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Research

Elevated atmospheric CO2 alters root symbiont

Blackwell Science Ltd

community structure in forest trees

Petra M. A. Fransson, Andrew F. S. Taylor and Roger D. Finlay
Department of Forest Mycology and Pathology, SLU, Box 7026, S-75007 Uppsala, Sweden

Summary

Author for correspondence: • Changes in below-ground ectomycorrhizal (ECM) community structure in

Petra M. A. Fransson response to elevated CO2 and balanced nutrient addition were investigated in a
Tel: +46 18 672797 37-yr-old Picea abies forest.
Fax: +46 18 309245
Email: petra.fransson@mykopat.slu.se
• Trees in whole-tree chambers were exposed to factorial combinations of ambient/
elevated CO2 (700 ppm) and fertilization (+/–). ECM fungal community structure
Received: 5 July 2001
was determined in 1997 and 2000 using a combination of morphotyping and
Accepted: 4 September 2001
molecular analyses. Samples were taken both from chambers and from reference
trees receiving the same fertilization treatments but without chambers.
• Significant effects on ECM community structure were found in response to ele-
vated CO2. Neither elevated CO2 nor fertilization altered species richness; however,
there was considerable variation among samples, which may have masked
treatment effects on individual species. After 3 yr, the effects of elevated CO2 on
community composition were of the same magnitude as those seen after 15 yr of
fertilization treatment.
• Our results show that increasing atmospheric CO2 concentrations affect the
community structure of root symbionts colonizing forest trees. The potential effects
of altered ECM community structure on allocation and turnover of carbon and
nutrients within forest ecosystems are discussed.

Key words: ectomycorrhizal fungi, community structure, elevated CO2, carbon,

ordination analysis.

© New Phytologist (2001) 152: 431–442

sequestration and emission of carbon. Boreal forest trees rely

Introduction
Within European forests there is a fine balance between the
quantity of carbon (C) fixed via photosynthesis and that
released through respiration (Valentini et al., 2000). The
result of these two processes determines the net exchange of C
between the terrestrial biosphere and the atmosphere and
whether forests act as a source or a sink for CO2. The balance
is affected by daily, seasonal and yearly variations in climatic
conditions (Hanson et al., 1993; Kirschbaum, 1995;
Goulden et al., 1998; Lindroth et al., 1998), and by changes
in land use patterns (Schimel, 1995; Falkowski et al., 2000).
Valentini et al. (2000) showed that total ecosystem respiration,
most notably that of roots and soil microorganisms, was the
main determinant of the C balance of a forest. Among soil
microorganisms, symbiotic mycorrhizal fungi are of particular
significance since they have a direct influence on both the
on ectomycorrhizal (ECM) fungi for their nutrient uptake,
and almost 100% of forest tree short roots are colonized by
ECM fungi (Taylor et al., 2000). The fungi, in turn, depend
strongly on current assimilates from their plant hosts (Högberg
et al., 2001), and up to 20% of the photosynthetically fixed
carbon may be allocated by the tree to the fungus (Finlay &
Söderström, 1992). Högberg et al. (2001) found a large and
almost immediate decrease in root associated respiration,
which constituted 54% of the total soil respiration, in
combination with a large decrease in ECM fruit body
production, following girdling of a pine stand illustrating the
importance of current assimilates to ECM fungi.
Individual ECM fungal species are likely to differ in how
they allocate plant derived C between growth, respiration and
exudation. The allocation patterns can in turn be affected by
the actual supply of C from the host plant to the fungus.

