Mycorrhizal fungal abundance s affected by long-term climatic manipulations in the field

Abstract

Climate change treatments - winter warming, summer drought and increased summer precipitation - have been imposed on an upland grassland continuously for 7 years. The vegetation was surveyed yearly. In the seventh year, soil samples were collected on four occasions through the growing season in order to assess mycorrhizal fungal abundance. Mycorrhizal fungal colonisation of roots and extraradical mycorrhizal hyphal (EMH) density in the soil were both affected by the climatic manipulations, especially by summer drought. Both winter warming and summer drought increased the proportion of root length colonised (RLC) and decreased the density of external mycorrhizal hyphal. Much of the response of mycorrhizal fungi to climate change could be attributed to climate-induced changes in the vegetation, especially plant species relative abundance. However, it is possible that some of the mycorrhizal response to the climatic manipulations was direct - for example, the response of the EMH density to the drought treatment. Future work should address the likely change in mycorrhizal functioning under warmer and drier conditions.

Full text

Mycorrhizal fungal abundance is affected by long-term
climatic manipulations in the field
PHILIP L. STADDON*{, KEN THOMPSON{, IVER JAKOBSEN{,J.PHILIPGRIME{,
ANDREW P. ASKEW{andALASTAIR H. FITTER*
*Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK, {Risù National Laboratory, Plant Research
Department, PO Box 49, Roskilde, DK-4000, Denmark, {Buxton Climate Change Impacts Laboratory (BCCIL), Department of
Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
Abstract
Climate change treatments ± winter warming, summer drought and increased summer
precipitation ± have been imposed on an upland grassland continuously for 7 years. The
vegetation was surveyed yearly. In the seventh year, soil samples were collected on four
occasions through the growing season in order to assess mycorrhizal fungal abundance.
Mycorrhizal fungal colonisation of roots and extraradical mycorrhizal hyphal (EMH)
density in the soil were both affected by the climatic manipulations, especially by
summer drought. Both winter warming and summer drought increased the proportion
of root length colonised (RLC) and decreased the density of external mycorrhizal hyphal.
Much of the response of mycorrhizal fungi to climate change could be attributed to
climate-induced changes in the vegetation, especially plant species relative abundance.
However, it is possible that some of the mycorrhizal response to the climatic manipula-
tions was direct ± for example, the response of the EMH density to the drought treatment.
Future work should address the likely change in mycorrhizal functioning under warmer
and drier conditions.
Keywords: arbuscular mycorrhizas, climate change, drought, global environmental change,
warming
Received 10 May 2002; revised version received 24 July 2002 and accepted 2 August 2002
Introduction
Human activity now has discernible effects on the Earth's
climate (Kerr, 2001). These effects will continue through
the next decades and will result in a substantially warmer
planet with altered weather patterns (Houghton et al.,
1995). Many global circulation models predict that tem-
perate areas will become warmer and drier (Cao &
Woodward, 1998), with more extreme events such as
severe droughts (Easterling et al., 2000). There is a con-
siderable amount of research, which aims at understand-
ing the effects of human-induced environmental change,
and climate change in particular, on the Earth's ecosys-
tems (e.g. Oechel et al., 2000). However, most of this
research is focused on the aboveground component of
the vegetation with rather little effort being targeted at
belowground aspects. The soil ecosystem is an integral
part of terrestrial ecosystems (Coleman & Crossley, 1996).
Soil, including its biota, has numerous ecosystem func-
tions (Killham, 1994) ± for example, nutrient cycling ±
and plays a crucial role in the terrestrial and global
carbon cycles (Schimel, 1995). Soil is, therefore, involved
in feedback mechanisms to global environmental change
(Davidson et al., 2000; but see also Luo et al., 2001), which
is driven primarily by anthropogenic greenhouse gases,
including carbon dioxide (CO
2
), released from the burn-
ing of fossil fuels (Wyman, 1991).
How soil organisms will respond to climate change
is not known. In fact, very little is known about soil
biodiversity and function per se (Killham, 1994). Of the
many various types of soil organisms, mycorrhizal fungi
could be considered as a key group. Mycorrhizal fungi
form symbiotic associations with plant roots: the fungus
receives its carbon from the plant and provides the plant
with nutrients, especially phosphorus (P), and other
Correspondence: Dr Philip L. Staddon, Risù National Laboratory,
Plant Research Department, Building 313, PO Box 49, Roskilde,
DK-4000, Denmark, tel. 45 46774289, fax 45 46774122, e-mail:
philip.louis.staddon@risoe.dk
Global Change Biology (2003) 9, 186±194
186 ß2003 Blackwell Publishing Ltd

