spinulosa M 2.6 — — — — — — —Cortinarius sp. 1 M, S; AJ937984 (95%) 5.1 — 1.4 — — — — —Cortinarius sp. 2 M, R — — — — 4.8 — — —Laccaria sp. 1 M, R 8.2 — 0.1 — — — — —Laccaria sp. 2 M, R — — 3.1 — — — — —Lactarius sp. 1 M 1.4 0.2 — — — — — —Pezizalean ECM M, S — 0.7 — — — — — —Russula sp. 1 M, S; AJ937992 (96%) 1.7 — — — — — — —Russula sp. 2 M, R 0.9 — — — — — — —Russula sp. 3 M — — 0.0 39.0 — — — —Russula sp. 4 M, R — — 0.4 — — — — —Russula sp. 5 M — — — — — — 0.9 —Russula sp. Nigricantinae M, R 0.1 0.4 — — — — — —Thelephora sp. 1 M, R — 0.6 — — — — — —Thelephora sp. 2 S; AJ937994 (93%) 0.3 2.2 — — — — — —Tomentella sp. 1 M, R — 2.3 — — — — — —Tricholoma sp. 1 M — — 7.4 — — — — —Tricholoma sp. 2 M — — — — 0.2 — — —AA 1 – 2209 M, R 1.2 — — — — — — —AA 2 – 2214 M 0.6 — — — — — — —AA 3 – 2223 M — 6.9 — — — — — —AA 4 – 2225 M, R — 0.1 — — — — — —

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ectomycorrhizal root tips, number of types of ectomycor-rhiza, percentage of vital ectomycorrhizal roots within allfine roots in a soil sample, Shannon–Weaver diversity index,and species richness index (Atlas and Bartha 1981) wereused as the main parameters for comparing gap and closedcanopy areas within and between different sites.

Ectomycorrhiza community composition and abundancewere explained by the set of environmental variables. To ob-tain ordination diagrams, direct gradient analysis after log-transformation of the data set was applied. Environmentalvariables and ectomycorrhiza occurrence data were de-trended by segment and subjected to canonical correspond-ence analysis using interspecific distances modified afterbiplot scaling, in a unimodal regression model, as an optionof the CANOCO software (Biometrics). Results were eval-uated for significance of obtained models using unrestrictedpermutations in the Monte Carlo test. The influence of thegap on the distribution of ectomycorrhizae at the studiedsites was assessed using the same statistical approach. Re-sults were used for generation of species response curves tofit multiple regression models to changes of environmentalvariables in a general linear model with Gaussian distribu-tion (option in CANOCO software). Species giving statisti-cally significant responses (p < 0.1) to the presence of agap after the Monte Carlo permutation test were included.

ResultsSporocarps of 67 ectomycorrhizal fungal species were re-

corded in 2 years of sampling, with a total of 521 recordsfor all four sites (supplemental Table S12). The number ofsporocarps was significantly lower in the canopy gap thanin the closed canopy in combined analysis for all sites(MANOVA, p < 0.001). Separate ANOVA indicated no gapeffect on sporocarp production, only in Rajhenavski Rog(ANOVA, p = 0.541). The exact positioning of sporocarpsrevealed the complete absence of sporocarps from the mid-dle of the 20 m wide newly made canopy gaps (Sneznajama and Ravnsholte Skov sites; data not shown) as earlyas 1 year after the clearcut was made, while vital ectomy-corrhizae were still present in the same area, at least in lownumbers.