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432 Research
Increased levels of CO2 in the atmosphere are known to
increase plant photosynthesis and subsequent C supply into
the soil (O’Neill, 1994; Rey & Jarvis, 1997; Ceulemans et al.,
1999). Whether this increased supply of C below-ground
actually leads to significant, long-term net carbon sequestra-
tion in forest soils is however, questioned (Schlesinger &
Lichter, 2001). Laboratory studies of tree seedlings have
demonstrated that Suillus bovinus and Laccaria bicolor
respond differently to elevated CO2 concentrations in the
way they partition assimilates between fungal biomass and
respiration (Gorissen & Kuyper, 2000). These authors also
showed that the fungi differed in their capacity to process and
transfer N to their tree hosts. In general, diversity in physio-
logy and function – both among and within different fungal
species – might be large (Cairney, 1999), but the dominance
of a few species is commonly seen in investigations of below-
ground ECM community composition (Gardes & Bruns,
1996; Erland et al., 1999; Peter et al., 2001). Changes within
these dominant species thus have a potentially large influence
on any physiological response changing community struc-
ture. The results of Gorissen & Kuyper (2000) also underline
the importance of nutritional effects and their likely interac-
tions with C allocation. Changed ECM community structure
may thus contribute to changes in the C balance of boreal
forest ecosystems. It is therefore important to establish how
individual species respond to elevated levels of CO2 under
field conditions.
There are few published studies of the effects of elevated
CO2 on ECM mycorrhizal community structure. Rey &
Jarvis (1997) found indications that the mycorrhizal species
composition based on morphology (morphotypes) shifted
towards those species characteristic of later successional stages
in young birch seedlings (Betula pendula) after growing in
open-top chambers under elevated levels of CO2 for 4.5 yr.
They interpreted this as an acceleration of tree ontogeny,
which may lead to the trees supporting ECM fungal species
with a higher carbon demand. Godbold et al. (1997) trans-
ferred paper birch (Betula papyrifera) and Eastern white pine
(Pinus strobus) saplings from a forest to growth chambers and
observed, after 24 wk of elevated CO2 exposure, an increase
in the proportion of morphotypes that produced rhizo-
morphs and large amounts of extraradical mycelium. Rygie-
wicz et al. (2000) planted 2-yr-old Douglas fir (Pseudotsuga
menziesii ) seedlings in chambers, and after 4 yr of elevated
CO2 exposure they found small overall effects on ECM fungal
diversity, based on gross morphological traits. The authors did
find an increase in ECM root tip proliferation during sum-
mer. Markkola et al. (1996) were unable to detect any signi-
ficant changes in ECM morphotype community structure
growing on roots of Pinus sylvestris as a result of CO2 enrich-
ment. The authors used pine seedlings, which were inculated
with Piloderma croceum, and then grown in pots with natural
mor humus for up to 11 months. As far as we know, there
are no studies of the effects of elevated CO2 on ECM fungi
associated with large forest trees growing under field conditions.
In the present study we investigated changes in the ECM fun-
gal community structure in a 37-yr-old Norway spruce forest
after factorial combinations of elevated CO2 and balanced
nutrient addition.
Materials and Methods
Study site
The study was conducted at the Flakaliden field site, northern
Sweden (64°07′ N; 19°27′ E; alt. 310 m above sea level (asl)).
The site was planted with Picea abies [L.] Karst. after clear-
felling in 1963. A fertilization experiment was started in 1987
in which plots were supplied with a balanced solution of macro-
and micronutrients on a daily basis throughout the growing
season. Since the start of the experiment the fertilization
treatment has included a total of 1125 kg N ha–1, added as
both ammonium and nitrate. In 1996, 12 whole tree chambers
were installed around trees. Six of the chambers were placed
in an unfertilized plot and six were placed in a fertlized plot.
The CO2-treatment started in 1998 and continued for 3 yr.
One half of the enclosed trees on each plot received ambient
levels of CO2 (350 ppm), the other half received elevated levels
of CO2 (700 ppm). The enclosed trees are hereafter referred
to as ambient trees and elevated trees. Three reference trees
(without chambers) on each plot were also included in this
analysis. The enclosed trees were irrigated via a tubing system
with amounts of water equivalent to those intercepted by the
same area outside the chambers. The mean distance between
individual trees in the plots was c. 3 m. Two of the chambers
(ambient tree no. 2 in the unfertilized plot and ambient tree
no. 2 in the fertilized plot) were moved to new trees in 1997 and
1998 as the trees died as a result of aphid and fungal attacks.
Sampling and identification of ectomycorrhizal fungi
In late August in 1997, before the CO2-treatment started, and
at the same time in 2000, before the final harvest of the
chamber trees, five soil cores (2.8 cm diameter) were taken
from the organic soil layer beneath each of the chamber trees
(6 + 6) and the reference trees (3 + 3). Forty-five soil cores
were thus taken from each plot, giving a total of 90 soil cores.
Soil samples were placed in separate plastic bags and
maintained at 4°C, until examined. The depth of the organic
layer in each core was measured before soaking the sample
in water for 30 min. Live root tips were then extracted by wet
sieving using a combination of 1.0 mm and 500 µm sieves.
Root tips were examined and classified into mycorrhizal
morphotypes, using macroscopic and microscopic features
(Agerer, 1986–98). The term morphotype is used here to
designate a recognizable group of mycorrhizal root tips.
Morphotypes were either identified to species or genus level,
or unidentified. The unidentified morphotypes were given
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numbers as they appeared in the processing. The total
abundance (total number of mycorrhizal root tips per core)
and relative abundance (number of each morphotype/total
no. of living mycorrhizal root tips) of each mycorrhizal type
were recorded. Nonmycorrhizal root tips were also recorded.
Subsamples of morphotypes were kept in the freezer for
internal transcribed spacer-restriction fragment length poly-
morphism (ITS-RFLP) analysis as a supplement to the
morphotyping to confirm consistency within and between
single morphotypes. DNA was extracted from the mycorrhizal
root tips according to Gardes & Bruns (1993), excluding the
initial freeze-thawing step. The ITS region of the rDNA was
amplified by PCR following the modified protocol by
Henrion et al. (1994). PCR was performed using High Fidelity
DNA polymerase and High Fidelity buffer (Roche). The
reaction mix had final concentrations of each nucleotide at
0.2 mM, each primer at 0.3 µM, polymerase at 0.026 µ µl–1,
Mg2Cl at 3.1 mM. DNA template was added as 25% of the
final reaction volume. Cycling parameters were modified to
1 cycle at 94°C for 3 min followed by 35 cycles of 30 s at
94°C, 45 s at 50°C, 1 min at 72°C ending with 1 cycle of 7 min
at 72°C. The primers used were ITS1-ITS4 as described by
White et al. (1990). PCR products were separated on a 1%
agarose gel in 0.5 X TBE-buffer at 4.7 Vcm–1, stained with
ethidium bromide and visualized by UV-light. The amplified