benefits such as protection from pathogens (Newsham
et al., 1995). Arbuscular mycorrhizas are the most
common type of mycorrhizas and are found in most
plant species (Smith & Read, 1997). They are particularly
common in temperate grasslands. Mycorrhizal fungi me-
diate competition between plant species (Hetrick, 1991)
and influence plant community structure (van der
Heijden et al., 1998). Perhaps, more importantly in terms
of the global carbon cycle, mycorrhizas can account for a
large proportion of the carbon fixed by the plants: up to
20% in some cases ( Jakobsen & Rosendahl, 1990). Also,
the extensive mycorrhizal hyphal network in the soil is
likely to provide an important pathway for the flow of
carbon from roots to bulk soil (Staddon, 1998). There has
been surprisingly little research undertaken concerning
the effects of climate change on arbuscular mycorrhizas
(Fitter et al., 2000). Virtually nothing is known on how
mycorrhizal fungi might respond to long-term environ-
mental change in the field. The response of mycorrhizal
fungi to environmental changes such as climate changes
must be better understood if we are to predict with
greater certainty how terrestrial ecosystems will respond
to future environmental changes.
The main objective of the work presented here is to
determine whether predicted climate change, specifically
increased winter temperature and altered summer pre-
cipitation, alters mycorrhizal abundance in the field, and
to what extent these effects of climate change on mycor-
rhizas are simply mediated by changes in the vegetation.
This research was based at the Buxton Climate Change
Impacts Laboratory (BCCIL), Derbyshire, UK (Grime
et al., 2000; www.shef.ac.uk/nuocpe/bccil/), where a
long-term climatic manipulation experiment is now in its
seventh year. We briefly assess the effects of climatic
manipulation on the vegetation and attempt to test the
following specific hypotheses:
.Climate change ± both winter warming and altered
summer precipitation ± alters mycorrhizal fungal
abundance.
.The effect of climate treatment on mycorrhizal fungal
abundance is mediated, at least in part, by a change in
the vegetation.
Materials and methods
Site, climate treatments and vegetation
The BCCIL site and climate treatments are fully described
in Grime et al. (2000). The site is an ancient upland lime-
stone sheep pasture at Buxton, Derbyshire, UK. The cli-
mate treatments were started in November 1993 and
were at the time of the work reported in this paper in
their seventh year.
From November to April inclusive, winter temperature
was elevated by 3 8C above ambient by heating cables
(Camplex Thermoforce Ltd) fastened to the soil surface.
The heating intensity was controlled by a computer
system linked to temperature probes. During July and
August, summer drought was imposed by excluding all
rainfall with automatically operated rainshelters, which
slide across the plot when it rains. From June to
September inclusive, summer water addition was im-
posed at regular intervals in order to reach the equivalent
of 20% increase in rainfall compared to the previous
10-year average; refer to Grime et al. (2000) for further
details. A fully randomised block design, each containing
nine 3 3m
2
plots, was used, replicated five times. Each
block contained the following treatments: (i) control,
(ii) summer drought, (iii) summer water addition, (iv)
winter warming (or heating), (v) winter warming and
summer drought, (vi) winter warming and summer
water addition, (vii) cable control (unconnected warming
cables) and two spare plots. The vegetation in the plots
was cut to a height of 4±5 cm at the end of each growing
season.
Vegetation surveys were performed at regular inter-
vals (see Grime et al., 2000), the latest point quadrat
sampling occurred in June 1999 and June 2000, immedi-
ately prior to the start of the yearly drought treatment.
Total quadrat point count was used as an estimate of
standing plant biomass. Two indices of plant diversity
were used:
.Simpson D1/SP2
i(richness, dominance) and
.Shannon±Wiener HSP
i
.lnP
i
(evenness),
where P
i
is the proportion of individual counts for
species `i' (calculations follow Begon et al., 1990).
Collection of belowground data
Soil cores were collected on four occasions over the grow-
ing season from the six main treatments (the cable control
treatment was excluded from the sampling as the previ-
ous unpublished data had shown that there was no de-
tectable difference between the cable control and the
control treatments). Harvests occurred on 3 May 2000,
immediately after the end of the winter warming treat-
ment; on 30 June 2000, prior to the onset of summer
drought; on 20 September 2000, immediately after the
end of summer drought; on 14 November 2000, at the
start of winter warming. Duplicate cores ± 2 cm in diam-
eter and up to 10 cm deep (when possible) ± were col-
lected from each of the treatments in each of the blocks
(in total 60 cores per harvest). Cores were sealed in plastic
bags and refrigerated at 5 8C until processed, which
varied from 1 to 14 days for individual cores. The storage
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
MYCORRHIZAS AND CLIMATE CHANGE 187

