mycological research 110 (2006) 734–748
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycres
Diversity and community structure of ectomycorrhizal
fungi in a wooded meadow
Leho TEDERSOOa,*,y, Triin SUVIa,y, Ellen LARSSONb, Urmas KÕLJALGa
a
Institute of Botany and Ecology, University of Tartu, 40 Lai Str., 51005 Tartu, Estonia
Botanical Institute, Göteborg University, PO Box 461, SE 40530 Göteborg, Sweden
b
article info
abstract
Article history:
Wooded meadows are seminatural plant communities that support high diversity of various
Received 1 September 2005
taxa. Due to changes in land use, wooded meadows have severely declined during the last
Received in revised form
century. The dominant trees in wooded meadows acquire mineral nutrients via ectomycor-
13 February 2006
rhizal fungi. Using anatomotyping and sequencing of root tips, interpolation and extrapola-
Accepted 16 February 2006
tion methods, we studied the diversity and community structure of ectomycorrhizal fungi in
Published online 12 June 2006
two soil horizons of both managed and forested parts of a wooded meadow in Estonia.
Corresponding Editor:
Species of Thelephoraceae, Sebacinaceae and the genus Inocybe dominated the whole ectomy-
John W. G. Cairney
corrhizal fungal community of 172 observed species. Forested and managed parts of the
wooded meadow harboured different communities of ectomycorrhizal fungi, whereas soil
Keywords:
horizon had a negligible effect on the fungal community composition. Diverse soil condi-
Deciduous forest
tions and host trees likely support the high richness of ectomycorrhizal fungi in the wooded
Ectomycorrhizal fungal community
meadow ecosystem. Direct sequencing integrated with interpolation and extrapolation
ITS sequencing
methods are promising to identify the fungi at the species level and to compare species rich-
Nature conservation
ness between communities of ectomycorrhizal fungi.
Rarefaction
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Seminatural ecosystems
Soil horizons
Species richness extrapolation
Introduction
Wooded meadows are seminatural, sparsely wooded ecosystems that have developed due to hay-making or sheep grazing
in low-productive areas in Europe. A few retained trees have
created heterogeneous light conditions and soil nutrient gradients. Such patchiness, together with shallow, nutrientpoor soils, support high small-scale species richness of plants
in wooded meadows (Kull & Zobel 1991).
During the last century, industrialization and urbanization have strongly affected land use and reduced the
importance of traditional farming methods, driving vast
countryside areas to abandonment (Vitousek 1994; DeFries
2002). Seminatural meadows and wooded meadows have
been most affected among seminatural ecosystems (Kukk
& Kull 1997; van Dijk 2002). Wooded meadows covered immense areas especially at low-productive coastal and mountainous sites throughout Europe. At present more than 99 %
of the wooded meadows have been abandoned, and have
developed naturally into thickets, bushlands and marshlands (Kukk & Kull 1997). Of similar ecosystems, North
American oak savannas have declined 5000-fold due to cessation of prescribed burning in the last few hundred years
(Nuzzo 1986).
* Corresponding author.
E-mail address: [email protected].
y These authors contributed equally to this work.
0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.04.007
Ectomycorrhizal fungi in a wooded meadow
In addition to high plant diversity, wooded meadows support many threatened orchids and agarics (Kukk & Kull 1997;
Kalamees 2004; Watling 2005). Of the rare fungi, fruit bodies
of calciphilous Boletus spp. and Entoloma spp. are prominent
in wooded meadows, but absent in nearby forests and
strongly overgrown wooded meadows. Autumnal fruiting of
both rare and common fungi is considerably reduced if the
meadows remain unmown in summer (Kalamees 1979). Similarly, plant species richness declines in a few years after cessation of management due to shading by rapidly growing tree
seedlings and bushes. Restoration of wooded meadows is
costly and plant species require decades to re-establish if
they are lost from the seed bank (Stampfli & Zeiter 1999).
Ectomycorrhizal (EcM) fungi provide mineral nutrition to
the dominant trees (Quercus robur, Betula spp., Tilia cordata,
etc.) in North European wooded meadows. EcM fungi differ in
enzymatic activities (Courty et al. 2005) and deliver speciesspecific benefits to their host plants (van der Heijden & Kuyper
2003), which render their biodiversity of high importance to
plant nutrition. EcM fungi are highly diverse in most ecosystems, comprising tens of observed species. Similar or even
larger number of species remains undetected due to rarity
and aggregated distribution of EcM fungal species on adjacent
root tips (Horton & Bruns 2001; Taylor 2002). The natural
mechanisms promoting high fungal diversity have remained
unclear, but it seems likely that differential preference for
soil conditions and host plants play the key role (Bruns 1995).
Rarefaction (interpolation) provides a powerful alternative
for species richness comparisons between sites and experimental conditions. When sample size is unequal among treatments, rarefaction facilitates biodiversity comparisons by
interpolating randomized species accumulation curves to
the same sample size, including calculation of confidence
intervals (Gotelli & Colwell 2001; Taylor 2002). Similarly,
extrapolation (i.e. estimation of the amount of unobserved
species) methods enable comparisons between sites and studies that employ different sampling schemes. Extrapolation
methods exploit the relative number or abundance of the rarest species, or predict the plateau of species accumulation
curves to estimate the total number of unseen species (Colwell
& Coddington 1994; Keating et al. 1998).
In this study we intended to determine the community
composition of EcM fungi in a single wooded meadow. We
hypothesized that the community structure and species richness differ between the managed and unmanaged (hereafter
forest) parts of the wooded meadow and between soil horizons. Using direct sequencing of EcM root tip DNA and species
richness extrapolation, we demonstrate an unusually high
below-ground diversity of EcM fungi. The forest and wooded
meadow harbour different communities of EcM fungi.
Materials and methods
Study site
Sampling was performed in a traditionally managed wooded
meadow and an adjacent forest at Tagamõisa, Saaremaa Island, northwestern Estonia (58 270 N, 22 000 E). The Tagamõisa
wooded meadow was selected, because it comprised
735
contrasting managed and forested areas with known history.
The study area arose from the Baltic Sea ca 5000 y before present and lies ca 8 m above sea level. The climate is dry maritime
with mean annual rainfall of 550 mm and mean annual
temperature of þ5.8 C. The Tagamõisa wooded meadow originates at least 300 y ago (T. Ojala, pers. comm.). Haymaking
and sheep grazing facilitated the development of a highly
diverse flora (up to 67 species m2), including several rare
orchids (Kukk & Kull 1997). The wooded meadow was
partly abandoned during World War II, resulting in rapid
forestation and decline in plant species richness. Several
inconsistent restoration attempts resulted in partial recovery of the wooded meadow during 1970 to 1990. Since the
mid-1990s, most of the area had been mown annually in
July and slightly grazed in late August (Kukk & Kull
1997). The forested area has remained unmanaged for
>60 y.
Vernal flooding has resulted in mound development especially in the forest. The soil is classified as a MollisihumiRendzic Leptosol (FAO et al. 1998), with a thick O-horizon (up
to 7 cm depth) and a sandy A-horizon (from 5–7 to 25–35 cm
depth) on maritime sand (below 25–35 cm depth) and limestone (below 90 cm depth). Both the wooded meadow and forest were dominated by downy birch (Betula pubescens),
common oak (Quercus robur), hazel (Corylus avellana), European
aspen (Populus tremula), black alder (Alnus glutinosa) and smallleaved lime (Tilia cordata). Of these, P. tremula, B. pubescens,
C. avellana, and A. glutinosa dominated the undergrowth.
Ground vegetation of the managed meadow was predominantly composed of a dense turf of Poaceae spp. and Cyperaceae
spp. The forested area was covered by sparse Vaccinium myrtillus, Hepatica nobilis, Oxalis acetosella, and Carex spp.