DNA was digested using the restriction enzymes Hinf 1, Mbo1
and Taq1 (Promega Corporation, Madison, WI, USA), for 2.5 h
in 37°C (Hinf 1, Mbo1) and 65°C (Taq1). Digestion products
were separated on a 2.3% Metaphor gel in 0.5 X TBE-buffer
at 4.7 Vcm–1, stained and visualized as previously. Fragment
lengths were estimated and band patterns analysed with
Taxotron® software system (Institute Pasteur, Paris, France).
Statistical analysis
To investigate community structure and possible treatment
effects, Detrended correspondence analysis (DCA) and
Canonical correspondence analysis (CCA) were carried out
on the total numbers of mycorrhizal root tips in each
morphotype (ter Braak & Smilauer, 1998). DCA represents
an indirect gradient analysis, based on the morphotype
abundance data, that examines the total variation in the
community samples. CCA represents a direct gradient
analysis, where the morphotype abundance data is explained
by environmental variables and part of the total variation
in the community samples is thus examined. In this case
the environmental variables are the treatments, that is no
fertilization – fertilization, no chamber – chamber and
ambient CO2 – elevated CO2. An interyearly comparison of
community structures was made by DCA analysis. The two
ambient trees where the chambers were moved between the
two samplings were excluded from the analysis. Arrows are
used in the DCA ordination graph to indicate the shift in
fungal community structure between 1997 and 2000. Data
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Research 433
were transformed (log10) before analysis, and the analyses
were performed with CANOCO 4.0 (ter Braak & Smilauer,
1998). Monte Carlo permutation tests (n = 199) were
performed to test the significance of the relationship between
morphotype data and treatments. In the CCA ordination
diagram vectors are included to make the presentation clearer,
although these are not strictly applicable since the environ-
mental variables are not continuous.
Possible treatment effects of elevated CO2 and chambers
on ECM morphotype richness, total root tip numbers and
individual ECM morphotype abundance (total) were ana-
lysed for each plot using one-way ANOVA. Morphotype
richness was tested using both the numbers of identified
morphotypes, that is taxa identified to species or genus level,
and the total numbers of morphotypes, that is both identified
and unidentified taxa. The data from 2000 were tested for
both the elevated CO2 and the chamber factors, while the
data from 1997 were only tested for the chamber factor.
To test whether the effects of CO2 and chamber treatments
were the same for both fertlized and unfertilized plots, that is
whether the possible effects of CO2/chamber and fertilization
treatments upon individual components of the community
structure interact, a two-way ANOVA was performed. Since
the fertilization treatment was not replicated, the significance
levels for the possible effects of the fertilization treatment
alone upon individual components cannot be used. It is however,
statistically correct to test the interaction and this part of the
results from the two-way ANOVA is reported (B. Vergerfors-
Persson, pers. comm.). The significance levels for the possible
effects of CO2/chamber treatment upon individual com-
ponents are not reported since they were already tested
through one-way ANOVA as described in the above section.
Mean values ± standard errors for total numbers of morpho-
types, number of identified and unidentified morphotypes
and levels of colonization are reported.
Results
Ordination analyses of community structure
The Canonical correspondence analysis showed significant
effects of elevated CO2 on ECM fungal community structure
in the year 2000 (Fig. 1a). 20.9% of the variation in the
species data set can be attributed to the fertilization-,
chamber- and CO2-treatments. The eigenvalues for CCA axes
1 and 2 were 0.371 and 0.303. The first two CCA axes
together display c. 15% of the total variance in the species
data set, and this part of the variation is shown in Fig. 1(a).
The Monte Carlo permutation tests showed that there were
statistically significant (P = 0.005) relationships between
ECM morphotype composition and environmental variables.
When the test was repeated for one environmental variable
at a time, the results were also significant for fertilization
and CO2 effects, but not for the chamber effect.
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The positioning of the samples relative to the end point of
the vectors in the CCA ordination diagram relates individual
trees to different treatments (Fig. 1a). The ordination diagram
shows the elevated CO2 trees clustered together in the upper
left part. For the unfertilized plot, elevated CO2 trees are
clearly separated from the ambient CO2 trees and reference
trees. The fertilized plot shows a similar pattern, although
the fungal communities on trees receiving elevated CO2
levels were not so clearly separated from ambient and refer-
ence trees.
Eleven taxa occurred on three or more of the trees exam-
ined and the CCA species scores are plotted together with the
environmental variables in Fig. 1(b). Of these, Amphinema
byssoides, Cenococuim geophilum, tomentelloid group 1 and
Tylospora fibrillosa, all seemed to be favoured by the fertiliza-
tion treatment. Piloderma croceum and tomentelloid group 2
Fig. 1 Ordination diagram based on
Canonical correspondence analysis (CCA) of
the ectomycorrhizal (ECM) community in a
37-yr-old Norway spruce stand subject to
elevated CO2 (700 ppm) and optimal
fertilization for 3 and 15 year, respectively.
The diagram shows the part of the variation
within the communities (21%) that can be
attributed to the treatments. Arrow length
indicates the relative importance of
environmental variables. (a) Open symbols,
unfertilized trees; closed symbols, fertilized
trees. Ambient CO2 trees with (open
diamonds) and without (open circles)
chamber and elevated CO2 with chamber
(open diamond within square). (b) Species
scores for ECM taxa that appeared on three
or more of the sampled trees. Abbreviation:
A. byssoides, Amphinema byssoides;
C. geophilum, Cenococcum geophilum;
P. gelatinosa, Piceirhiza gelatinosa;
P. rosa-nigrescens, Piceirhiza rosa-nigrescens;
P. byssinum, Piloderma byssinum;
P. croceum, Piloderma croceum; Russula
spp.; tomentelloid group 1, clamped
tomentelloids; tomentelloid group 2,
unclamped tomentelloids; T. fibrillosa,
Tylospora fibrillosa; Unknown, unknown
basidiomycete.
are grouped near the origin and between the two plot treat-
ment clusters, and can thus not be related to any of the treat-
ments. Piloderma byssinum seemed to be negatively affected
by the fertilization treatment, and the unknown basidio-
mycete appeared to be favoured by the CO2 treatment.
In 1997, before the CO2 treatment started, the CCA ana-
lysis showed a clear separation between the unfertilized and
fertilized plots, and 14.4% of the total variance of the species
could be explained by the fertilization and chamber treat-
ments (figure not shown). The significance of the treatments
was tested as previously and the ECM morphotype distribu-
tion was significantly (P = 0.01) related to fertilization and
chamber treatments. Variation in community structure in the
unfertilized plot was larger compared to the variation in the
fertilized plot. Eigenvalues for CCA axes 1 and 2 were 0.50
and 0.38, respectively. Seven taxa in 1997 occurred on three
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Fig. 2 Ordination diagram based on