of the cores up to 2 weeks was not a problem as we were
not assessing the vitality of mycorrhizas but rather `total'
mycorrhizal parameters, which we have previously
shown to remain relatively constant under such condi-
tions (see Staddon & Fitter, 2001).
Each core was processed for measurements of extrara-
dical mycorrhizal hyphal length (EMH) density, percent
root length colonised (RLC) and root weight density as
follows. A subsample of soil was fresh-weighed and then
placed in 500 mL of water. This was then mixed using a
magnetic stirrer, and diluted accordingly depending on
the concentration of the soil solution. From the final
solution, duplicate 5 mL samples were taken and passed
through 0.45 mm filters under vacuum for extraradical
mycorrhizal hyphae (EMH) collection (for a full descrip-
tion of the procedure, see Staddon et al., 1999). Roots were
extracted from the soil and dried at 75 8C for a minimum
of 3 days. A small random subsample of roots was separ-
ated from the main bulk of the roots prior to drying and
stained with acid fuchsin for internal mycorrhizal assess-
ment. The timing of the staining procedure was 2±3 min
in potassium hydroxide (KOH) at 80 8C, 1 min in hydro-
chloric acid (HCl) at room temperature, 25 min in acid
fuchsin at 80 8C, followed by destaining in lactoglycerol
(for a full description of the procedure, see Staddon et al.,
1998). Both the filters containing the EMH and the stained
roots were mounted in lactoglycerol onto microscope
slides.
In order to allow for conversion of soil fresh weight
(FW) to soil dry weight (DW), the remaining soil from
each core was fresh-weighed and then oven-dried at
75 8C. This involved all the cores from harvests 3 and 4,
but only a random subset from harvests 1 and 2. Soil
moisture (percent water content) data is, therefore, avail-
able for harvests 3 and 4, immediately after the end of the
summer drought and 2 months after.
Extraradical mycorrhizal hyphal density was assessed
using a compound microscope (Zeiss Jenamed 2) fitted
with a 1 1cm
2
100-grid graticule (Graticules Ltd, UK) at
250 with a minimum of 30 grids per filter being ob-
served. The grid-line intercept method (see Tennant,
1975) was used in order to obtain a length of hyphae
per filter. The mean value of the four filters (two filters
per duplicate) per replicate was used. Hyphal density is
finally expressed in m hyphae per g soil DW. Hyphae
were counted as mycorrhizal if they showed the
following typical characteristics: dichotomous branching,
angular projections, absence of septa (Nicolson, 1959).
This standard method (e.g. Miller et al., 1995; Kabir et al.,
1997; Schweiger & Jakobsen, 1999; Rillig et al., 2002) may
slightly underestimate EMH density by excluding some
hyphae, which may actually be mycorrhizal. Internal
mycorrhizal colonisation was assessed using a com-
pound microscope (Nikon EFD-3 Optiphot-2) fitted with
a cross-hair graticule at 200 with epifluorescence
(Merryweather & Fitter, 1991); a minimum of 80 intersec-
tions were assessed per core. Scoring followed McGonigle
et al. (1990). The mean value for the two cores per replicate
was used. Further details on the methods used for collec-
tion of mycorrhizal data are available in Staddon et al.
(1998, 1999).
Statistical analysis
All statistical analyses were performed using SPSS 10.0.
All data was checked for normality and where necessary
was ln transformed (EMH density, root density, EMH/
root ratio) or square root arcsine transformed (RLC, per-
cent species cover). Correlation analysis was used in
order to test for relationships between variables (EMH,
RLC, root density, plant diversity, plant species cover,
soil moisture, etc.). Repeated measures analysis of vari-
ance (anova) and repeated measures analysis of covar-
iance (ancova) were used in order to test for climate
treatment effects on the various measured variables, in-
cluding plant diversity.
Results
Effects on the vegetation
Climate treatments had no effect on plant biomass.
However, plant diversity (both Simpson Dand Shannon±
Wiener Hindices) was significantly decreased by both
winter warming and summer drought (P<0.001); summer
water addition had little effect (Fig. 1). For example, winter
warming decreased the June 2000 Simpson Dindex from
8.1 (nonheated plots) to 5.3 (heated plots); similarly,
summer drought decreased the June 2000 Simpson D
index from 7.8 (plots with no water treatment) to 4.6
(droughted plots). Both winter warming and summer
drought increased Festuca cover (P<0.001) and
0
Control Drought Water Heat H + D H + W
2
4
6
Simpson D index
8
10
12
Fig. 1 Plant diversity index as affected by climatic manipula-
tions. Simpson Dindex of diversity data is presented, but a
similar pattern is seen with the Shannon±Wiener Hindex.
Empty bars: June 1999; hashed bars: June 2000; error bars repre-
sent standard errors.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
188 P. STADDON et al.