A total of 53 739 short roots were counted from all sites,

of which 14 548 were vital ectomycorrhizal roots that werefurther identified, while the remaining short roots weremainly old (nonturgescent) and unidentifiable ectomycorrhi-zal roots, while nonmycorrhizal root tips were present onaverage at less than 1% frequency per site. Vital ectomycor-rhizae were separated into 53 distinct morphotypes–anatomotypes (Table 2). Twenty-four morphotypes (45%)were identified at the species level on the basis of one ofthe applied approaches for identification: Byssocorticiumatrovirens (Fr.) Bond. & Sing. ex Sing.; Clavulina cinerea(Bull.) J. Schrot; Cenococcum geophilum Fr.; Entolomarhodopolium Berk. & Broome; Genea hispidula Berk. exTul. & C. Tul.; Hebeloma sinapizans (Fr.) Sacc.; Laccariaamethystina Cooke; Lactarius acris (Bolton) Gray; L. blen-nius (Fr.) Fr.; L. camphoratus (Bull.) Fr.; L. pallidus Pers.;L. rubrocinctus Fr.; L. salmonicolor R. Heim & Leclair; L.subdulcis (Bull.) Gray; L. subsericatus Kuhner & Romagn.;Russula cyanoxantha (Schaeff.) Fr.; R. fellea (Fr.) Fr.; R.illota Romagn.; R. lepida Fr.; R. mairei Sing.; R. ochro-leuca (Pers.) Fr.; Tomentella terrestris (Berk. & Broome)M.J. Larson; Tricholoma sciodes (Pers.) C. Martın; Xeroco-mus chrysenteron (Bull.) Quelet. A further 20 anatomo-types, including anatomotypes described as recognizableectomycorrhizae on beech (Fagirhiza fusca, Fagirhiza pal-lida, Fagirhiza setifera, and Fagirhiza spinulosa,) wereidentified at the genus or group level, and nine (17%) re-mained unidentified after comparison with the PCR–RFLPdatabase based on reference material of local origin anddistant samples or after sequencing if the minimum se-quence similarity for the genus level was set at 98%.

From the group of Ascomycotina, only three types of ec-tomycorrhizae were identified (Cenococcum geophilum, Ge-nea hispidula, and an ectomycorrhiza from the order ofPezizales (AJ969438) — Pezizalean ECM), with an overalllow percentage of vital ectomycorrhizal root tips. PezizaleanECM was only found in one soil sample from the regenerat-ing area in the natural forest site.

Four types of ectomycorrhizae were common to all foursites (C. geophilum, R. ochroleuca, L. pallidus, and L. sub-dulis), while C. geophilum was the only ectomycorrhizaconstantly present both in gaps and under closed canopy.Thirty-six types of ectomycorrhiza were site-specific(Table 2). In addition to the four species in common, Rus-

Table 2 (concluded).

RajhenavskiRog Snezna jama Rude Skov

RavnsholteSkov

Type of ectomycorrhiza Method of identification Canopy Gap Canopy Gap Canopy Gap Canopy Gap

AA 5 – 2271 M, R 0.9 — — — — — — —AA 6 – 2193 M — — — 6.3 — — — —AA 7 – 2297 M — — — 4.0 — — — —FS 1 – 2169 M, R — — — — — — — 15.6

Note: Ectomycorrhizae were identified by morphology (M), PCR–RFLP analysis (R), and sequencing (S). The accession number for sequences depositedin European Molecular Biology Laboratory (EMBL), as well as percentage of similarity data computed through BLAST searches, were included in ‘‘Methodof identification’’. The rDNA internal transcribed spacer (ITS) genes were targeted for molecular analysis. ‘‘AA’’ indicates unidentified type of ectomycor-rhizae in Abies alba; ‘‘FS’’ indicates unidentified type of ectomycorrhizae in Fagus sylvatica.

2 Supplementary data for this article are available on the journal Web site (cjfr.nrc.ca) or may be purchased from the Depository of Unpub-lished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON K1A 0R6,Canada. DUD 3982. For more information on obtaining material refer to cisti-icist.nrc-cnrc.gc.ca/eng/ibp/cisti/collection/unpublished-da-ta.html.

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Table 3. List of 65 variables included in ordination analysis, span of their values taken from canopy gap to closed canopy (dataobtained from Vilhar et al. 2005), and significance of their contribution to the distribution of ectomycorrhizae in studied sites.