Detrended correspondence analysis (DCA) of
the ectomycorrhizal (ECM) community in a
37-yr-old Norway spruce stand, showing the
interyearly variation between 1997 and
2000. The Norway spruce stand was subject
to elevated CO2 and optimal fertilization
treatments. The direction of arrows in the
ordination diagram indicates the shift in
community structure for individual trees
between the two years, and long arrows
indicates larger differences than small arrows.
(a) All trees in the unfertilized plot remained
separated from the trees in the fertilized plot,
with one exception. The fungal community
structures of elevated trees, that are marked
in red, seem to become more similar to
those of the fertilized plot (b). The division
between the unfertilized plot and fertilized
plot is indicated by the dashed line. Red/blue,
elevated trees in unfertilized/fertilized
plot; orange/turqoise, ambient trees in
unfertilized/fertilized plot; black,
reference trees.

or more of the trees. Cenococcum geophilum, T. fibrillosa and
tomentelloid group 1 were favoured by the fertilization treat-
ment, while Piloderma species and Inocybe spp. were mainly
associated with the trees in the unfertilized plot.
Inter-yearly variation in community composition
A comparison of the ECM community structure between
years was made using DCA analysis. The results showed that
the composition of fungi colonizing tree roots in the
unfertilized plot remained different from that in the fertilized
plot (Fig. 2a), with an exception. The elevated CO2 treatment
seemed to drive the community structure in the unfertilized
plot towards that in the fertilized plot (Fig. 2a). This is
explained by changes in the relative abundance of some taxa,
but the specific changes, that is which of the fungal taxa
are responsible for the shift in community structure, are not
easily distinguishable. In this analysis no apparent shift
in community structure was seen as a result of the CO2
treatment in the fertilized plot. All trees in the fertilized plot
are grouped together in the DCA diagram (Fig. 2b). Despite
the variation in ECM community structure between years,
especially for the reference trees in the unfertilized plot,
ambient trees and reference trees in the unfertilized plot
remain separated from the fertilized plot. In general the
variation both within and between years was considerable.
Effects of elevated CO2 and fertilization treatment on
individual components of the community structure
In 1997, a total of 58 ECM morphotypes was found, 14
which were identified to species or genus level and 44 that
were unknown (Table 1). In 2000, a total of 42 morphotypes
was found, 14 were identified and 27 were unknown types
(Table 2). For all investigated trees in 2000, the number of
identified ECM morphotypes per tree (5.3 ± 0.5) exceeded
the number of unknown morphotypes (1.7 ± 0.4). In 1997,
the number of unidentified morphotypes was higher

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Table 1 Relative abundance of ectomycorrhizal (ECM) morphotypes, sampled in 1997 from Norway spruce plots receiving no treatment or fertilization. Individual trees were either covered with
whole tree chambers, not recieving CO2 treatment in 1997, or free-standing reference trees receiving no additional treatment

Control plot Fertilization plot

Chamber trees Reference trees Chamber trees Reference trees

Tree treatment
Replicate tree no. 1 21 3 4 5 6 1 2 3 1 22 3 4 5 6 1 2 3

Amphinema sp. 3.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.4 0 0

Cenococcum 0 0 8.8 14.3 0 <1 1.8 14.3 1.9 0 3.6 14.9 1.2 7.5 12.1 19.6 8.6 14.7
Cortinarius spp. 0 0 3.0 0 64.2 0 25.5 9.3 <1 0 0 0 0 0 0 0 1.7 0
Dermocybe spp. 0 0 0 0 0 <1 0 1.8 0 0 0 0 0 0 0 0 3.4 2.0
L. rufus 0 25.0 0 0 0 0 34.1 0 0 0 0 0 0 0 0 0 0 0
Lactarius spp. 88.5 0 0 0 0 0 <1 0 0 <1 0 40.4 0 0 11.0 0 0 0
Piceirhiza bicolorata 0 0 0 0 0 0 8.1 0 0 0 0 0 0 0 0 1.4 0 0
P. rosa-nigrescens 0 0 0 0 0 0 0 3.3 0 0 0 0 0 0 0 0 0 0
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P. byssinum 0 0 0 11.1 0 0 0 5.0 24.5 0 0 0 0 0 0 8.7 0 0