decreased Carex cover (P<0.05), although the effects
were much stronger for Festuca than for Carex.
Festuca ovina and Carex species ± C. flacca,
C. caryophyllea,C. panicea and C. pulicaris in declining
order of abundance ± were the dominant species at the
site, with Festuca cover ranging from 19% in the summer
water addition (W) treatment to 51% in the winter warming
and summer drought (HD) treatment and Carex cover
ranging from 12 to 32% in the HD and W treatments, re-
spectively. Festuca and Carex cover were negatively correl-
ated (P<0.001). Festuca cover was negatively correlated
with plant diversity (P<0.001) and positively correlated
with plant biomass (P0.029), whereas Carex cover was
positively correlated with plant diversity (P0.001) and
negatively correlated (although only a trend) with plant
biomass (P0.066).
Two measures of soil moisture are available: 20
September (H3) and 14 November 2000 (H4). Note that
the H3 measure taken in September immediately after the
end of the summer drought treatment was strongly
linked to the water regime, whereas this was not the
case for the H4 (November) measure (Fig. 2). Plant diver-
sity was positively correlated with H3 soil moisture
(P<0.01) but not significantly so with H4 soil moisture.
In contrast, plant biomass was negatively correlated with
H4 soil moisture (P<0.01) but not with H3 soil moisture.
Festuca cover was negatively correlated with soil mois-
ture (P0.004 at H3 and P0.061 at H4), whereas Carex
cover was not (positive trend only). In other words, it is
not that Carex species were strongly associated with soil
moisture (as might be expected), but rather that Festuca
ovina was strongly negatively correlated with soil
moisture.
In June 1999, there was no correlation between plant
biomass and plant diversity; however, in June 2000 there
was a significant negative correlation between plant
biomass and the Shannon±Wiener Hdiversity index
(P0.050; P0.068 for Simpson D). Plant diversity was
strongly correlated between the two years (P<0.001);
however, there was surprisingly no relationship between
the estimate of plant biomass for the two years.
Belowground effects: mycorrhizas and roots
There were significant effects of the climate treatments on
both mycorrhizas and roots (Table 1). Percent root length
colonised RLC was increased by both summer drought
(P<0.001) and winter warming (P<0.05) (Fig. 3).
However, EMH length density was decreased by both
summer drought (P<0.001) and winter warming
(P<0.01) (Fig. 4). Similarly, root weight density was
decreased by both summer drought (P<0.05) and winter
0
Fraction soil moisture content
Control Drought Water Heat H + D H + W
0.1
0.2
0.3
0.4
0.5
Fig. 2 Soil moisture content (SMC) as affected by climatic ma-
nipulations. Empty bars: 20 September 2000; hashed bars: 14
November 2000; the September measure was immediately after
the end of the summer drought treatment; error bars represent
standard errors.
Table 1 The level of significance for the between-subjects
effects of water and temperature treatments on mycorrhizal and
root parameters as obtained by repeated measures analysis of
variance (anova). The water factor has three levels: control,
water addition and drought; the temperature factor has two
levels: control and heating. Post hoc tests showed that the
drought treatment was responsible for most of the water factor's
significant effects ± that is, the control and water addition treat-
ments were very similar
Water Temperature Interaction
Percent root length
colonised (RLC)
*** * *
Extraradical mycorrhizal
hyphal (EMH) length density *** ** *
Root weight density * * NS
EMH length to root weight
ratio NS NS NS
*P<0.05; **P<0.01; ***P<0.001; NS not significant.
20
Percent root length colonised
15/Apr./2000 4/Jun./2000 24/Jul./2000 12/Sept./2000 1/Nov./2000
30
40
50
60
Fig. 3 Root length colonised (RLC) (percent) by arbuscular
mycorrhizal fungi as affected by climatic manipulations. Treat-
ments are: control ( ), summer drought ( ), summer
water addition ( ), winter warming ( ), warming and
drought ( ), warming and water addition  ; error
bars represent standard errors.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
MYCORRHIZAS AND CLIMATE CHANGE 189

warming (P<0.05) (Fig. 5). Neither treatment type (tem-
perature or precipitation) had any obvious effect on the
ratio of EMH length to root weight (Fig. 6). There was a
significant interaction (P<0.05) between the winter
warming and summer water manipulation treatments
on EMH and RLC, possibly involving differential effects
of the water addition treatment depending on the winter
warming treatment. Inclusion of moisture as a covariate
minimally altered these findings, as did plant biomass
(when covariate significant). Plant diversity was never
significant as a covariate and generally nor was Festuca
or Carex cover.
Extraradical mycorrhizal hyphal length and root
weight density were positively and percent RLC nega-
tively correlated with plant diversity (Table 2). However,
none of the mycorrhizal and root variables were correl-
ated with plant biomass. Extraradical mycorrhizal
hyphal length and root density were generally positively
correlated with Carex cover and negatively with Festuca
cover, but for RLC the opposite was true (Table 3). The
ratio of EMH length to root weight was not significantly
correlated with either Carex or Festuca cover. The only
exception to this was the 3 May harvest, where there was
a negative correlation between the ratio of EMH length to
root weight and Festuca cover. Of note is that the increase
in percent RLC and decrease in EMH length density by
the summer drought and winter warming treatments
corresponded with an increase in the cover of Festuca
and Koeleria macrantha and a decrease in that of Carex
and Potentilla erecta.Festuca and Koeleria cover increased
from a combined cover of 23 and 21% in the control (C)
and the summer water addition (W) treatments, respect-
ively, to a maximum of 56% in the winter warming and
summer drought (HD) treatment; Carex and Potentilla
cover decreased from a combined maximum cover of
33 and 39% in C and W to a minimum of 13% in HD.
Extraradical mycorrhizal hyphal length density was
positively correlated with root weight density (P<0.001),
but negatively correlated with per cent RLC (P<0.001),
100
15/Apr./2000 4/Jun./2000 24/Jul./2000 12/Sept./2000 1/Nov./2000
1000
mm hyphae g−1 soil dw
10 000
100 000
Fig. 4 Extraradical mycorrhizal hyphal (EMH) density (mm
hyphae g
1
soil dw) as affected by climatic manipulations. Note
the log 10 scale. Treatments are: control ( ), summer
drought ( ), summer water addition ( ), winter
warming ( ), warming and drought ( ), warming and
water addition ( ); error bars represent standard errors.
0
15/Apr./2000 4/Jun./2000 24/Jul./2000 12/Sept./2000 1/Nov./2000
10
20
mg root g−1 soil dw
30
40
50
60
Fig. 5 Root weight density (mg root g
1
soil dw) as affected by
climatic manipulations. Treatments are: control ( ), summer
drought ( ), summer water addition ( ), winter
warming ( ), warming and drought ( ), warming and
water addition ( ); error bars represent standard errors.
200
mm hyphae
/
mg root
0
15/Apr./2000 4/Jan./2000 24/Jul./2000 12/Sept./2000 1/Nov./2000
400
600
800
Fig. 6 Ratio of extraradical mycorrhizal hyphae (EMH) to root
(mm/mg) as affected by climatic manipulations. Treatments are:
control ( ), summer drought ( ), summer water add-
ition ( ), winter warming ( ), warming and drought
(), warming and water addition ( ); error bars repre-
sent standard errors.
Table 2 Correlations between mycorrhizal and root parameters
and plant diversity based on vegetation data from June 2000
( June 1999 data gave similar results). All dates refer to 2000
Percent
root length
colonised
(RLC)
Extraradical
mycorrhizal
hyphal (EMH)
density
Root
weight
density
EMH
length to
root weight
ratio
3May NS *** ** **
30 June ±*** NS *NS
20 September ±* *** *NS
14 November ±* ** NS NS
*P<0.05; **P<0.01; ***P<0.001; NS not significant; positive
correlation; ± negative correlation.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
190 P. STADDON et al.