Rajhenavski Rog Snezna jama

Environmental factors used in CCA Span of values in subplots p Span of values in subplots pRegeneration by beech (0–5 years) 1–6 0.0400 1–12 0.0010Regeneration by beech (6–10 years) 0–15 NS 0–1 0.0010Regeneration by silver fir (0–10 years) 0–4 0.0472 0 NSPresence of gap 0–1 NS 0–1 0.0010Estimation of grass cover 0%–0.05% 0.0144 0.05%–0.95% 0.0016Thickness of soil horizon O 2.2–4 cm NS 2.3–5.7 cm 0.0066Thickness of soil horizon A 4.2–9.7 cm 0.0006 8.5–11.8 cm 0.0252Thickness of soil horizon B 15–49 cm NS 17.3–34 cm 0.0228pH of litter 5.23–5.85 0.0010 4.73–5.34 0.0134pH of soil at 5 cm depth 4.83–5.46 0.0734 4.88–5.76 0.0102pH of soil at 10 cm depth 4.90–5.44 0.0008 4.89–5.81 0.0070pH of soil at 20 cm depth 5.06–6.38 0.0870 5.07–5.99 0.0286Total nitrogen in litter 0.84%–1.26% NS 0.96%–1.31% NSTotal nitrogen in soil at 5 cm 0.34%–0.54% NS 0.40%–0.60% 0.0444Total nitrogen in soil at 20 cm 0.13%–0.16% NS 0.17%–0.32% 0.0010C:N ratio of litter 29.84–41.17 0.0850 25.36–40.76 0.0946C:N ratio at 5 cm depth 15.19–16.41 NS 15.26–17.46 0.0470C:N ratio at 10 cm depth 13.28–14.69 0.0155 13.65–15.57 NSC:N ratio at 20 cm depth 12.26–15.44 0.0514 12.86–13.17 0.0740Percentage of humus in litter 59.31%–67.41% NS 57.06%–67.67% 0.0202Percentage of humus in soil at 5 cm depth 8.96%–15.31% NS 10.53%–18.02% 0.0536Percentage of humus in soil at 10 cm depth 4.67%–6.78% NS 6.95%–8.08% 0.0050Percentage of humus in soil at 20 cm depth 2.72%–3.71% 0.0492 3.70%–5.09% 0.0010Deposition of Ca2+ 6.63–12.3 mg/L NS 6.69–10.49 mg/L 0.0602Deposition of Cl– 2.27–4.00 mg/L 0.0298 1.73–2.32 mg/L 0.0696Deposition of K+ 4.94–14.19 mg/L 0.0880 2.14–10.72 mg/L 0.0676Deposition of Mg2+ 0.91–1.94 mg/L 0.0250 0.86–1.91 mg/L 0.0604Deposition of NH4

+ 3.25–7.2 mg/L NS 1.44–5.73 mg/L 0.0344Deposition of NO3

– 8.08–15.88 mg/L 0.0124 6.53–12.18 mg/L 0.0044Deposition of SO4

2– 12.44–18.32 mg/L 0.0532 9.22–12.67 mg/L 0.0416Leaching of Al3+ from substratea,c 0.03–0.05 mg/L NS 0.01–0.04 mg/L 0.0060Leaching of Ca2+ from substratea,c 38.52–59.40 mg/L 0.0544 25.89–44.08 mg/L 0.0058Leaching of Cl– from substratea,c 0.56–0.99 mg/L NS 1.15–1.68 mg/L NSLeaching of K+ from substratea,c 0.08–0.28 mg/L NS 0.03–0.34 mg/L 0.0114Leaching of Mg2+ from substratea,c 0.66–0.99 mg/L 0.0004 1.50–9.00 mg/L 0.0675Leaching of NH4

+ from substratea,c 0.06–0.69 mg/L NS 0.05–0.85 mg/L 0.0046Leaching of NO2

– from substratea,c 0.00–0.30 mg/L 0.0490 0.12–1.05 mg/L 0.0740Leaching of NO3

– from substratea,c 1.57–19.33 mg/L NS 1.21–47.93 mg/L 0.0632Leaching of SO4

2+ from substratea,c 3.14–5.28 mg/L NS 2.35–3.80 mg/L 0.0616Leaching of Al3+ from substrateb,c 0.03–0.06 mg/L NS 0.01–0.02 mg/L NSLeaching of Ca2+ from substrateb,c 33.81–68.09 mg/L NS 1.86–114.71 mg/L NSLeaching of Cl– from substrateb,c 0.32–1.49 mg/L NS 0.19–4.84 mg/L NSLeaching of K+ from substrateb,c 0.02–0.24 mg/L NS 0.03–0.40 mg/L 0.0008Leaching of Mg2+ from substrateb,c 0.38–1.32 mg/L 0.0738 0.62–16.46 mg/L NSLeaching of NH4