To obtain reference sequences for species-level identification of EcM fungi, we performed irregular fruit body surveys
in summer and autumn, 2003 to 2005. The most abundant
agarics included Lactarius pubescens, L. evosmus, Leccinum
spp., Boletus luridus, Inocybe rimosa and I. maculata. Of taxa
with resupinate fruit bodies, Tomentella terrestris, T. cinerascens,
and Amphinema byssoides were the most common. Peziza
michelii and Hymenogaster spp. were the most abundant cup
fungi and taxa with hypogeous fruit bodies, respectively. Boletus spp. fruited exclusively in the wooded meadow, whereas
T. cinerascens, and A. byssoides were found only in the forest.
Sampling
Eight plots (6 m diam) were established in both the wooded
meadow and forest (Fig 1). Plots were randomly distributed
between 15–60 m from the well-defined community edge and
at least 25 m from each other. Half of the plots were established
around randomly selected individual birch trees >50 y old
(hereafter designated birch plots) and the rest around randomly selected spots. Five to six 15 15 cm soil cores were
taken at random from each plot using a spade and a sharp
knife. The soil cores were separated into two root samples:
the O-horizon sample to 5 cm depth and the A-horizon sample
from 15–20 cm depth. In cases where root samples comprised
an insufficient amount (<30 cm in length) of living roots,
another soil core was taken instead. EcM roots were manually
separated from the soil and non-EcM roots after soaking the
736
L. Tedersoo et al.
Three soil samples (125 cm3) were taken from each birch
plot from both O-horizon and A-horizon, and pooled by horizon. Soil pH was determined in 1 N potassium chloride solution. Concentrations of calcium and magnesium were
measured optically in 1 M ammonium acetate (Tecator ASTN
90/92; Page et al. 1982). Phosphorus and potassium were determined in ammonium lactate (Tecator ASTN 9/84; AOAC 1990).
Organic matter content was determined based on the loss on
ignition for 2 h at 360 C.
DNA extraction, PCR and sequencing
Fig 1 – Scheme of the study area at Tagamõisa including
the managed and unmanaged (forest) wooded meadow.
Randomly selected plots are shown as circles. B, only birch
roots sampled.
root samples in tap water. Only birch roots were further analysed in birch plots, whereas the roots of all EcM trees were
studied in other plots. Birch roots were recognized by a characteristic bright red bark, mild taste, and frequent ramification of
long roots. No attempt was made to confirm the host species
using molecular techniques. Roots were cut into 2–3 cm fragments and laid onto large Petri dishes filled with water. All
EcM root tips were assigned to morphotypes on 12 randomly
selected root fragments, and counted. Two individual root
tips of each morphotype from each sample were mounted
into CTAB lysis buffer (100 mM Tris-HCI (pH 8.0), 1.4 M NaCl,
20 mM EDTA, 2% cetyl-trimethyl-ammonium-bromide) and
kept at room temperature until molecular analyses. An additional root tip cluster of each morphotype was stored in FEA
(90 % formalin, 5 % ethanol, 5 % acetic acid). Morphotypes
were further separated into anatomotypes based on the structure of mantle, cystidia, emanating hyphae and rhizomorphs
(Agerer 1991). One or two root tips of each anatomotype per
plot were subjected to DNA extraction and sequencing.
DNA was extracted according to Gardes & Bruns (1993), including UltraClean 15 (Mo Bio Laboratories, West Carlsbad, California) treatment. PCR was performed using Ready To Go
beads (Amersham Pharmacia Biotech., Piscataway, New Jersey), 0.5 ml of 20-mM primers, 6 ml DNA extract and 18 ml distilled
water. By default, primer pairs ITS1F (50 cttggtcatttagaggaagtaa
30 ) and LR21 (50 acttcaagcgtttcccttt 30 ) or ITS1F and TW13
(50 ggtccgtgtttcaagacg 30 ) were used to selectively amplify the
fungal rDNA ITS and partial LSU (Fig 2). If multiple or no PCR
products were obtained, the DNA of another root tip was
extracted or the same DNA was reamplified, using the
following primers in several combinations: ITS1 (50 tccgtaggtg
aacctgcgg 30 ), ITS4 (50 tcctccgcttattgatatgc 30 ), ITS4B (50 caggag
acttgtacacggtccag 30 ), LR5 (50 tcctgagggaaacttcg 30 ), LR7 (50 tacta
ccaccaagatct 30 ). Well-defined double bands were cut from agarose gels and melted in a Tris–borate–ethylenediaminetetraacetate (TBE) solubilizer and sodium iodide (2:1:9 v/v/v) at 55 C,
followed by UltraClean treatment and PCR amplification using
internal primers. As some anatomotypes consistently resulted
in multiple PCR products (Leccinum spp.), the LSU was amplified
using a primer pair LR0R (50 acccgctgaacttaagc 30 ) and TW13.
PCR included an initial 3 min at 95 C, followed by 35 cycles
of 30 s at 95 C, 30 s at 55 C (50 C when primer ITS4B was included) and 1 min at 72 C (2 s increment time for each following cycle; final cycle, 10 min). PCR products (2 ml) were run with
bromophenol blue (1 ml) on 1 % agarose gels with ethidium bromide for ca 1 h and visualized under the UV light. Single PCR
products were purified using Exo-Sap enzymes (Sigma, St
Louis, Missouri) according to the manufacturer’s instructions.
Sequencing was performed in a CeqTM 8000 Genetic Analysis System (Beckman Coulter, Fullerton, California) using
a primer ITS3 (50 gcatcgatgaagaacgcagc 30 ) to obtain sequences
up to 600 bases in length, including both the variable ITS2 and
conserved flanking LSU region. The LSU of Leccinum spp. was
sequenced using a primer ctb6 (50 gcatatcaataagcggagg 30 ). Sequences were checked for possible machine errors and
grouped by similarity (>99 % sequence identity) using
Sequencher 4.2 software (Gene Codes, Ann Arbor, Michigan).
Identity groups were further sequenced using primers ITS1 or
ITS4. The ITS1 rDNA was omitted from further analyses due
to poor sequence quality. To reveal the taxonomic affinities
Fig 2 – Map of primers used for amplification and sequencing rDNA ITS and partial LSU (28 S).
Ectomycorrhizal fungi in a wooded meadow
737
of fungal species, blastn or fasta3 searches of the ITS2 (without
any bases of flanking conserved regions) were carried out
against the public sequence databases National Center for Biotechnology Information (NCBI), European Molecular Biology
Laboratory (EMBL) and UNITE (Kõljalg et al. 2005). We also performed blastn searches of partial LSU sequences if the ITS2 sequences could not be fully matched to any fruit body
sequences. We selected 98 % of ITS2 sequence identity as
a value of a molecular species criterion, because no sequences
obtained in this study displayed pair-wise identity between 97–
98.5 %. Moreover, many species of basidiomycetes exhibited
equally intense double peaks in sequence chromatograms,
which never included >1.5 % of the bases. These double peaks
probably represent polymorphism in ITS alleles (see Hibbett
2001; Selosse et al. 2002). To improve the identification at the
species level, we sequenced the ITS2 or the whole ITS region
of 16 fruit bodies from 12 fungal species as described above.
Both fruit body (Table 1) and root tip (Table 2) sequences
were submitted to EMBL. Throughout the text we prefer using
the major clades of Homobasidiomycetes (sensu Larsson et al.
2004) rather than orders, because there is much polyphyly
among taxa with resupinate and hypogeous fruit bodies.
To demonstrate the phylogenetic position of sebacinoid
EcM species among Sebacinaceae (Fig 3), we manually aligned
all congeneric fruit body and a few best-matching root tip
sequences. We performed a parsimony analysis using PAUP
4.0d81 (Swofford 2002) with tree bisection–reconnection (TBR)
as a branch-swapping algorithm, gaps as the fifth character,
and 1000 random start replications. Efibulobasidium rolleyi was
selected as an outgroup taxon based on LSU phylogeny. BS
support was calculated based on 1000 permutations.
the forest and wooded meadow and between soil horizons,
area-based rarefaction curves with 95 % confidence intervals
were calculated, using a computer program EstimateS (Colwell
2004). In these analyses, plots were sampled randomly with replacement, because otherwise confidence intervals are meaningless in the upper end of the rarefaction curve (Colwell et al.