which was also negatively correlated with root weight
density (P<0.001). Extraradical mycorrhizal hyphal and
root density were both positively correlated with soil
moisture content (P<0.001 and P0.001, respectively),
but RLC was negatively correlated with soil moisture
(P<0.001).
Discussion
The vegetation
As reported previously for vegetation data until 1996
(Grime et al., 2000), there was no effect of the various
climatic manipulations, which started in November
1993, on total plant biomass in 1999 or 2000. Grime et al.
(2000) also reported minimal changes in the vegetation
until 1996. However, by 1999, there were highly signifi-
cant effects of the imposed climate treatments on plant
diversity as measured by both the Simpson Dindex of
dominance and the Shannon±Wiener Hindex of even-
ness. Both winter warming and summer drought
substantially decreased plant diversity. A similar phe-
nomenon has been reported for a subalpine Colorado
meadow (Harte & Shaw, 1995). However, at the Colorado
site it was an increase in a shrub, Artemisia tridentata,
which resulted in lower plant diversity under the drier
and warmer conditions.
This change in plant diversity at the Buxton site was
correlated with a strong increase in the percentage cover
of the dominant grass Festuca ovina and a decrease in the
cover of the sedges ± principally Carex flacca ± in both the
winter warming and the summer drought treatments.
Soil moisture appeared to be a determining factor for
Festuca cover, but not for Carex cover. This would suggest
that, in the case of the Buxton site, the soil would still be
sufficiently wet after the climatic manipulations to sup-
port Carex over its entirety but that the winter warming
and summer drought allowed Festuca to become more
competitive. The winter warming treatment ± particularly
when combined with summer drought ± shifted the grow-
ing season away from the summer towards the spring and
autumn. Grasses seemed to be able to take advantage of this
shift, whereas sedges did not. This is likely to be because
sedge growth is confined to the warmest parts of the year,
whereas grass growth is not (Grime et al., 1985). The in-
creased competitive ability of F. ovina and some of the
other grasses ± for example, Koeleria macrantha ± under
the warmer and drier conditions was likely to be the
main cause for the decrease in occurrence of many of
the less common forbs such as Lotus corniculatus and
Plantago lanceolata. It also resulted in the overall decrease
of the number of rarer species.
The location of the site was likely to have played a
major role in the measured outcomes of the climate treat-
ments on plant diversity. The availability of a pool of
species, which can take advantage of changing climatic
conditions when they occur, is a crucial factor in deter-
mining how an ecosystem will respond (e.g. Buzas &
Culver, 1994). In a manipulation experiment such as the
one reported here, there may well be no available pool of
species with the potential to invade. This, it could be
argued, reflects the current environmental conditions of
fragmented habitats (see Casagrandi & Gatto, 1999).
Mycorrhizas and roots
There were significant effects of the climate treatments on
both the mycorrhizas and roots. The overall level of RLC
by arbuscular mycorrhizal fungi at the site was stimu-
lated by both summer drought and winter warming. This
observation that drought promoted more extensive
mycorrhizal colonisation has been noted in other field
studies (Auge
Â, 2001). However the opposite was true for
the density of both EMH and roots. Similar effects of
drought on RLC and EMH have been noted previously
(unpublished data) during an experiment performed in
growth chambers. Both in the field and in the laboratory,
soil moisture content (SMC) was a key factor in determin-
ing the relative abundance of mycorrhizal fungi within
and outside plant roots. This does not mean that SMC
affects mycorrhizal fungi directly; the effect could be via
a change in root density and or vegetation composition
(see next section). Overall, RLC at the Buxton site was
negatively correlated with EMH suggesting that there
Table 3 Correlations between mycorrhizal and root parameters
and Carex and Festuca cover based on vegetation data from June
2000. All dates refer to 2000
Carex cover Festuca cover
Percent
root length
colonised (RLC)
03 May NS NS
30 June 
*