+ from substrateb,c 0.09–1.42 mg/L NS 0.00–1.01 mg/L 0.0282Leaching of NO2

– from substrateb,c 0.00–0.35 mg/L 0.0118 0.00–0.09 mg/L 0.0676Leaching of NO3

– from substrateb,c 2.13–36.94 mg/L NS 0.02–279.71 mg/L NSLeaching of SO4

2+ from substrateb,c 1.79–6.55 mg/L NS 0.20–8.21 mg/L NSAverage precipitation 124.8 mm NS 124.8 mm NSTotal throughfall measured 99–277 mm NS 137–270 mm 0.0150Average drainagea,c 11.868–33.085 mm 0.0190 12.651–31.679 mm 0.0112Monthly drainage fluxb,c 71.21–198.51 mm NS 75.90–190.07 mm 0.0150Actual evapotranspirationb,c 88.07–93.48 mm NS 81.12–98.60 mm NSPotential evapotranspirationb,c 101.698 mm NS 90.81–121.08 mm NSDirect SF (irradiance) 8.86%–24.10% 0.0920 3.51%–47.43% 0.0488

Grebenc et al. 1381

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sula sp. 3 and anatomotype FS1–2196 were also recognizedto dominate with a high percentage (>15%) in gaps. In Raj-henavski Rog, a high number of anatomotypes (29) occurredin low percentages (<5%) summed up for gap and closedcanopy treatment (Table 2). The number of types of ectomy-corrhiza, the Shannon–Weaver diversity index, and speciesrichness index were significantly reduced in samples takenin gaps from all sites except Ravnsholte Skov. The other pa-rameters changed only in some sites (Table 4). In relation tothe management practices applied on sites, with data joinedfor gap and closed canopy, the number of described ectomy-corrhiza fungal taxa was lower in the managed forest sitesSnezna jama and Ravnsholte Skov (19 and 14) comparedwith the natural forest sites Rajhenavski Rog and RudeSkov (32 and 16).

Species response curves analysing the influence of canopygap on the occurrence of ectomycorrhizae at the two Slov-enian sites revealed a significant positive correlation of sev-eral ectomycorrhizal types with gap formation. Theectomycorrhiza of Russula cyanoxantha showed preferentialoccurrence in the transition zone between gap and canopy,while other ectomycorrhizae analysed in the general lineardistribution model showed a negative correlation with thepresence of the canopy gap (Fig. 2).

The effect of measured or modelled environmental factorschanging through the gap was significant (p < 0.05) for 15variables in the natural forest Rajhenavski Rog and for 33variables in the managed forest in Snezna jama (Table 3).The highest significance (p < 0.001) for the change in theectomycorrhizal population in the natural forest was shownin litter and soil pH, thickness of horizon A, and leachingof Mg ions at 40 cm depth; a significant influence (p <0.05) was shown also for grass cover, C:N ratio, and deposi-tion through precipitation of several ions (Cl–, Mg2+, NO3

–).For the managed forest site with the clear-cut gap, the grasscover; the presence of the gap; the thickness of all soil hori-zons; soil nitrogen content; percentage of humus at all soildepths; site substrate composition; deposition of NH4

+,NO3

– and SO42–; or leaching of Ca2+, K+, NH4

+, and NO2–

with soil solution at 40 cm depth were significantly corre-lated with the drift of the ectomycorrhizal population. Eightvariables with significant influence were common for both

sites (deposition of NO3, drainage, gap, grass cover, humus,pH, thickness of horizon A, and young beech regeneration;Table 2) and were used for ordination analysis (Figs. 3Aand 3B).

The distribution of ectomycorrhiza anatomotypes alongthe gradients of environmental factors showed severalgroups of species with a common response to changes. Atleast three groups of species were recognized at the naturalsite in Rajhenavski Rog. Group I, including Tricholoma sci-odes and Fagirhiza fusca, was negatively correlated with thecanopy gap, while Group II showed a positive correlation toincreasing soil pH. Russula ochroleuca ectomycorrhizashowed a comparable response for both sites. A positive cor-relation with an increasing number of beech seedlings in thesite was modelled for Laccaria amethystina, Russula illota,Cortinarius sp., and Lactarius vellereus (Group III). Allthree observed groups were less substantially expressed forthe managed forest site. The opposite directions of gradientsfor the gap and beech seedlings for both sites indicate theabsence of regeneration in Snezna jama.

DiscussionThe Rajhenavski Rog area is beech- and silver fir-

dominated virgin forest with a well-known history of main-tenance and continuity. Such elements are an importantreference for research and education and also give impor-tant and rare information about untouched forest ecosys-tems.