P. croceum 0 0 10.0 0 1.1 0 4.1 9.0 9.2 0 0 10.6 0 6.8 21.1 0 0 0
Russula spp. 0 75.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Tomentelloid 0 0 7.1 0 0 0 0 0 0 85.2 0 4.2 10.8 17.3 5.3 18.1 0 7.3
Tylospora sp. 0 0 0 0 10.5 0 0 0 3.2 4.9 88.0 6.4 71.1 48.1 0 34.1 67.4 43.3
Unknown tips 7.7 0 14.0 12.7 21.1 68.9 11.6 51.0 49.6 0 6.0 6.4 0 10.5 45.8 16.7 18.9 10.7
Nonmyco. tips 0 0 57.1 61.9 3.1 29.9 14.3 6.3 11.1 9.6 2.4 17.1 16.9 9.8 4.7 0 0 22.0
Total no. tips 78 56 170 63 95 241 440 335 216 312 167 47 83 133 190 138 175 150
Types id. 2 2 5 2 3 2 5 6 6 3 2 4 3 4 4 6 4 4
Types unid. 1 0 1 3 2 2 5 1 8 0 2 2 0 5 4 3 3 6
Colonization (%) 100 100 42.9 38.1 96.9 70.1 85.7 93.7 88.9 90.4 97.6 82.9 83.1 90.2 95.3 100 100 78.0

Abundances are expressed as percentage of root tips examined. 1,2The chamber was moved to a new tree in 1997 and 1998, respectively.
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Table 2 Relative abundance of ectomycorrhizal (ECM) morphotypes, sampled in 2000 from Norway spruce plots receiving no treatment or fertilization. Individual trees were treated with ambient
or elevated levels of CO2 in whole tree chambers, or were free-standing reference trees recieving no additional treatment

Control plot Fertilization plot

Ambient Elevated Reference trees Ambient Elevated Reference trees

Tree treatment
Replicate tree no. 1 21 3 1 2 3 1 2 3 1 22 3 1 2 3 1 2 3

Amphinema sp. 0 0 0 0 0 4.5 0 6.0 19.9 26.9 1.4 0 0 8.1 0 1.0 6.5 8.7
Cenococcum 0 0 11.1 0 3.7 10.5 3.7 13.1 3.7 20.1 0 18.0 35.0 18.3 10.2 31.4 8.8 21.4
Cortinarius spp. 69.2 0 10.2 0 0 10.9 5.6 6.1 25.0 1.5 0 0 0 0 0 1.0 0 0
Dermocybe cf. 0 0 0 0 0 0 0 6.6 0 0 0 0 0 0 0 0 0 <1
cinnamomeus
Dermocybe spp. 0 0 0 0 0 1.4 <1 0 0 0 0 0 0 0 0 0 0 0
L. deterrimus 0 0 0 0 0 0 0 0 3.2 0 0 0 0 0 0 0 0 0
Lactarius spp. 0 0 0 0 0 25.0 0 0 0 0 0 0 0 0 1.3 0 0 0
Piceirhiza gelatinosa 0 0 0 0 7.4 1.4 0 0 0 0 0 14.6 10.0 0 31.5 6.9 <1 2.4
P. rosa-nigrescens 0 0 6.5 0 0 0 0 1.6 2.8 0 21.4 0 0 0 0 0 0 0
P. byssinum 0 80.0 0 0 5.1 0 10.0 0 3.7 0 0 0 0 8.6 0 0 0 0
P. croceum 11.6 0 0 20.5 4.4 0 20.7 0 0 0 0 12.6 0 0 43.4 0 0 21.0
Russula spp. 0 0 0 0 0 <1 <1 0 5.1 0 0 0 0 0 0 0 0 0
Tomentelloid 0 0 20.4 56.8 0 0 0 3.8 0 0 6.9 <1 0 1.1 1.3 0 <1 1.6
Tylospora sp. 15.4 0 50.0 0 4.4 4.1 51.8 12.0 25.9 42.3 60.7 45.1 40.0 40.9 8.8 20.5 51.8 37.7
Unknown 3.8 20.0 1.8 9.1 74.3 34.5 7.6 49.7 10.7 9.2 9.6 8.7 15.0 21.5 3.5 38.2 17.6 6.8
Non-myco. tips 0 0 0 13.6 <1 7.3 0 1.1 0 0 0 <1 0 1.5 0 1.0 14.1 0
Totals (no. tips) 26 5 108 44 136 220 301 183 216 130 145 206 20 186 226 102 170 252
Types is 3 1 6 3 5 8 7 8 8 4 4 6 3 5 7 5 5 8
Types unid 1 0 1 1 5 6 0 2 2 2 2 2 0 3 1 1 1 1
Colonization (%) 100 100 100 86.4 100 92.7 100 98.9 100 100 100 100 100 98.5 100 99.0 85.9 100

Abundances expressed as percentage of root tips examined. CO2 concentrations: ambient (350 p.p.m) and elevated ( 700 p.p.m).
1,2
The chamber was moved to a new tree in 1997 and 1998, respectively.