2004). To demonstrate the total EcM fungal diversity of the
study site and to estimate the sufficiency of sample size, we
calculated area-based species accumulation curves, sampling
plots randomly without replacement. Incidence-based species
richness estimates Jackknife2 (Burnham & Overton 1979) and
Chao2 (Chao 1987) were calculated to evaluate their performance on this particular data set and to estimate the number
of unseen species. We always used area-based rarefaction and
species incidence, because EcM root tips do not represent fungal individuals. As discussed by Colwell et al. (2004), area-based
rarefaction better reflects the true distribution of species,
whereas individual-based methods assume non-aggregated
distribution of species and individuals.
Detrended correspondence analysis (DCA) and canonical
correspondence analysis (CCA) were used to reveal trends
within the ectomycorrhizal fungal community and in relation
to measured environmental variables, respectively. Species
frequency with down-weighted rare species was used in
both analyses. Wooded meadow versus forest, soil horizon,
plot type (birch plot or all-tree plot) and ordinated plot (by
position, see Fig 1) were fitted in the species space to demonstrate their relative importance and correlation with the
main axes. Birch plots were subjected to CCA. Ordination analyses were performed using PC-ORD (McCune & Mefford 1999).
Statistical analyses
Results
The plot was selected as a sampling unit, because samples
from such a small area are likely strongly autocorrelated and
not independent (Taylor 2002; Lilleskov et al. 2004). To compare
the large-scale differences in total species diversity between
Identification of EcM fungi
We distinguished 172 species of EcM fungi based on morphotyping 26,547 and sequencing 468 root tips. Among these, ten
Table 1 – List of fruit bodies sequenced for this study
Species
Collection
number
Forest type and potential hosts
Boletus radicans
Entoloma sinuatum
Geastrum triplex
Gyrodon lividus
TAA185095
TAA185096
TAA158536
TAA185097
Inocybe maculata
Lactarius evosmus.
Rhizopogon roseolus
Russula velenovskyi
Sebacina dimitica
Sebacina epigaea
Sebacina epigaea
Sebacina helvelloides
Sebacina incrustans
Sebacina incrustans
Sebacina incrustans
Sebacina sp.
TAA185093
TAA185092
TAA185325
TAA185076
TAA169135
TAA185250
TAA167039
TAA164447
TAA180827
TAA185123
TAA185242
TAA185327
Calcareous wooded meadow: Quercus robur, Betula sp.
Mixed forest: Pinus sylvestris, Q. robur, Corylus avellana
Mixed forest: Picea abies
Wet wooded meadow: Alnus glutinosa, B. pubescens,
Salix sp., Q. robur
Wetland forest: B. pubescens, A. glutinosa
Wetland forest: B. pubescens, A. glutinosa, Q. robur
Mixed forest: P. sylvestris, Q. robur, C. avellana
Wetland forest: A. glutinosa, B. pubescens, Q. robur
Wetland forest: Alnus sp., Betula sp.
Old-growth forest: P. abies, Tilia cordata, Populus tremula
Wetland forest: P. tremula
Mixed forest: P. abies, B. pendula
Old-growth forest: P. abies, Tilia cordata, Populus tremula
Forest plantation: P. abies
Forest plantation: P. abies
Wooded meadow: Q. robur, C. avellana, P. tremula, Betula sp.
Locality
EMBL
accession
number
Tagamõisa, Saare Co, Estonia
Kudjape, Saare Co, Estonia
Laulasmaa, Harju Co, Estonia
Tagamõisa, Saare Co, Estonia
AJ966742
AJ966745
AJ966746
AJ966743
Tagamõisa, Saare Co, Estonia
Tagamõisa, Saare Co, Estonia
Karuse, Lääne Co, Estonia
Tagamõisa, Saare Co, Estonia
Restu, Valga Co, Estonia
Järvselja, Tartu Co, Estonia
Rokka, Jõgeva Co, Estonia
Hobusemägi, Valga Co, Estonia
Järvselja, Tartu Co, Estonia
Aovere, Tartu Co, Estonia
Kärde, Tartu Co, Estonia
Kalli-Nedrema, Lääne Co, Estonia
AJ966749
AJ966747
AJ966744
AJ966748
AJ966757
AJ966754
AJ966756
AJ966750
AJ966751
AJ966752
AJ966753
AJ966755
738
L. Tedersoo et al.
Table 2 – Relative abundance of EcM fungal species arranged in descending order of root tip abundance in the whole
community
Ectomycorrhizal fungal species
Best match
Root tips colonized (%)
Wooded meadow
Species
EMBL
accession
Cenococcum geophiluma
Lactarius pubescens
Inocybe maculata
Boletus luridus
Thelephoraceae sp1a
Thelephoraceae sp12a
Cortinarius sp6
Sebacinaceae sp4
Sebacinaceae sp7
Lactarius evosmus
Lactarius torminosus
Sebacinaceae sp5
Meliniomyces spa
Paxillus sp1
Thelephoraceae sp10a
Sebacinaceae sp6
Thelephoraceae sp6a
Paxillus sp3
Hebeloma sp3
Elaphomyces spb
Pyronemataceae sp
Tomentella pilosa
Leccinum rigidipes
Melanogaster variegatusb
Sebacinaceae sp1
Sebacinaceae sp11
Sebacinaceae sp8
Inocybe sp14
Inocybe sp1
Genea sp1a,b
Pachyphloeus sp1b
Thelephoraceae sp7
Thelephoraceae sp26
Sebacinaceae sp15
Thelephoraceae sp17
Tomentellopsis sp3
Thelephoraceae sp24
Hebeloma sp5
Thelephoraceae sp11
Wilcoxina sp
Sebacinaceae sp9
Inocybe sp3
Sebacinaceae sp3
Thelephoraceae sp30
Inocybe sp11
Boletus radicans
Inocybe sp4
Thelephoraceae sp38
Cortinarius sp4
Tuber rapaeodorumb
Thelephoraceae sp36a
Hygrophorus cossus
Inocybe sp8
Thelephoraceae sp2
Thelephoraceae sp5a
Russula claroflava
Thelephoraceae sp50a
Cortinarius sp8
Gyrodon lividus
ndc
AJ893208
AJ893277
AJ893197
AJ893292
AJ893303
AJ893233
AJ893256
AJ893259
AJ893210
AJ893209
AJ893257
nd
AJ893205
AJ893301
AJ893258
AJ893297
AJ893207
AJ893222
AJ893252
AJ893243
AJ893299
AJ893200
AJ893204
AJ893253
AJ893263
AJ893260
AJ893284
AJ893271
AJ893239
AJ893241
AJ893298
AJ893317
AJ893267
AJ893308
AJ893355
AJ893315
AJ893224
AJ893302
AJ893249
AJ893261
AJ893273
AJ893255
AJ893321
AJ893281
AJ893198
AJ893274
AJ893329
AJ893231
AJ893250
AJ893327
AJ893288
AJ893278
AJ893293
AJ893296
AJ893214
AJ893341
AJ893235
AJ893199
Forest
Species
Accession
number
Identity
(%)
O-horizon
A-horizon
O-horizon
A-horizon
nd
Lactarius pubescens
Inocybe maculata
Boletus luridus
Tomentella coerulead
Tomentella badiad
Cortinarius pulchellus
Sebacina epigaead
Sebacina incrustansd
Lactarius evosmusd
Lactarius torminosus
Sebacina epigaead
nd
Paxillus involutus
Tomentella atramentariad
Sebacina helvelloidesd
Tomentella bryophilad
Paxillus involutus