The highest species richness of ectomycorrhizae wasfound in the natural forest, supporting the idea that mycor-rhizal fungi are best adapted to prevailing conditions in nat-ural forest sites (Meyer 1973). The occurrence of sporocarpssharply followed the gap edge line a few metres into thegap. Such a decrease was more pronounced in the managedsites without any natural regeneration. Two intensive sam-pling years of only by sporocarp mapping were not suffi-cient to obtain a good overview of the population structureof ectomycorrhizal fungi, but it was sufficient to obtain theirdistribution. To obtain insight into species population com-position, intensive sampling should take place for a mini-mum of 3–8 years, as proposed by Gardes and Bruns

Table 3 (concluded).

Rajhenavski Rog Snezna jama

Environmental factors used in CCA Span of values in subplots p Span of values in subplots p

Indirect SF (irradiance) 11.50%–27.40% NS 12.89%–30.00% NSTotal SF (irradiance) 9.87%–23.30% 0.0920 4.67%–44.16% 0.0518Quantity of skeletal elements 40 363–189 687 kg/ha NS 117 710–258 156 kg/ha 0.0044Quantity of total coarse roots 779.5–7 448.9 kg/ha NS 2 702.4–14 668.2 kg/ha NSQuantity of ground component 879 142–1 445 603 kg/ha NS 580 753–822 409 kg/ha NSSurface rock cover 1%–5% NS 1%–50% 0.0066Inclination 7%–15% NS 19%–45% 0.0100No. of tree species in the site 3 NS 2 NSShading by tree canopies 0%–70% 0.0760 0%–100% 0.0542Shading by shrubs 15%–90% NS 0%–6% NS

Note: SF, site factor; NS, not significant (p > 0.1).aValues calculated with chlorine balance method.bValues calculated with WatBal method.cFor details on modelling, see Vilhar et al. 2005.

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(1996). In our study, sporocarps disappeared in the centre ofgaps, while vital ectomycorrhizal root tips were still present.Mycelium of ectomycorrhizal fungi from the gap were pre-sumably connected to a common mycelial network at theedge of the gap and were exchanging the nutrients necessaryto sustain themselves with mycelia from the closed canopyarea (Dighton and Boddy 1989). The presence of ectomycor-rhizae in a small canopy gap area can in some sites facilitatesubsequent establishment and mycorrhization of natural re-generation, common for southern beech-dominated forests(Mlinsek 1985). The sporocarp production of ectomycorrhi-zal species was not affected significantly at the site withsome natural regeneration (Rajhenavski Rog, Table 1) butthe species composition changed there in favour of early-stage and multistage succession fungi. Only a few specieswere in common above and below ground, as reported pre-viously by several authors from different forest types andstands (Gardes and Bruns 1996; Kraigher et al. 1996).

In the canopy gaps, the diversity and richness of the ec-tomycorrhiza community decreased, except in the gap withpronounced regeneration. An effect similar to gap forma-tion was also observed for fire-disturbed sites but withsome important differences, such as faster liberation of nu-trients, altered soil pH (Cairney and Bastias 2007), fertil-ization effect of fires (Tuininga and Dighton 2004), etc. Inan 8-year-old lodgepole pine clear-cut stand, Byrd et al.(2000) found no significant difference in root-tip abun-dance or the ascomycetes–basidiomycetes ratio betweenclear-cut and undisturbed sites, while species richness waslower in the cut sites. The results indicate a similarity onlyfor the regenerating area at the Rajhenavski Rog site,where diversity indices for the 6-year-old regenerating areawere similar to those of closed canopy, but the speciescomposition of ectomycorrhizae was different. The reduc-tion of ectomycorrhizae in small gaps is in accordancewith the results of Parsons et al. (1994), who detected asignificant reduction in ectomycorrhizae in a gap as smallas 9 m in diameter. The observed reduction in the numberof ectomycorrhizae and short roots support the idea of anapproximately linear reduction of fine roots away from thetree trunk and, on the other hand, for sites with abundantnatural regeneration, indicate that the soil space releaseddue to the removal of trees (gap) is quickly filled by newroots (Jones et al. 2003b).