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(2.7 ± 0.5), and the number of identified morphotypes
lower (3.7 ± 0.3). Between 1 and 14 morphotypes were found
per tree with a mean value of 6.4 ± 0.8 morphotypes per
tree in 1997 and 7.1 ± 3.0 in 2000. ITS-RFLP analyses
showed satisfactory consistancy between and among tested
morphotypes.
The level of colonization was lower and more variable in
1997, with a mean of 85.2% ± 4.3, compared to 2000 when
the mean colonization level was 97.9% ± 1.1. ECM morpho-
type richness and total root tip numbers were generally not
affected by the treatments, with a few exceptions (see below).
In Tables 1 and 2, the relative abundances of Norway spruce
root tips colonized by each ECM morphotype are listed for
each tree. Unknown morphotypes were assigned numbers as
they occurred during processing, and the category called
tomentelloid fungi includes both Tomentella and Pseudoto-
mentella species, that is both clamped and unclamped tomen-
telloids. No individual ECM morphotype, except P. croceum
and C. geophilum (see below), showed a significant response to
the different treatments in either of the years. This apparent
lack of response by individual ECM morphotypes to treat-
ments was mainly due to the high variability within the data.
Significantly (F1,7 = 6.27, P = 0.04) more nonmycorrhizal
root tips and unidentified ECM morphotypes (F1,7 = 7.00,
P = 0.03) were found on the elevated CO2 trees compared
to ambient trees and reference trees in the unfertilized plot in
2000. No significant effects of elevated CO2 were detected for
the fertilized plot.
Significantly fewer root tips were found in the chambers in
the unfertilized plot both in 1997 (F1,7 = 12.25, P = 0.01) and
in 2000 (F1,7 = 7.17, P = 0.03). Significantly fewer identified
ECM morphotypes (F1,7 = 15.75, P = 0.005) and fewer Pilo-
derma croceum root tips (F1,7 = 16.99, P = 0.004) were found
in the chambers compared to reference trees in the unferti-
lized plot in 1997. Significantly fewer Cenococcum geophilum
root tips (F1,7 = 6.08, P = 0.04) were found in the chambers
in the fertilized plot in 1997.
Interactions between elevated CO2 and fertilization
treatments
In 1997, significant interactions were found between the
fertilization treatment and the chamber treatment for the
total number of root tips (F3,14 = 6.69, P = 0.02) and for root
tips colonized by P. croceum (F3,14 = 10.91, P = 0.005). No
significant interactions were found between elevated CO2
treatment and fertilization treatment in 2000.
Discussion
Increased levels of CO2 in the atmosphere are known to affect
both host plants (Saxe et al., 1998; Ceulemans et al., 1999;
Norby et al., 1999) and mycorrhizal fungi (Hodge, 1996;
Fitter et al., 2000; Treseder & Allen, 2000). The effects of
elevated CO2 on ECM community structure is important
to establish since individual ECM fungal species may differ
greatly in both physiology and function (Cairney, 1999). In
our study the ECM community structure of Norway spruce
trees changed in response to elevated levels of CO2. Canonical
correspondence analysis was performed in order to compare
the response of the fungal community as well as single ECM
taxa to elevated CO2 and fertilization. Almost 21% of the
total variation in the species data set could be attributed to
the CO2 and fertilization treatments, reflecting the fact that
the fungal community composition was affected by other
environmental variables than the present treatments. Using
CCA analysis, this is as large a part of the total variation one
can normally expect to explain (ter Braak & Verdonschot,
1995). High variablility among individual samples was
obvious here and is a general feature of ECM community data
(Horton & Bruns, 2001), but despite this a significant effect
of the CO2 treatment could be detected. Samples from
individual trees and chambers may have contained tree roots
from other, nearby trees, but we consider the samples to have
been representative of the conditions experienced by the trees.
Changes in ECM community structure due to elevated CO2
levels have been reported previously for seedlings and saplings
(Godbold et al., 1997; Rey & Jarvis, 1997; Rygiewicz et al.,
2000), but not for large forest trees grown under field
conditions as far as we are aware.
The shift in community structure in the present study was
mainly due to a change in abundance of a few common spe-
cies. Similar responses were reported by Godbold et al. (1997)
for paper birch saplings exposed to elevated levels of CO2,
where the frequencies of dominant morphotypes changed
significantly. Responses to elevated CO2 in the present study
were stronger in the unfertilized plot, compared with the
fertilized plot, as shown in Fig. 1. This was possibly due to the
same ECM morphotypes responding in a similar way both to
elevated CO2 and fertilization, thus making the actual effect
of CO2 on community composition in the fertilized plot less
pronounced. Fifteen years of fertilization may already have
shifted the community in the fertilized plot so that the CO2
treatment had little effect once started. The DCA ordination
diagram of interyearly differences in ECM community structure
showed that morphotype composition for two out of three
elevated trees in the unfertilisated plot became more similar to
those found in the fertilized plot, indicating such a response.
In the present study the morphotype richness was high and
this is in accordance with previous reports on below-ground
ECM diversity (Erland et al., 1999; Rygiewicz et al., 2000;
Peter et al., 2001). Richness was not affected by elevated CO2
in accordance with the studies by Rygiewicz et al. (2000) and
Godbold et al. (1997). Rey & Jarvis (1997) did not report on