Hebeloma ammophilum
Elaphomyces muricatusd
Pyronemataceae spe
Tomentella pilosa
Leccinum rigidipes
Melanogaster variegatus
Sebacina incrustansd
Tremellodendron pallidum
Sebacina dimiticad
Inocybe pudica
Inocybe quetiodorf
Genea spe
Pachyphloeus spe
Tomentella cinerascensd
Tomentella viridula
Sebacina epigaead
Tomentella lilacinogrisead
Tomentellopsis echinospora
Tomentella ferruginea
Hebeloma incarnatulum
Tomentella cinerascensd
Wilcoxina mikolae
Sebacina epigaea
Inocybe rimosad
Sebacina epigaead
Tomentella punicead
Inocybe rimosad
Boletus radicansd
Inocybe rimosad
Thelephora caryophyllead
Cortinarius heterosporus
Tuber rapaeodorum
Tomentella bryophilad
Hygrophorus cossus
Inocybe flocculosa
Tomentella bryophilad
Tomentella bryophilad
Russula claroflava
Tomentella bryophilad
Cortinarius atrocaerulaeus
Gyrodon lividusd
nd
AY336958
AJ534933
AY278766
UDB000266
UDB000238
AY083192
UDB000975
UDB000979
UDB000983
AY336959
UDB000977
nd
AY525980
UDB000235
UDB000972
UDB000253
AY585921
AY308585
UDB000092
nd
AJ421252
AF454584
AJ555534
UDB000979
AF384862
UDB000974
AY228341
EL115_04
nd
nd
UDB000232
AF272914
UDB000977
UDB000272
AJ410767
AF272909
AF124684
UDB000232
AY219841
AF490397
UDB000103
UDB000977
UDB000959
UDB000103
UDB000980
UDB000103
UDB000119
AF268894
AJ557525
UDB000253
AY242852
AY228534
UDB000253
UDB000253
AY061665
UDB000253
AY083178
UDB000981
nd
100.0
100.0
99.6
92.3
93.0
91.0
95.5
89.4
100.0
98.1
90.7
nd
100.0
91.2
88.7
91.8
100.0
100.0
82.6
nd
100.0
100.0
99.6
88.9
87.7
93.4
71.1
100.0
nd
nd
92.5
99.5
92.8
99.6
94.4
90.6
100.0
98.7
80.4
86.5
94.0
88.2
99.8
86.8
100.0
88.3
94.7
82.0
100.0
93.3
99.5
80.7
92.8
93.3
100.0
91.8
97.8
100.0
19.74
9.68
1.02
5.43
8.32
7.96
0.00
0.00
0.14
2.71
0.00
0.00
1.16
0.00
0.00
0.00
0.00
2.65
0.00
0.00
2.01
1.21
0.87
1.23
4.02
0.00
0.00
0.00
2.71
2.06
2.84
0.00
0.00
2.59
0.80
0.00
0.00
0.00
0.00
0.19
0.00
0.00
0.00
2.06
0.03
0.77
0.05
0.00
0.00
0.00
0.00
0.00
0.00
0.73
0.00
0.00
0.00
1.38
0.83
3.84
18.95
0.83
24.21
4.93
5.92
0.00
0.00
0.00
0.55
0.00
0.00
2.73
0.00
7.88
0.90
0.00
0.00
0.07
0.00
3.21
1.72
0.55
3.71
0.00
0.00
0.02
0.00
0.31
0.92
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.71
0.00
0.00
0.00
0.11
0.11
1.55
0.09
2.34
0.00
0.00
0.00
0.00
0.00
1.09
0.00
0.00
0.00
0.02
0.70
20.34
7.73
3.28
0.00
0.10
0.00
4.10
5.43
1.88
2.92
4.04
3.70
1.75
3.51
0.01
3.02
3.16
1.62
3.14
1.01
0.16
0.30
1.63
0.00
0.00
1.96
1.72
0.46
0.00
0.00
0.00
1.60
0.32
0.00
0.90
1.07
0.22
1.31
0.67
0.00
0.21
1.00
0.24
0.00
0.87
0.00
0.41
0.00
0.64
0.61
0.00
0.94
0.26
0.00
0.12
0.22
0.62
0.00
0.00
22.48
0.52
20.90
0.00
1.57
0.00
4.58
1.79
6.47
0.95
0.19
0.50
0.53
0.64
0.07
0.00
0.34
0.33
0.00
3.53
0.45
1.48
0.09
0.00
0.00
0.00
0.31
2.43
0.02
0.12
0.00
0.00
2.10
0.00
0.14
0.64
2.10
0.09
1.19
0.00
1.89
0.48
1.81
0.00
0.40
0.00
1.08
0.00
0.64
0.67
1.75
0.00
1.17
0.00
1.38
1.07
0.34
0.00
0.00
Ectomycorrhizal fungi in a wooded meadow
739
Table 2 (continued)
Ectomycorrhizal fungal species
Best match
Root tips colonized (%)
Wooded meadow
Species
Thelephoraceae sp52
Pachyphloeus sp2b
Sebacinaceae sp2
Thelephoraceae sp13
Russula firmula
Thelephoraceae sp4
Russula velenovskyi
Lactarius deterrimus
Thelephoraceae sp47
Sebacinaceae sp18
Cortinarius sp7
Thelephoraceae sp21
Thelephoraceae sp35a
Geopora sp
Alnicola sp2
Lactarius scrobiculatus
Russula fuscorubroides
Inocybe sp2
Amphinema sp2
Thelephoraceae sp41
Thelephoraceae sp39
Thelephoraceae sp23
Hebeloma sp1
Alnicola sp1
Tricholoma muricatum
Peziza michelii
Thelephoraceae sp9
Inocybe sp5
Laccaria sp1
Thelephoraceae sp14
Leccinum sp3
Thelephoraceae sp34
Melanogaster sp1b
Inocybe sp16
Cortinarius sp3
Genea sp2a,b
Leccinum sp2
Thelephoraceae sp51a
Thelephoraceae sp31a
Thelephoraceae sp20
Helvella sp
Unknown sp2
Thelephoraceae sp55a
Lactarius camphoratus
Sebacinaceae sp12
Pezizaceae sp2
Amphinema sp3
Pezizaceae sp1
Sebacinaceae sp13
Inocybe sp13
Thelephoraceae sp25a
Thelephoraceae sp58a
Thelephoraceae sp53
Paxillus sp2
Tuber sp2b
Thelephoraceae sp27a
Thelephoraceae sp16a
Hebeloma sp2
Thelephoraceae sp33a
Thelephoraceae sp28a
EMBL
accession
AJ893343
AJ893242
AJ893254
AJ893304
AJ893215
AJ893295
AJ893216
nd
AJ893338
AJ893270
AJ893234
AJ893312
AJ893326
AJ893248
AJ893227
AJ893213
AJ893218
AJ893272
AJ893290
AJ893332
AJ893330
AJ893314
AJ893220
AJ893226
AJ893287
AJ893246
AJ893300
AJ893275
AJ893237
AJ893305
AJ893202
AJ893325
AJ893203
AJ893285
AJ893230
AJ893240
AJ893201
AJ893342
AJ893322
AJ893311
AJ893247
nd
AJ893346
AJ893212
AJ893264
AJ893245
AJ893291
AJ893244
AJ893265
AJ893283
AJ893316
AJ893349
AJ893344
AJ893206
AJ893251
AJ893318
AJ893307
AJ893221
AJ893324
AJ893319
Species
Tomentella ellisiid
Pachyphloeus spe
Sebacina dimiticad
Tomentella lapidum
Russula firmula
Tomentella sublilacinad
Russula velenovskyid
nd
Tomentella subclavigera
Tremellodendron pallidum
Cortinarus saturninus
Tomentella umbrinosporad
Tomentella coerulead
Geopora spe
Alnicola bohemica
Lactarius scrobiculatus
Russula fuscorubroides
Inocybe flocculosa
Amphinema byssoides
Tomentella lapidad
Tomentella lateritia
Tomentella bryophilad
Hebeloma brunneifolium
Alnicola scolecina
Tricholoma muricatum
Peziza micheliid
Tomentella viridula
Inocybe nitidiuscula
Laccaria amethystea
Thelephora anthocephala
Leccinum rotundifoliaeg
Tomentella ferruginea
Melanogaster variegatus
Inocybe godeyid
Cortinarius teraturgus
Genea spe
Leccinum variicolorg
Thelephora anthocephala
Tomentella badiad
Thelephora caryophyllead
Helvella elastica
nd
Tomentella lapida
Lactarius camphoratusd
Sebacina epigaea
Pezizaceae spe
Amphinema byssoides
Pezizaceae spe
Sebacina helvelloidesd
Inocybe flocculosa
Tomentella lapidad
Tomentella coerulead
Tomentella subclavigera
Paxillus involutus
Tuber rufum
Tomentella ferruginea
Tomentella bryophilad
Hebeloma hiemale
Tomentella botryoides
Tomentella bryophilad
Forest
Accession Identity O-horizon A-horizon O-horizon A-horizon
number
(%)
UDB000219
nd
UDB000974
AF272941
AF418631
UDB000230
UDB000982
nd
AF272939
AF384862
AY083189
UDB000233
UDB000951
nd
AF124712