***
20 September 
{

*
14 November 
*

*
Extraradical
mycorrhizal
hyphal (EMH)
length density
03 May NS 
***
30 June 
*
NS
20 September 
*

***
14 November 
**

**
Root weight
density
03 May 
{

**
30 June NS 
*
20 September NS 
*
14 November 
*

{
EMH length to
root weight ratio
03 May NS 
**
30 June NS NS
20 September NS NS
14 November NS NS
*
P<0.05;
**
P<0.01;
***
P<0.001;
{
P<0.10; NS not significant;
positive correlation; ± negative correlation.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
MYCORRHIZAS AND CLIMATE CHANGE 191

was a trade-off on the part of the mycorrhizal fungus
between investing in root occupation and soil explor-
ation. This fits well with the theory that under stressed
conditions ± for example, drought ± a mycorrhizal fungus
will invest more resources in storage capacity in roots
and less in external hyphae (Smith & Smith, 1996).
However, an alternative explanation for the negative
correlation between RLC and EMH could be the
change in relative abundance of roots belonging to
mycorrhizal versus nonmycorrhizal plant species (see
next section).
The climatic manipulations interacted with each other
to some degree in their effects on mycorrhizal param-
eters. This was particularly so for the heating and water
addition treatments: water addition decreased RLC only
under nonheated conditions and increased EMH density
only under heated conditions. Presumably these inter-
actions would have been because of changes in the vege-
tation, which would have occurred under warmer and
wetter conditions (see next section).
Root weight density exhibited the same pattern of re-
sponse to the climatic variables as EMH length density.
This resulted in a relatively stable EMH to root ratio
across all treatments. Although this ratio is of a length
to a biomass (m/g) it is nonetheless informative in terms
of carbon partitioning between host plants and mycor-
rhizal fungi (see Read, 1992). A stable ratio would indi-
cate that, despite severe climatic manipulation, the
proportion of belowground carbon invested in standing
root or EMH structures was unchanged; or in other
words, carbon partitioning between root and EMH was
not affected by the climate treatments. A caveat to this is
that root turnover may have been altered by the climatic
manipulation, as has been reported previously for a dif-
ferent upland grassland (Fitter et al., 1998); if this oc-
curred at the Buxton site then EMH turnover might also
be altered. Nonetheless, this observation that the carbon
partitioning between root and EMH was rather stable has
been noticed previously in the context of elevated atmos-
pheric CO
2
: any change in mycorrhizal parameters was
proportional to changes in plant size (Staddon & Fitter,
1998). This would also support the theory that the mycor-
rhizal fungi simply responded to the amount of carbon
available to them and had rather little control over the
amount they can obtain from the host plant (Tester et al.,
1986).
As noted above, a caveat to this is that in this climate
change experiment, the quantity of mycorrhizal fungi
inside the roots was affected inversely to that outside
the roots. This would indicate that the fungi were altering
their within carbon allocation pattern between structures
inside and outside of the roots leading to an increase in
the ratio of internal to extraradical mycorrhizal carbon in
the summer drought and winter warming treatments.
This would also mean that, although the ratio of standing
EMH to root was unaffected by the climate treatments,
the amount of carbon partitioned to the fungi in the
mycorrhizal associations was increased in the drought
and warming treatments. A question that arises here is
whether, or more likely how, the turnover of root and
mycorrhizal structures was affected by the climatic ma-
nipulations. Indeed, fine roots have been shown to alter
their turnover under changed climatic conditions
(Fitter et al., 1997). As far as we are aware, nothing is
known about the environmental effects on mycorrhizal
turnover.
Are mycorrhizal effects dependent on vegetation changes?
Mycorrhizal parameters were correlated with plant di-
versity and the percent cover of the dominant grasses and
sedges. Generally the direction of the correlations with
vegetation parameters was opposite for RLC and EMH.
The increase in RLC with increasing cover of Festuca and
Koeleria and decreasing cover of Carex and Potentilla (and
overall decrease in plant diversity) is not surprising
when the mycorrhizal status of the plants is taken into
account: both Festuca and Koeleria are mycorrhizal,
F. ovina can be quite strongly so, whereas Carex species
are generally nonmycorrhizal (occasionally, some
species can be moderately mycorrhizal) and Potentilla is
known to be facultatively mycorrhizal (Harley & Harley,
1987).
The negative correlation between RLC and EMH
could, therefore, be explained in terms of the change in
relative abundance of roots belonging to mycorrhizal
versus nonmycorrhizal plant species under the climatic
manipulations. Although the summer drought and
winter warming treatments resulted in an increase in
percent Festuca cover, and therefore also a likely increase
in the proportion of Festuca roots, the overall root weight
density decreased. So, under the drought and warming
treatments, the greater proportion of Festuca roots
resulted in the overall increase in the percentage of
RLC; and the lower total root density (and concomitant
lower absolute RLC) resulted in the decrease in EMH
density. However, assuming no major change in specific
root length at the site, an increase in percent root length
colonisation with no change in the ratio of EMH to root
would mean that the ratio of internal to external mycor-
rhizal structures was increased. If this is indeed what is
happening, this would support the theory of increased
mycorrhizal fungal carbon allocation to structures inside
roots under conditions of environmental stress (Smith &
Smith, 1996). This could be either as a result of changes in
carbon partitioning within an `individual' mycorrhizal
fungus or as a result of possible changes in the mycor-
rhizal fungal community.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
192 P. STADDON et al.