the total numbers of taxa found, or the abundance of these,
in their study of CO2 effects on ECM fungi. In the present
study, balanced fertilization also had no effect on morphotype
richness. Different fertilization treatments has earlier been
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NPH_276.fm Page 439 Friday, November 2, 2001 10:02 AM
shown to either decrease ECM richness (Alexander & Fairley,
1983; Rühling & Tyler, 1991; Peter et al., 2001) or not to
affect richness at all (Kårén & Nylund, 1997; Jonsson et al.,
1999).
Godbold et al. (1997) suggested that birch saplings grown
under elevated CO2 levels could support mycorrhizal species
with a higher C-demand. Twelve morphotypes were found by
Godbold et al. (1997) on paper birch and Eastern white pine,
with an average of six morphotypes per individual birch
sapling. They observed an increase in the number of mor-
photypes forming extensive extraradical mycelia and rhizo-
morphs. Increased production of mycelium due to elevated
CO2 has been reported for several different ECM species
(Ineichen et al., 1995; Rouhier & Read, 1998a) as well as for
arbuscular mycorrhizal (AM) fungi (Rouhier & Read, 1998b;
Sanders et al., 1998). The increase in fungal biomass is sus-
tained by the extra C fixed by and supplied from the host
plant under enriched CO2 regimes (Godbold et al., 1997).
However, the turnover of mycelium is likely to be faster com-
pared to roots, and an increase in the amount of mycelium
could lead to a faster turnover of C within the plant-fungus
system (Fitter et al., 2000). In the present study it was
impossible to say what happened with the extraradical myce-
lial production.
Rey & Jarvis (1997) found indications that the mycorrhizal
species composition in birch shifted towards later successional
stages after CO2 treatment, and they interpreted this as an
acceleration of tree ontogeny. This may lead to the trees sup-
porting ECM fungal species with a higher carbon demand, in
accordance with the conclusions of Godbold et al. (1997).
Rey & Jarvis (1997) also reported that trees grown in elevated
CO2 invested more C into fine roots, and did not experience
any decline in nutrient concentrations in plant tissues. The
authors interpret the increased fine root density as likely to
have met the nutrient demands of the trees. In addition, the
above mentioned increased production of mycelia under
elevated CO2 may also contribute to more efficient nutrient
acquisition.
Due to the dry conditions in the chambers in our study, the
number of root tips decreased compared to reference trees,
although attempts were made to irrigate. This also affected the
colonization levels to some extent (see below), but we may
consider that the irrigation problem should not have affected
the overall response of the ECM community structure to
elevated CO2 because in the CCA ordination diagram, the
chamber effect was small and not significant. Irrigation has
previously been shown not to affect the community compo-
sition at the Flakaliden field site (Fransson et al., 2000), since
the site is not water limited. Water limitation may influence
the response of host plants and associated mycorrhizal fungi
to increasing CO2, as reported for longleaf pine (Runion
et al., 1997).
An increase in colonization of ECM root tips due to ele-
vated levels of CO2 has often been reported in the literature.
© New Phytologist (2001) 152: 431– 442 www.newphytologist.com
Research 439
This has been demonstrated both from different types of pot
studies (O’Neill et al., 1987; Godbold & Berntson, 1997)
and from studies conducted in field soil (Tingey et al., 1997),
although Lewis et al. (1994) and Walker et al. (1997) found
no effect. The degree of ECM root tip colonization in boreal
forests, however, is often close to 100% (Taylor et al., 2000)
and in those types of systems elevated CO2 will have no effect
on colonization levels. The response of seedlings germinated
and grown according to nursery practices or other seminatural
growth conditions might be expected to differ from those seen
with rooted trees in a more undisturbed environment. What
may happen in the field is that the production of fine roots
increases as a result of elevated CO2, fine roots being more
responsive to CO2 compared to the rest of the root system,
but it is not clear if this response would persist (Norby et al.,
1999). Norby et al. (1999) did not find any support for an
increased root : shoot ratio in their analysis of open-top
chamber field experiments. In the present study the degree of
colonization in 1997 before the CO2 treatment started was
variable, due to irrigation problems in some of the chambers.
Increases in the numbers of nonmycorrhizal root tips can be
observed after dry periods and subsequent addition of water
when the root tips start to proliferate. In the sampling in 2000
all levels of colonization were high as expected, and in an ear-
lier study at the same field site high levels of colonization were
also observed (Fransson et al., 2000).
Elevated CO2 affected few individual components of the
ECM community, only nonmycorrhizal root tips and uniden-
tified ECM morphtypes in the unfertilized plot increased sig-
nificantly with CO2 treatment. The high variablility among
samples may have masked significant effects of CO2 on indi-
vidual taxa. The below-ground ECM community is often
dominated by a few common species (Gardes & Bruns, 1996;
Erland et al., 1999; Peter et al., 2001) with a long tail of rare
species in the distribution curve. Although there is still little
known about differences in carbon use efficiency between
ECM fungal species, there may be differences in their parti-
tioning of C, and in their response to elevated CO2 concen-
trations (Dosskey et al., 1990; Rouhier & Read, 1998a;