AF140263
AF418624
AY228534
AY838271
UDB000250
AF272926
UDB000253
AY309959
AF325629
AF458440
UDB000986
AF272914
AJ534934
AF539737
AF272927
AF139704
AF272909
AJ555534
UDB000101
AF389151
nd
AF139706
AF272927
UDB000952
UDB000119
AF335455
nd
AF272941
UDB000387
AF490397
AF092098
AY838271
nd
UDB000972
AY228534
UDB000250
UDB000951
AF272939
AY585917
AF106892
AF272909
UDB000253
AF124669
AF272912
UDB000253
97.7
nd
96.2
90.1
100.0
88.4
100.0
nd
93.6
95.2
98.3
94.5
92.6
nd
83.0
99.6
100.0
66.8
91.3
96.4
88.8
92.4
100.0
100.0
99.1
100.0
96.9
77.0
78.0
95.0
99.2
88.8
87.2
91.2
92.5
nd
98.8
99.6
100.0
92.0
86.2
nd
91.6
100.0
88.8
72.3
96.0
nd
89.1
68.7
97.3
96.4
92.9
100.0
65.5
95.1
96.9
100.0
93.3
92.0
0.00
1.34
0.00
0.00
0.00
1.23
0.00
0.00
0.00
0.00
1.09
0.56
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.56
0.77
0.10
0.73
0.00
0.00
0.32
0.66
0.07
0.00
0.00
0.36
0.07
0.07
0.00
0.00
0.53
0.44
0.00
0.00
0.32
0.39
0.00
0.00
0.00
0.17
0.00
0.32
0.39
0.36
0.00
0.10
0.00
0.00
0.00
0.00
0.32
0.00
0.00
0.31
0.00
0.00
0.00
0.22
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.63
0.00
1.29
0.00
0.00
0.00
1.03
0.00
0.28
0.00
0.35
0.00
0.00
0.90
0.46
0.00
0.48
0.00
0.00
0.35
0.70
0.70
0.00
0.00
0.00
0.00
0.00
0.00
0.24
0.11
0.00
0.00
0.00
0.35
0.57
0.00
0.02
0.04
0.00
0.00
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.00
0.39
0.04
0.00
0.67
0.63
0.74
0.03
0.35
0.33
0.61
0.64
0.00
0.00
0.12
0.00
0.19
0.51
0.48
0.00
0.30
0.00
0.00
0.22
0.00
0.41
0.00
0.00
0.00
0.00
0.17
0.08
0.00
0.00
0.00
0.18
0.06
0.00
0.05
0.03
0.30
0.00
0.00
0.21
0.13
0.08
0.00
0.00
0.06
0.00
0.00
0.00
0.16
0.00
0.21
0.21
0.04
0.00
0.18
0.17
0.00
0.00
1.32
0.00
0.00
0.19
0.00
0.00
0.67
0.64
0.07
0.00
0.00
0.00
0.83
0.00
0.57
0.00
0.00
0.02
0.29
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
0.22
0.38
0.52
0.00
0.00
0.00
0.29
0.45
0.00
0.00
0.48
0.00
0.00
0.00
0.10
0.26
0.33
0.00
0.00
0.00
0.00
0.00
0.40
0.00
0.38
0.00
0.00
0.28
0.00
0.00
0.03
0.00
0.00
(continued on next page)
740
L. Tedersoo et al.
Table 2 (continued)
Ectomycorrhizal fungal species
Best match
Root tips colonized (%)
Wooded meadow
Species
Pseudotomentella sp1
Thelephoraceae sp32a
Thelephoraceae sp3a
Thelephoraceae sp29
Thelephoraceae sp18
Hebeloma sp4
Russula sphagnophila
Thelephoraceae sp46a
Amphinema sp1
Thelephoraceae sp22
Sebacinaceae sp17
Lactarius cf. necator
Thelephoraceae sp48a
Unknown sp3
Thelephoraceae sp43
Thelephoraceae sp54
Thelephoraceae sp45
Thelephoraceae sp60a
Hebeloma sp6
Thelephoraceae sp40a
Inocybe sp12
Inocybe sp6
Inocybe sp9
Thelephoraceae sp57a
Unknown sp4
Unknown sp1a
Thelephoraceae sp37
Inocybe sp10
Sebacinaceae sp16
Rhizopogon luteolus
Thelephoraceae sp42a
Sebacinaceae sp14
Cortinarius sp5
Thelephoraceae sp56a
Cortinarius sp2
Thelephoraceae sp59a
Thelephoraceae sp49
Thelephoraceae sp15
Inocybe sp17
Tomentellopsis sp1
Sebacinaceae sp10
Hebeloma sp7
Cortinarius sp10
Thelephoraceae sp44
Thelephoraceae sp19
Cadophora finlandica
Unknown sp5
Tomentellopsis sp2
Russula laricina
Pseudotomentella sp2
Laccaria sp2
Cortinarius sp9
Cortinarius sp1
EMBL
accession
AJ893351
AJ893323
AJ893294
AJ893320
AJ893309
AJ893223
AJ893219
AJ893337
AJ893289
AJ893313
AJ893269
AJ893211
AJ893339
nd
AJ893334
AJ893345
AJ893336
nd
AJ893225
AJ893331
AJ893282
AJ893276
AJ893279
AJ893348
nd
nd
AJ893328
AJ893280
AJ893268
AJ893196
AJ893333
AJ893266
AJ893232
AJ893347
AJ893229
AJ893350
AJ893340
AJ893306
AJ893286
AJ893353
AJ893262
AJ893238
nd
AJ893335
AJ893310
nd
nd
AJ893354
AJ893217
AJ893352
nd
AJ893236
AJ893228
Species
Pseudotomentella tristis
Tomentella lilacinogrisead
Tomentella ferruginea
Tomentella galzinii
Tomentella umbrinospora
Hebeloma nigellum
Russula sphagnophila
Tomentella lapidad
Amphinema byssoides
Tomentella lilacinogrisead
Sebacina epigaead
Lactarius necatord
Tomentella atramentariad
nd
Tomentella bryophilad
Tomentella botryoidesd
Tomentella ramosissima
nd
Hebeloma aestivale
Tomentella atramentaria
Inocybe flocculosa
Inocybe maculta
Inocybe aff. boltoniif
Tomentella stuposad
nd
nd
Tomentella lilacinogrisead
Inocybe flocculosa
Sebacina spd
Rhizopogon luteolus
Tomentella viridula
Tremellodendron schweinitzii
Cortinarius casimirid
Tomentella terrestris
Cortinarius acutovelatus
Tomentella stuposad
Tomentella lateritiad
Tomentella subclavigerad
Cortinarius subtortus
Tomentellopsis submollis
Sebacina dimiticad
Hebeloma cavipes
nd
Tomentella lapida
Tomentella ferruginea
nd
nd
Tomentellopsis echinospora
Russula laricina
Pseudotomentella tristis
nd
Cortinarius cedriolens
Cortinarius dionyseae
Forest
Accession Identity O-horizon A-horizon O-horizon A-horizon
number
(%)
AF274772
UDB000272
AF272909
AF272928
AF272920
AY311524
AY061719
UDB000249
AY838271
UDB000953
UDB000975
UDB000361
UDB000955
nd
UDB000253
UDB000255
U83480
nd
AY308582
AF272904
AY228534
AF534933
EL 71_03
UDB000246
nd
nd
UDB000272
AY228534
UDB000976
AF062936
AF272914
AY296258
UDB000062
AF272901
AY083175
UDB000248
UDB000963
UDB000259
AY174859
AY641459
UDB000974
AF124670
nd
AF272941
AF272909
nd
nd
AJ410758
AY061685
AF274772
nd
AY083179
AY174813
82.8
94.4
93.6
99.1
99.5
99.5
100.0
96.9
100.0
99.6
94.6
98.4
97.3
nd
91.5
94.8
96.9
nd
98.6
89.8
65.9
72.4
100.0
100.0
nd
nd
93.8
72.4
100.0
100.0
93.3
86.3
94.3
99.1
99.1
97.3
92.9
93.3
65.1
91.7
95.2
95.3
nd
90.6
95.9
nd
nd
100.0
99.1
78.4
nd
97.9
90.0
0.31
0.00
0.29
0.29
0.29
0.00
0.00
0.24
0.15
0.00
0.00
0.00
0.00
0.10
0.17
0.00
0.17
0.00
0.00
0.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.09
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.03
0.03
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
EMBL accession numbers and the best ITS2 sequence matches to fruit body sequences are indicated.
a Species with melanized cell walls.
b Species with presumably hypogeous fruit bodies.
c nd, Not determined.
d Identified according to UNITE database (Kõljalg et al. 2005).
e Identified based on phylogenetic analysis in Tedersoo et al. (2006).
f E. Larsson, unpublished sequence data.
g Identification based on LSU sequence.