Another possible explanation as to why EMH density
does not follow RLC is that EMH were more directly
affected by the edaphic conditions, especially soil mois-
ture (Auge
Â, 2001: Table 6) but also soil temperature. In
other words, there are two mechanisms operating: the
direct effects of edaphic conditions on EMH and the
indirect effects mediated by altered plant community
composition. So, in the case of this experiment, summer
drought ± for example ± favoured strongly mycorrhizal
plants on the one hand but on the other hand resulted in
a decline in EMH density relative to the amount of
mycorrhizal colonised root (as the ratio of EMH length
to root weight was unchanged and assuming no change
in specific root length). Any decrease in EMH growth as a
direct result of edaphic factors would have resulted in
more mycorrhizal carbon being available for intraradical
mycorrhizal structures. However, from our data it is not
possible to distinguish whether there were truly any
direct edaphic effects on the EMH. There is also the
possibility that various mycorrhizal fungal species were
favoured in the drought and warming treatments either
as a result of altered plant species abundance or as a
direct result of altered edaphic conditions.
Conclusions
Mycorrhizal fungi responded to long-term climatic ma-
nipulations in the field, including a change in both soil
temperature and moisture content. The most significant
mycorrhizal fungal response was to drought, where the
proportion of root length colonised (RLC) was increased
and the extraradical mycorrhizal hyphal (EMH) density
was decreased. Much of the mycorrhizal fungal response
to climate change was attributed to vegetation changes.
However, changes in plant species composition could not
account for all of the mycorrhizal fungal response to the
altered climatic conditions. Future work should attempt
to separate the direct effects of climate change on mycor-
rhizas from the more indirect effects via vegetation
changes. Also, the likelihood that future climate change
will result in an altered mycorrhizal fungal community
needs to be addressed. However, because of the role of
mycorrhizal fungi in the global carbon cycle, how mycor-
rhizal functioning will be affected by climate change
is perhaps the key area to concentrate most research
effort.
Acknowledgements
This research was funded by NERC and the European
Commission. Philip Staddon is currently in receipt of a Marie
Curie Individual Fellowship. Thanks to all the staff at BCCIL for
making this work possible. Thanks to NERC for core funding to
BCCIL.
References
Auge
ÂRM (2001) Water relations, drought and vesicular±
arbuscular mycorrhizal symbiosis. Mycorrhiza,11, 3±42.
Begon M, Harper JL, Townsend CR (1990) Ecology: Individuals,
Populations and Communities, 2nd edn. Blackwell, Oxford.
Buzas MA, Culver SJ (1994) Species pool and dynamics of
marine paleocommunities. Science,264, 1439±1441.
Cao M, Woodward FI (1998) Dynamic responses of terrestrial
ecosystem carbon cycling to global climate change. Nature,
393, 249±252.
Casagrandi R, Gatto M (1999) A mesoscale approach to extinc-
tion risk in fragmented habitats. Nature,400, 560±562.
Coleman DC, Crossley DA (1996) Fundamentals of Soil Ecology.
Academic Press, San Diego.
Davidson EA, Trumbore SE, Amundson R (2000) Soil warming
and organic carbon content. Nature,408, 789±790.
Easterling DR, Meehl GA, Parmesan C et al. (2000) Climate
extremes: observations, modeling and impacts. Science,289,
2068±2074.
Fitter AH, Graves JD, Self GK et al. (1998) Root production,
turnover and respiration under two grassland types along an
altitudinal gradient: influence of temperature and solar radi-
ation. Oecologia,114, 20±30.
Fitter AH, Graves JD, Wolfenden J et al. (1997) Root production
and turnover and carbon budgets of two contrasting grass-
lands under ambient and elevated atmospheric carbon dioxide
concentrations. New Phytologist,137, 247±255.
Fitter AH, Heinemeyer A, Staddon PL (2000) The impact of
elevated CO
2
and global climate change on arbuscular my-
corrhizas: a mycocentric approach. New Phytologist,147,
179±187.
Grime JP, Brown VK, Thompson K et al. (2000) The response of
two contrasting limestone grasslands to simulated climate
change. Science,289, 762±765.
Grime JP, Shacklock JML, Band SR (1985) Nuclear DNA con-
tents, shoot phenology and species co-existence in a limestone
grassland community. New Phytologist,100, 435±445.
Harley JL, Harley EL (1987) A check-list of mycorrhiza in the
British flora. New Phytologist (Suppl), 105, 1±102.
Harte J, Shaw R (1995) Shifting dominance within a montane
vegetation community ± results of a climate-warming experi-
ment. Science,267, 876±880.
van der Heijden MGA, Klironomos JN, Ursic M et al. (1998)
Mycorrhizal fungal diversity determines plant biodiversity,
ecosystem variability and productivity. Nature,396, 69±72.
Hetrick BAD (1991) Mycorrhizas and root architecture.
Experientia,47, 355±362.
Houghton JT, Meira Filho LG, Bruce J et al. (1995) Climate Change
1995: the Science of Climate Change. Intergovernmental Panel on
Climate Change. Cambridge University Press, Cambridge.
Jakobsen I, Rosendahl L (1990) Carbon flow into soil and external
hyphae from roots of mycorrhizal cucumber plants. New
Phytologist,115, 77±83.
Kabir Z, O'Halloran IP, Fyles JW et al. (1997) Seasonal changes of
arbuscular mycorrhizal fungi as affected by tillage practices
and fertilization: hyphal density and mycorrhizal root colon-
ization. Plant and Soil,192, 285±293.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
MYCORRHIZAS AND CLIMATE CHANGE 193