Gorissen & Kuyper, 2000). Changes in abundance of domi-
nating fungi may thus affect the C-allocation pattern of the
ECM community.
Variation in ECM community composition both within
seasons and among years has been reported (Malajczuk &
Hingston, 1981; Wu et al., 1993), and was also evident in the
present study. Rygiewicz et al. (2000) found seasonal patterns
in the number of ECM root tips and in two of the dominant
morphotypes (the Rhizopogon group and C. geophilum) to
respond differentially to CO2. Effects of elevated CO2 on
ECM fungi have also been shown to vary over time by O’Neill
et al. (1987). Samples in our study were taken at the end of the
growth season at both sampling occasions to avoid some of
the variation attributed to seasonal changes in community
structure. Despite the considerable interyearly variation in
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440 Research
community composition a clear pattern was revealed in the
DCA analysis. The ECM community structure of all fertilized
trees was clearly separated from that of the unfertilized trees,
with the exception of the elevated trees in the unfertilized
plot. These became more similar to the fertilized plot after
three years of CO2 treatment.
After 3 yr, the effects of CO2 on community composition
were of the same magnitude as those seen after 15 yr of ferti-
lization treatment (indicated in Fig. 1 by the similar length of
CO2 and fertilization vectors). Trees are seen to respond
within hours-days to elevated CO2, and the response of the
below-ground symbiont will be mediated through the host
plant. The dependence of ECM fungi on current assimilates
have been shown both in laboratory and field experi-
ments (Söderström & Read, 1987; Lamhamedi et al., 1994;
Högberg et al., 2001). Physiological responses by ECM fungi
to changes in C-supply are therefore likely to take place shortly
after the plant responds. Shifts in community composition,
however, may take a longer time to be induced. We do not
know if the induced change in ECM community structure
observed in the present study is transient in the present
study, but evidence from long-term studies on the effects of
N-fertilization upon ECM communities suggests that once
initiated, changes in community structure may be more
enduring compared to physiological responses.
C- and N-nutrition within ECM fungi are intimately
linked. The response by the fungus to elevated CO2 levels is
most likely to be affected by availability, uptake and allocation
of N. We found no detectable interactions between elevated
CO2 and fertilization for individual components of the com-
munity, as might have been expected. Interactions between
elevated CO2 and N treatments were reported by Runion
et al. (1997), with increased colonization, fine root lengths
and numbers of ECM root tips under CO2 enrichment only
under high N conditions. The authors suggested that sink/
source relationships were a major factor regulating ECM
fungal responses to elevated CO2.
Klironomos et al. (1997) found increased growth of extra-
matrical mycelium of AM fungi colonizing Populus tremuloides
under low N-availability/high CO2 treatment, and a decrease
under high N-availability. Körner et al. (1997) suggested that
microbial activity of a late successional alpine grassland eco-
system was colimited by C supply and N availability. We still
know very little about individual species responses.
The issue of whether or not trees will experience a down-
regulation or acclimation of photosynthesis as a response to
long-term exposure to elevated CO2 has been debated over
the years. If a down-regulation occurs in the field it might
have large effects on the capacity of forests to sequester
CO2 and act as the predicted sink for atmospheric C. Results
from short-term pot studies have shown considerable down-
regulation of photosynthetic rates within days-weeks (Curtis
& Wang, 1998; Ward & Strain, 1999). This down-regulation
seems to be a result either of sink-limitations (accumulation
of starch and succrose) or environmental limitations (root
restrictions due to pot size, nutrient availability). For trees in
the field, the situation seems to be quite different, and down-
regulation has usually not been seen in longer-term experi-
ments (Saxe et al., 1998; Curtis & Wang, 1998; Norby et al.,
1999). But although down-regulation in the field may not
generally be considered to be as pronounced as for short-term
pot experiments, it cannot be dismissed as a potentially
important factor in the over-all C-cycling. In the Flakaliden
experiment, down-regulaton of net photosynthesis was
observed (G. Wallin, pers. comm.).
Fitter et al. (2000) suggested that the response of mycor-
rhizal fungi could counteract down-regulation by providing
the plants with more nutrients, but only up to a certain point.
Eventually the whole system will come to a new equilibrium
since some factor will always be limiting in ecosystem pro-
cesses. In conclusion, our results show that elevated CO2 can
have an impact on the community structure of ECM fungi
in mature forests, and potentially alter carbon and nutrient
allocation and turnover within forest ecosystems. Since soil
fertility has been shown to limit C sequestration by forest
ecosystems in an enriched CO2 environment (Oren et al.,
2001) the role of ECM fungi could be even more important
than earlier believed.
Acknowledgements
This work was supported by the Swedish National Energy
Administration (STEM) and the Knut and Alice Wallenberg
foundation. We wish to thank Jörg Brunet for valuable help
with the ordination analysis, Birgitta Vegerfors-Persson for
evaluating the statistical analysis and Björn Lindahl for
discussing the ordination results.
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