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.11
0.02
0.00
0.00
0.00
0.00
0.07
0.20
0.02
0.00
0.00
0.00
0.00
0.00
0.13
0.00
0.00
0.11
0.00
0.11
0.00
0.00
0.09
0.00
0.00
0.00
0.00
0.00
0.04
0.00
0.02
0.00
0.02
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.15
0.14
0.00
0.05
0.00
0.13
0.00
0.12
0.00
0.00
0.00
0.00
0.10
0.10
0.00
0.00
0.08
0.00
0.08
0.00
0.00
0.07
0.00
0.01
0.06
0.00
0.00
0.00
0.05
0.03
0.00
0.03
0.04
0.03
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.01
0.01
0.01
0.00
0.01
0.01
0.01
0.00
0.31
0.00
0.00
0.00
0.03
0.03
0.00
0.00
0.22
0.00
0.22
0.00
0.00
0.00
0.19
0.00
0.00
0.00
0.00
0.00
0.00
0.15
0.00
0.12
0.12
0.00
0.00
0.09
0.00
0.00
0.00
0.00
0.00
0.03
0.00
0.02
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
0.00
0.00
0.00
Ectomycorrhizal fungi in a wooded meadow
741
Fig 3 – One of the three most parsimonious trees demonstrating the phylogenetic position of sebacinoid ectomycorrhizal root
tip sequences (in bold) among identified fruit body sequences as inferred from parsimony analysis of ITS2 sequences.
Numbers above branches indicate BS support.
species were identified solely based on mantle anatomy, because amplification or sequencing failed. Five of these species
remained unknown, possessing insufficient anatomical similarity to any published descriptions. Nine additional root tip sequences (incl. Phialocephala fortinii, Lachnum sp., Irpex sp.,
Polyporus sp., Exidia sp. and Verticillium sp.) were considered
non-EcM saprobes, root or soil inhabiting taxa. Resequencing
of the respective anatomotypes always revealed true EcM fungal species. Based on the high similarity to database sequences,
EcM fungi of Russula and Lactarius spp. were easily identified at
the species level. Conversely, sequences from EcM root tips
corresponding to the thelephoroid clade, Pezizales, Inocybe, Cortinarius and Sebacinaceae (Fig 3), were rarely matched to any
fruit body sequences with >95 % identity. Sequence databases
742
EMBL and NCBI taken together, and UNITE contributed to 55.0
and 39.4 % of the best ITS2 sequence matches, respectively.
L. Tedersoo et al.
Discussion
Diversity of EcM fungi
Diversity and community structure of EcM fungi
This study revealed a few abundant and a large number of
rare species (Fig 4). Cenococcum geophilum was the most common species, occupying 17.8 % of root tips, 30.2 % of root
fragments, 59.0 % of samples and 93.8 % of plots. While C.
geophilum dominated from the root tip to plot scale, other
species were differentially represented at these scales (Fig
4), indicating more or less aggregated distribution. Of these
species, Lactarius pubescens (8.5 %), Inocybe maculata (6.2 %)
and Boletus luridus (5.4 %) exceeded 5 % in relative root tip
abundance. Apart from C. geophilum, I. maculata colonized
the largest number of root fragments (12.5 %) and samples
(28.3 %), while L. pubescens and Tomentella sp1 occupied the
largest number of plots (both 68.8 %), followed by I. maculata
(62.5 %) and Pyronemataceae sp. (56.3 %). Of the rare fungi, ten
species (5.8 %) colonized a single root tip, 22 species (12.8 %)
colonized a single root fragment, 60 species (34.9 %) occurred
only in one sample, and 101 species (58.7 %) were present in
a single plot.
Thelephora/Tomentella was the dominant taxon (60 spp.),
followed by Sebacina/Tremellodendron (18 spp.), Inocybe (16
spp.), Russula/Lactarius (13 spp.), Cortinarius (10 spp.) and Hebeloma/Alnicola (9 spp.). The thelephoroid clade comprised 65
species, followed by euagaric (39 spp.), russuloid (13 spp.)
and bolete (12 spp.) clades. Pezizales comprised 13 species.
Basidiomycetes and ascomycetes accounted for 150 and 17
of the identified species, respectively.
We calculated area-based rarefaction curves and species
richness estimates for the whole EcM fungal community (Fig 5).
The rarefaction curve did not reach a plateau when all
plots were randomly sampled. Chao2 and Jackknife2 functions estimated the total richness of 325 and 329 species,
respectively. Neither of these functions levelled off with
increasing sample size. Jackknife2 produced an estimate
of the observed 172 spp. when ca 35 % of plots and ca
50 % of species were sampled, whereas Chao2 produced
high, but unstable total richness estimates at very small
sample sizes.
The forest and wooded meadow comprised 116 and 88 species, respectively, with no significant difference based on the
overlapping confidence intervals (not shown). Only 32
(18.6 %) species were shared between the two communities.
Species richness in O-horizon (146 spp.) was significantly
higher than in A-horizon (118 spp.; Fig 6). The two soil horizons shared 92 (53.5 %) species. DCA axis1 separated the forest
plots from wooded meadow plots based on their fungal community composition (Fig 7). Axis2 was correlated with weak
effects of plot position and birch plot. EcM fungal community
structures was affected least by soil horizon. Fungal communities of the two soil horizons of the same plot nearly always
clustered together. The first two axes (eigenvalues 0.551 and
0.367) explained 18.5 and 11.4 % of the variance, respectively.
However, all the environmental variables were strongly correlated with CCA axis1 and revealed no distinct patterns based
on species frequency (not shown).
A total of 172 species of EcM fungi were observed in this
snapshot study of 166 root samples and 16 plots. Similarly,
high richness of EcM fungi has been reported in sequencing
studies in a Corsican old-growth Quercus ilex stand (140 species, 120 samples; Richard et al. 2005) and a Californian mixed
old-growth forest (101 species, 144 samples over three years;
Izzo et al. 2005). Using sequencing and extrapolation, Walker
et al. (2005) estimated the total EcM species richness of oak
seedlings between 116 and 143 species in a mature mixed
forest in the Appalachian Mountains (75 observed species,
259 seedlings, 120 plots, two years). Anatomotyping and
RFLP-typing, the most frequently used identification methods
in most previous studies (reviewed in Horton & Bruns 2001),
tend to underestimate the diversity of EcM fungi due to poor
resolution of closely related species (Kårén et al. 1997; Edwards
& Turco 2005).
Unlike plants (Kukk & Kull 1997), communities of EcM
fungi were highly diverse in both the wooded meadow and
forest. However, DCA revealed a substantial difference in
the fungal community composition in the wooded meadow
versus forest, which confirms the observation of only 18.6 %
of shared species. We believe that much of the difference
in species occurrence is attributable to an insufficient sampling effort, where most of the rare species have remained
below the detection limit. Still, habitat quality may play
a substantial role in influencing the frequency and abundance of dominant species. In general, a diverse array of
habitats and hosts likely contribute to the high species richness at the study site. In particular, the wooded meadow soil
has a higher pH, but lower nutrient concentrations than forest soil (L. Tedersoo et al. unpublished). However, CCA
revealed no obvious effect of the measured environmental
variables on the EcM fungal community, suggesting that
the most important soil parameters have remained unmeasured. Alternatively, plot size may be considerably larger
than the size of a nutrient patch (Agerer & Göttlein 2003),
or fungal species possess wide ecological amplitudes. Reich
et al. (2001) demonstrated lower nitrogen mineralization
and litter nitrogen concentration, lower shoot and fine root
net primary production, but higher fine root biomass and
lifespan when canopy openness increased in an American
oak savanna. Grassland soils are more compacted and
drought sensitive because of direct sun exposure (Wilson
1993), whereas differences in soil patch quality are more
pronounced in forests due to abundant dead wood, pit and
mound structure, and nutrient uptake by extensive tree
root systems. Stem flow and litter fall account for higher nutrient concentrations, creating local hotspots of enzymatic
and microbial activity below tree canopies (Dahlgren et al.