The most common ascomycete ectomycorrhiza in thestudied sites was Cenococcum geophilum, a species knownto indicate stressed conditions in sites (LoBuglio 1999). As-comycetes (Pezizalean ectomycorrhiza) constitute a consid-erable proportion of the mycobionts in ectomycorrhizalcommunities in mature boreal deciduous and coniferous for-ests in several soil types (Tedersoo et al. 2006). Accordingto some authors, ascomycete ectomycorrhizae become evenmore common after fire disturbance (Fujimura et al. 2005)and under other environmentally stressful conditions. Theoverall percentage of ascomycetes in the ectomycorrhizaefrom our sites was relatively low (5.7%–6.1%) and far lowerthan observed in the provenance test trial for beech (planted5 years before harvesting of roots for analysis), which hadabout 40% (Pucko et al. 2004), and the clear-cut gap inlodgepole pine forest with 38.8% observed increase of asco-mycetes after clear-cut disturbance (Byrd et al. 2000). TheT

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Grebenc et al. 1383

Published by NRC Research Press

generally low percentage of ascomycetes, their uneven dis-tribution among canopy and gap sites, and low percentageof C. geophilum in particular reflect the continuity of matureforests and indicate extensive management of the research

sites in the past and only poorly reflect the influence of asmall canopy gap stress effect on the ectomycorrhizal com-munity.

Information based on fine roots and ectomycorrhizae

Fig. 2. Ectomycorrhiza species response curves based on multiple regression models and fit to the presence of canopy gap in a generallinear model with Gaussian distribution. Only species with a significance level after the Monte Carlo test at p < 0.1 are presented.

Fig. 3. Ordination diagram for the first and the second ordination axis, including gradients of environmental variables with significant (atleast p < 0.05) contribution to the occurrence of ectomycorrhizae for the Rajhenavski Rog natural forest site with regenerating canopygap (A) and the Snezna jama managed forest site with clear-cut canopy gap (B). AA, unidentified type of ectomycorrhizae in Abies alba.

1384 Can. J. For. Res. Vol. 39, 2009

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compared with sporocarp production better reflects the ecto-mycorrhiza fungal community on site because of the fasterturnover and constant presence of fine roots and ectomycor-rhizae in the soil (Kraigher et al. 1996; Majdi et al. 2001).In general, very little is known about the ecology of differ-ent species in situ. The combined approach — sporocarpand ectomycorrhiza mapping and ordination with environ-mental parameter gradients as used in our study — revealedthe affinities of species to specific environmental conditionsand gave a better insight into the biology of each of them.Although only a small number of observed species show re-sponse curves and ordination diagrams that were statisticallysignificant, we believe Russula ochroleuca, Tomentella ter-restris, and Thelephora sp. 2 are species adapted to distur-bances such as small gaps or forest edges in beech- andsilver fir-dominated mature forests. This is important infor-mation for the biology of ectomycorrhizal species and theirresponse to the presence of small canopy gaps and changesof other environmental factors. Our results confirmed thatthe species composition of a mycorrhizal fungal communitydepends on host plant age and environmental conditions(Wilcox 1996), with pH, C:N ratio, percentage of humus,drainage, grass cover, and deposition of some anions andcations as the most significant changes in the gap directingectomycorrhizal community drift, and direct and total sitefactor (irradiance) as the key element determining and dis-tinguishing gap from closed canopy conditions.

AcknowledgementsThe study was co-financed by the 5th Framework Pro-

gramme (FP) EU project Nat-Man (QLRT1-CT99-1349);EU BIOD’IBERIA projects A9 and A64; the Ministry ofHigher Education, Science and Technology (MEST) throughthe Young Researchers Scheme (for T. Grebenc andU. Vilhar); the Research Programme Forest Biology, Ecol-ogy and Technology (P4-0107); and research projects onCarbon Sequestration in Beech Forests (L4-6232) and Car-bon Sequestration and Ozone (L1-6706), co-financed byMEST and the Ministry for Agriculture, Forestry and Food.We thank Prof. Reinhard Agerer for checking several prob-lematic ectomycorrhizal identifications; Tomaz Hartmanfrom the Slovenian State Forest Service, Regional Unit Ko-cevje, for general information on studies of the RajhenavskiRog Forest Reserve; Mihej Urbancic, Igor Smolej, and LadoKutnar for soil, site, vegetation, and light regime studies;Jana Jansa and Matej Rupel for technical support; MichelleBozzano from Bioversity International for preparation of themap on beech distribution in Europe (obtained with permis-sion from EUFORGEN and part of the Technical Guidelinefor Conservation of Beech Genetic Resources in Europe);and the Associate Editor of the Canadian Journal of ForestResearch and three anonymous reviewers for providing use-ful comments to improve the manuscript.

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