Kerr RA (2001) It's official: humans are behind most of global
warming. Science,291, 566.
Killham K (1994) Soil Ecology. Cambridge University Press,
Cambridge.
Luo Y, Wan S, Hui D et al. (2001) Acclimatization of soil respir-
ation to warming in a tall grass prairie. Nature,413, 622±625.
McGonigle TP, Miller MH, Evans DG et al. (1990) A new method
which gives an objective measure of colonization of roots by
vesicular±arbuscular mycorrhizal fungi. New Phytologist,115,
495±501.
Merryweather JW, Fitter AH (1991) A modified method for
elucidating the structure of the fungal partner in vesicular±
arbuscular mycorrhiza. Mycological Research,95, 1435±1437.
Miller RM, Reinhardt DR, Jastrow JD (1995) External hyphal
production of vesicular±arbuscular mycorrhizal fungi in pas-
ture and tallgrass prairie communities. Oecologia,103, 17±23.
Newsham KK, Fitter AH, Watkinson AR (1995) Multi-
functionality and biodiversity in arbuscular mycorrhizas.
Trends in Ecology and Evolution,10, 407±411.
Nicolson TH (1959) Mycorrhiza in the Gramineae. I.
Vesicular±arbuscular endophytes, with special reference to
the external phase. Transactions of the British Mycological
Society,42, 421±438.
Oechel WC, Vourtilis GL, Hastings SJ et al. (2000) Acclimation of
ecosystem CO
2
exchange in the Alaskan Arctic in response to
decadal climate warming. Nature,406, 978±981.
Read DJ (1992) The mycorrhizal mycelium. In: Mycorrhizal
Functioning (ed. Allen MF), pp. 102±133. Chapman & Hall,
New York.
Rillig MC, Wright SF, Shaw MR et al. (2002) Artificial climate
warming positively affects arbuscular mycorrhizae but de-
creases soil aggregate water stability in an annual grassland.
Oikos,97, 52±58.
Schimel DS (1995) Terrestrial ecosystems and the carbon cycle.
Global Change Biology,1, 77±91.
Schweiger PF, Jakobsen I (1999) Direct measurement of arbuscu-
lar mycorrhizal phosphorus uptake into field-grown winter
wheat. Agronomy Journal,91, 998±1002.
Smith SE, Read DJ (1997) Mycorrhizal Symbiosis, 2nd edn.
Academic Press, San Diego.
Smith FA, Smith SE (1996) Mutualism and parasitism: diversity
in function and structure in the `arbuscular' (VA) mycorrhizal
symbiosis. Advances in Botanical Research,22, 1±43.
Staddon PL (1998) Insights into mycorrhizal colonisation at ele-
vated CO
2
: a simple carbon partitioning model. Plant and Soil,
205, 171±180.
Staddon PL, Fitter AH (1998) Does elevated atmospheric carbon
dioxide affect arbuscular mycorrhizas? Trends in Ecology and
Evolution,13, 455±458.
Staddon PL, Fitter AH (2001) The differential vitality of intrar-
adical mycorrhizal structures and its implications. Soil Biology
and Biochemistry,33, 129±132.
Staddon PL, Fitter AH, Graves JD (1999) Effect of elevated at-
mospheric CO
2
on mycorrhizal colonisation, external hyphal
production and phosphorus inflow in Plantago lanceolata and
Trifolium repens in association with the arbuscular mycorrhizal
fungus Glomus mosseae.Global Change Biology,5, 347±358.
Staddon PL, Graves JD, Fitter AH (1998) Effect of enhanced
atmospheric CO
2
on mycorrhizal colonisation by Glomus mos-
seae in Plantago Lanceolata and Trifolium repens.New Phytologist,
139, 571±580.
Tennant D (1975) A test of a modified line intersect method of
estimating root length. Journal of Ecology,63, 995±1001.
Tester M, Smith SE, Smith FA et al. (1986) Effects of photon
irradiance on the growth of shoots and roots, on the rate of
initiation of mycorrhizal infection units in Trifolium subterra-
neum L. New Phytologist,103, 375±390.
Wyman RL (1991) Global Climate Change and Life on Earth.
Chapman & Hall, New York.
ß2003 Blackwell Publishing Ltd, Global Change Biology,9, 186±194
194 P. STADDON et al.