1997; Waldrop & Firestone 2004). Nutrient gradients influence EcM fungal species composition (Lilleskov et al. 2002)
and partly explain the differential preference of EcM fungi
for different distance to tree trunk (Mason et al. 1982; Cline
et al. 2005). Conversely, large gaps and sparse root distribution create establishment opportunities for early stage fungi,
Ectomycorrhizal fungi in a wooded meadow
743
Fig 4 – Abundance of EcM fungal species. (A) Based on colonized root tips; (B) Based on colonized root fragments (2–3 cm
diam); (C) Based on root samples; (D) Based on plots. Open bars, unmanaged wooded meadow (forest); closed bars, managed
wooded meadow. Species are arranged in descending order of root tip abundance (see Table 2).
744
Fig 5 – Rarefied species accumulation curve with 95 %
confidence intervals (CI), Chao2 and Jackknife2 species
richness estimates of the whole fungal community. Plots
were sampled randomly without replacement using 1000
permutations for each sample size. Sobs, rarefied species
richness.
including Hebeloma spp., Laccaria spp., and many Pezizales
spp., especially in the wooded meadow (Mason et al. 1982;
Tedersoo et al. 2006).
In contrast, fungal species composition was nearly identical in topsoil and the bottom of A-horizon. The accumulating species richness decreased in A-horizon probably due to
lower root density and root tip to fragment ratio. Only Wilcoxina sp. and Sebacinaceae sp4 were found in a single root
cluster penetrating cracks in the limestone bedrock
(L. Tedersoo, personal observation). These two species
were locally abundant in O- and A-horizons above. Vertical
niche differentiation among EcM fungi may occur in mature
forests with more developed soil stratification. However, insufficient sample size and conflicting results (Tedersoo et al.
2003; Rosling et al. 2003), or the inability to distinguish saprobes from EcM fungi (Dickie et al. 2002) provide little evidence for vertical niche differentiation among EcM fungal
species.
Species richness extrapolation
To evaluate the sufficiency of sample size, we built accumulation curves for rarefied and estimated species richness.
Neither of the curves levelled off with increasing sample
L. Tedersoo et al.
Fig 6 – Plot-based rarefied species accumulation curves with
95 % confidence intervals (CI) of O-horizon and A-horizon.
Plots were sampled randomly with replacement using 1000
permutations for each sample size. Sobs, rarefied species
richness.
size, indicating that sampling effort was too low to fully characterize species composition of the wooded meadow, forest
and the whole site. Jackknife2 and Chao2 estimated the total
richness of EcM fungi between 325 and 329 species. Jackknife2 produced an estimate of 172 species (the number of
observed species) when ca 35 % of samples and ca 50 % of
species were randomly sampled. However, Chao2 produced
high richness estimates already at minute sample sizes,
though finally resulting in a similar estimate. Chao2 and
Jackknife2 are considered the best incidence-based richness
estimators and they usually require that ca 50 % of species
and 25–33 % of sampling units are sampled to stabilize the
estimate values (Colwell & Coddington 1994; Melo & Froelich
2001). Sampling half of the species is probably never
achieved in EcM community studies due to poor resolution
of species using conventional identification techniques and
low sample size. The precise estimation of species richness
using extrapolation methods has been questioned for hyperdiverse communities of taxa, which comprise an endless tail
of rare species. Therefore, these estimates are considered
conservative, reflecting just a lower bound of the true species
richness (Mao & Colwell 2005). Indeed, the high discrepancy
between profound agaric surveys and below-ground studies
confirms the presence of a large number of unobserved
EcM fungal species on root tips (Dahlberg et al. 1997; Richard
et al. 2005).
Ectomycorrhizal fungi in a wooded meadow
745
Fig 7 – Detrended correspondence analysis indicating the effects of wooded meadow management, soil horizon, birch
plots and ordinated plots (arrows) on ectomycorrhizal fungal community structure (see legend). Samples were pooled by
plot and by horizon (triangles). Species (circles) frequency with down-weighted rare species was used in the analysis.
Two soil horizons of the same plot are connected with a pointed line. Plots are numbered according to Fig 1. Names of
only the most frequent species are indicated.
Distribution of taxa
Based on the relative frequency of higher taxa, the EcM fungal
community at Tagamõisa resembles most strongly EcM communities in temperate deciduous woodlands in alkaline soils.
In particular, the thelephoroid clade, Sebacinaceae and Inocybaceae were dominant, and there was an apparent under-representation of cantharelloid and athelioid clades (Murat et al.
2005; Richard et al. 2005). The thelephoroid clade accounted
for 37.8 % of species and colonized 19.8 % of root tips in this
study. Thelephoroid fungi are also a substantial, but rarely
the dominant, component in European and North American
coniferous forests (Kõljalg et al. 2000; Horton & Bruns 2001).
However, Kaldorf et al. (2004) demonstrated that a single species, Tomentella aff. ellisii, dominated an aspen plantation in
Germany. Species richness of the thelephoroid clade decreased by 10.8 %, when 95.0 % was used as the value of a molecular species criterion, which corresponds to the
approximate limit of a morphospecies (U. Kõljalg et al. unpublished). Most Thelephoraceae spp. and Sebacinaceae spp. were
not matched to fruit bodies based on ITS2 sequences, which
suggests high cryptic diversity in these families. Thelephoraceae spp. together with C. geophilum and Meliniomyces sp.
accounted for the majority of taxa with melanized cell walls.
Melanized fungi were relatively more abundant in the
wooded meadow than forest (Table 2). Melanin protects fungi
against environmental stress, including desiccation (Butler &
Day 1998). The sun-exposed shallow meadow soils with reduced organic matter content are more sensitive to drought
compared with forests (Wilson 1993), thus potentially favouring fungi with melanized cell walls.
Sebacina/Tremellodendron was more abundant in the forest
than wooded meadow (Table 2; and unpublished data), comprising 18 species. Sebacinaceae spp. are common in deciduous
and mixed forests in Europe and eastern North America (Avis
et al. 2003; Weiß et al. 2004; Richard et al. 2005). The observed
746
preference of Sebacinaceae spp. for the forest plots was unexpected, because orchids that commonly associate with Sebacinaceae spp. inhabit predominately well-managed wooded
meadows (Kukk & Kull 1997).
The genus Inocybe comprised 16 species on root tips. Inocybe
was among the most species rich genera in a Corsican deciduous forest (Richard et al. 2005), and in Oregon and Californian
coniferous forests [Cullings et al. 2001; Izzo et al. 2005 (sequences reanalysed by us using fasta3 queries and compared
with our unpublished fruit body sequences)]. We believe
that high variation in EcM anatomy and ITS sequences, low
alignment power of blastn algorithm, and the lack of public
sequences have generally hampered identification of Inocybe
spp. In this study, Inocybe spp. appeared more common in
the A-horizon, especially in forest pits. This may be related
to a high tolerance to elevated ground water and seasonal
flooding or preference for soil mineral particles. Inocybe spp.
most often fruit on organic-poor and damp soils (L. Tedersoo,
pers. obs.).
In contrast to the dominant genera, nearly all Russula and
Lactarius spp. were identified to species. Both R. velenovskyi
and R. sphagnophila were sequenced from root tips representing typical beige russuloid and dark brown thelephoroid anatomotypes. This finding indicates either specific relationships
between fungi, unnoticed double colonization, or DNA handling mistakes.
Boletus radicans, a locally red-listed species, both fruited
and formed EcM only in well-managed parts of the wooded
meadow. In addition, B. luridus occurred in abundance both
above and below ground exclusively in the wooded meadow.
These results corroborate with several observations that Boletus spp. fruit predominantly in the managed wooded
meadows (Kalamees 2004). Calciphilous Boletus spp. may prefer wooded meadows because of more alkaline soils or more
abundant sunlight as Estonia is the northernmost habitat for
several Boletus spp. We anticipate that the fungi we found
only in the wooded meadow are also likely to inhabit the forest soil, but below our detection limits.
Conclusions and future considerations
Integrating anatomotyping and sequencing enabled us to distinguish EcM fungi from saprobes and endophytic fungi and
provided species level identification for many EcM fungi on
root tips. The results show that traditionally managed areas
differ most strongly from forested areas, with no apparent dependence on soil variables or soil horizon. However, replicated wooded meadows are needed to reveal the
characteristic EcM fungi and to address the effects of management per se on changes in community structure and diversity.
Rarefaction and extrapolation offer promising alternatives to
species richness comparisons between sites and treatments,
but high sampling effort is unavoidable. To compare the fungal diversity and community composition between studies,
considering the presence of cryptic species, molecular species
criteria need to be established together with appropriate software (Schloss & Handelsman 2005). Due to differences in the
rate of evolution in the ITS region, molecular taxonomists
should develop molecular species criteria separately for each
genus.
L. Tedersoo et al.
Acknowledgements
We thank John W. G. Cairney and referees for comments. We
thank Saaremaa Keskkonnateenistus for permission to sample in Tagamõisa wooded meadow; Teele Jairus, Sergei Põlme,
Marko Peterson, Eele Õunapuu, Erki Laaneoks and Helena
Faust for assistance during root sampling; Tiina Ojala and
Mari Reitalu for providing information on history of the study
site; Martin Ryberg and R. Henrik Nilsson for commenting on
an earlier draft of the manuscript. This study was funded by
ESF grants nos 5232 and 6606 and WFS.
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Unpublished supplementary information (lost during the revising process)
Table 1. The effect of wooded meadow management and soil horizon on diversity and frequency of
ectomycorrhizal fungi. Values represent means ± SE. Different letters indicate significant differences (based on
Benjamini and Hochberg´s sharpening modification to Bonferroni correction; Benjamini & Hochberg, 2000 as
implemented in a spreadsheet program of Verhoeven et al. (2005)) for both factors separately based on threeway mixed ANOVAs. Neither block effects nor interactions were significant. Species with melanized cell walls
(see table 2) were distinguished microscopically; species with hypogeous fruit bodies were distinguished based
on sequence similarity and phylogenetic analyses (table 2).
Wooded meadow management
Soil horizon
Diversity variables / taxa
The number of observed
species per plot
The number of observed
species per sample
The number of observed
species per root fragment
Jackknife2 richness estimate
for plot
Chao2 richness estimate for
plot
Shannon diversity index for
plot
The number of root tips per
root fragment
Managed
Unmanaged
O-horizon
A-horizon
14.3 ± 0.9
16.4 ± 0.9
16.9 ± 1.0x
13.8 ± 0.7y
4.63 ± 0.36
5.30 ± 0.28
5.65 ± 0.32x
4.27 ± 0.23y
1.50 ± 0.07a
1.83 ± 0.07b
1.82 ± 0.07x
1.50 ± 0.07y
25.3 ± 1.8
29.6 ± 1.9
30.1 ± 2.0x
24.8 ± 1.6y
24.1 ± 2.19
31.0 ± 3.3
29.8 ± 2.5
25.3 ± 3.2
2.49 ± 0.08
2.65 ± 0.06
2.65 ± 0.08x
2.49 ± 0.05y
10.8 ± 0.8a
15.3 ± 1.7b
15.9 ± 1.6x
10.3 ± 0.8y
Cenococcum geophilum
3.06 ± 0.48
2.81 ± 0.39
3.80 ± 0.36x
2.07 ± 0.38y
Genea sp1
0.75 ± 0.19a
0.05 ± 0.05b
0.50 ± 0.18
0.30 ± 0.15
Genea sp3
0.94 ± 0.23
0.31 ± 0.18
0.56 ± 0.22
0.69 ± 0.22
Inocybe maculata
0.25 ± 0.14a
2.49 ± 0.35b
1.16 ± 0.34x
1.58 ± 0.43y
Lactarius pubescens
1.13 ± 0.20
0.81 ± 0.32
1.25 ± 0.31
0.69 ± 0.20
0.98 ± 0.24
b
0.62 ± 0.25
0.35 ± 0.15
0.56 ± 0.24
b
1.44 ± 0.43
1.19 ± 0.28
0.68 ± 0.22x
0.30 ± 0.12y
0.00 ± 0.00
a
Thelephoraceae sp1
2.06 ± 0.36
a
Tomentella pilosa
0.69 ± 0.22
0.29 ± 0.11
Boletus spp. (%)
5.92 ± 0.90a
0.00 ± 0.00b
2.24 ± 0.75
3.69 ± 1.16
a
b
3.75 ± 1.14
7.12 ± 1.50
Sebacinaceae sp4
Cortinariaceae spp. (%)
6.75 ± 1.47
4.12 ± 1.25
Inocybe spp. (%)
8.78 ± 2.05
14.64 ± 1.59
8.36 ± 1.46x
15.06 ± 2.06y
Pezizales spp. (%)
18.1 ± 1.7a
4.1 ± 1.2b
10.1 ± 2.4
12.0 ± 2.3
Russulaceae spp. (%)
6.89 ± 0.85
9.47 ± 1.27
7.03 ± 1.46
13.79 ± 1.12
9.36 ± 1.45
8.95 ± 1.82
9.61 ± 1.73
a
b
Sebacinaceae spp. (%)
4.52 ± 1.14
Thelephorales spp. (%)
Taxa with hypogeous fruit
bodies (%)
Taxa with melanized cell walls
(%)
Taxa with melanized cell walls
(excl. C. geophilum; %)
32.3 ± 2.2
26.9 ± 2.1
30.9 ± 2.3
28.3 ± 2.1
15.8 ± 1.93a
4.6 ± 1.3b
10.2 ± 2.3
10.2 ± 2.1
24.5 ± 2.2a
19.7 ± 1.8b
21.8 ± 2.1
22.3 ± 2.1
17.7 ± 2.0a
14.3 ± 1.7b
16.1 ± 1.8
15.9 ± 2.0
Table 2. The effect of wooded meadow management and soil horizon on soil variables. Values represent means
± SE. Different letters indicate significant differences (based on Benjamini and Hochberg´s sharpening
modification to Bonferroni correction; Benjamini & Hochberg, 2000 as implemented in a spreadsheet program
of Verhoeven et al. (2005)) for both factors separately based on three-way mixed ANOVAs. Neither block
effects nor interactions were significant.
Soil variable
Wooded meadow management
Managed
Unmanaged
a
O-horizon
b
6.40 ± 0.21y
6.53 ± 0.14
P (mg kg-1)
14.6 ± 6.8a
42.3 ± 2.7b
40.3 ± 8.6x
16.6 ± 4.7y
K (mg kg-1)
84.0 ± 119.9a
207.1 ± 29.5b
226.3 ± 42.1x
64.8 ± 17.7y
Mg (mg kg-1)
337.7 ± 74.6 a
865.8 ± 39.6 b
746.5 ± 132.7
457.0 ± 143.5
8.65 ± 1.57
6.31 ± 1.82
43.9 ± 9.6x
20.5 ± 6.9y
Ca (g kg )
4.64 ± 1.07
Organics (g kg-1)
15.7 ± 6.7a
a
10.33 ± 0.29
48.8 ± 1.9b
5.74 ± 0.22
A-horizon
x
pHKCl
-1
5.61 ± 0.12
Soil horizon
b
References
Benjamini Y, Hochberg Y. 2000. On the adaptive control of the false discovery rate in multiple testing with
independent statistics. Journal of Educational and Behavioral Statistics 25: 60-83.
Verhoeven KJF, Simonsen KL, McIntyre LM. 2005. Implementing false discovery rate control: increasing
your power. Oikos 108: 643-647.