Effects of three years of soil warming and shading on therate of soil respiration: substrate availability and notthermal acclimation mediates observed response

I A I N P. H A R T L E Y 1 , A N D R E A S H E I N E M E Y E R and P H I L I N E S O N

Biology Department, Stockholm Environment Institute (SEI-York centre), University of York, York YO10 5YW, UK

Abstract

In a number of recent field studies, the positive response of soil respiration to warming

has been shown to decline over time. The two main differing hypotheses proposed to

explain these results are: (1) soil microbial respiration acclimates to the increased

temperature, and (2) substrate availability within the soil decreases with warming so

reducing the rate of soil respiration. To investigate the relative merits of these two

hypotheses, soil samples (both intact cores and sieved samples) from a 3-year grassland

soil-warming and shading experiment were incubated for 4 weeks at three different

temperatures under constant laboratory conditions. We tested the hypothesis that sieving

the soils would reduce differences in substrate availability between warmed and control

plot samples and would therefore result in similar respiration rates if microbial activity

had not acclimated to soil warming. In addition, to further test the effect of substrate

availability, we compared the respiration rates of soils taken from shaded and unshaded

plots. Both soil warming and shading significantly reduced respiration rates in the intact

cores, especially under higher incubation temperatures. However, sieving the soil greatly

reduced these differences suggesting that substrate availability, and not microbial

acclimation to the higher temperatures, played the dominant role in determining the

response of heterotrophic soil respiration to warming. The effect of shading appeared to

be mediated by reduced plant productivity affecting substrate availability within the soil

and hence microbial activity. Given the lack of evidence for thermal acclimation of

microbial respiration, there remains the potential for prolonged carbon losses from soils

in response to warming.

Keywords: acclimation, CO2, heterotrophic respiration, incubation, microbial community, positive

feedback, root biomass, soil warming, substrate availability, temperature

Received 8 November 2006; revised version received 8 February 2007 and accepted 5 February 2007

Introduction

Only approximately 45% of the CO2 released by anthro-

pogenic activities has ended up in the atmosphere and

it is believed that increased photosynthesis in terrestrial

ecosystems has absorbed a large proportion of the

remaining emissions (Houghton et al., 1998; Intergo-

vernmental Panel on Climate Change, 2001). However,

Cox et al. (2000) suggested that the positive effect of

increasing global temperatures on soil respiration may

result in terrestrial ecosystem losing soil carbon (C) and

so enhance the rate of climate change by �35%. If these

results are correct, then C-cycle feedbacks have the

potential to undermine international attempts to curb

global CO2 emissions (Bellamy et al., 2005). Therefore,

it is necessary to undertake a critical evaluation of the

assumptions underlying this modelling study, the key

one being that soil organic matter (SOM) decomposition

will respond to long-term warming with a Q10 of 2 (i.e.

heterotrophic soil respiration will increase exponen-

tially, with its rate doubling for every 10 1C rise in

temperature). A number of recent studies have chal-

lenged this assumption.

Long-term monitoring in the field has identified

temperature as a key determinant of soil respiration

1Present address: School of Biological and Environmental Sciences,

University of Stirling, Stirling FK9 4LA, UK.

Correspondence: Iain P. Hartley, fax 144 1786 467843,

e-mail: i.p.hartley@stir.ac.uk

Global Change Biology (2007) 13, 1761–1770, doi: 10.1111/j.1365-2486.2007.01373.x

r 2007 The AuthorsJournal compilation r 2007 Blackwell Publishing Ltd 1761

rates (e.g. Raich & Schlesinger, 1992; Lloyd & Taylor,

1994; Davidson et al., 1998; Rodeghiero & Cescatti,

2005), and laboratory incubations of both intact cores

(e.g. Fang & Moncrieff, 2001; Reichstein et al., 2005) and

sieved root-free samples (e.g. Bowden et al., 1998; Fang

et al., 2005) have also demonstrated a strong tempera-

ture dependence. Further, a meta-analysis of the results

from 17 different long-term field-warming experiments

identified a statistically significant increase in the rate of

soil respiration following warming (Rustad et al., 2001),

yet the magnitude of this increase was not as large as

predicted from short-term experiments. In addition, a

number of studies have observed that the initial in-

crease in the rate of soil respiration declines over time

(Rustad et al., 2001; Eliasson et al., 2005). These observa-

tions have led authors to conclude that soil respiration

may ‘acclimate’ to warming and so dampen any

potential positive feedback to global climate change

(Luo et al., 2001).

Two main explanations for soil respiration ‘acclima-

tion’ have been proposed. Firstly, soil warming may

induce a change in the composition of the microbial

community within soil (Zogg et al., 1997; Andrews et al.,

2000; Zhang et al., 2005) with the decomposition activ-

ities associated with the new community having a

different temperature dependence (Luo et al., 2001).

Alternatively, modelling studies have suggested that

soil respiration responses to increased temperature are

likely to be limited by substrate availability, with labile

C pools becoming depleted by increased activity in

response to warming, and so leading to a subsequent

reduction in the rate of soil respiration (Kirschbaum,

2004; Eliasson et al., 2005; Knorr et al., 2005). These two

conflicting hypotheses may have very different conse-

quences. If acclimation/adaptation of the microbial

community is central to the reduction in the rate

of respiration in warmed plots, then soil C stocks may

be preserved and the potential for C sequestration

in terrestrial ecosystems maintained. However, if sub-

strate limitation is the key driver then there remains

the potential for reduced sequestration of recently

plant-derived C and, ultimately, C losses from soils

(Eliasson et al., 2005).

A further complication exists when investigating the

responses of soil microbes to soil warming due to the

potentially large contribution (�50%) of root respiration

to belowground respiration (Hanson et al., 2000). Root

respiration and SOM decomposition may respond dif-

ferently to long-term changes in temperature (Fitter

et al., 1998; Huang et al., 2005) and difficulties associated

with separating these two terms (Hanson et al., 2000)

mean that direct measurements of the response of

heterotrophic respiration to increases in soil tempera-

ture are often difficult in situ. However, removing soil

samples from the field severs all connections to assim-

ilate supply and results in a rapid decline in the rate of

root respiration (Langley et al., 2005). Therefore, the

incubation of soils taken from soil warming experi-

ments can allow the effects on heterotrophic respiration

to be measured directly.

In addition, by breaking up aggregates and making

previously physically protected SOM available, sieving

can reduce differences in substrate availability between

soil samples and generally causes an increase in the rate

of respiration (Franzluebbers, 1999). In terms of redu-

cing substrate availability differences between samples,

sieving has an advantage over single substrate addi-

tions which, although providing an estimate of micro-

bial biomass, tend to promote the proliferation of

microbes especially adapted to the particular substrate

added (Pennanen et al., 2004). In addition the very rapid

pulse of respiration associated with substrate-induced

respiration was not suitable for the type of investigation

being carried out here.

The main aim of our study was to determine whether

microbial community acclimation/adaptation or sub-

strate depletion is the key driver of the reduction in the

positive warming response of soil respiration observed

in many long-term warming experiments (Rustad et al.,

2001). Intact cores and sieved samples taken from a full

factorial, 3-year grassland soil-warming and shading

experiment were incubated at three different tempera-

tures. The shading treatment altered plant growth rates

(Edwards et al., 2004), and hence, the amount of new C

entering the soil. It, therefore, provided a further op-

portunity to investigate the importance of substrate

availability in determining the respiration rates in the

intact cores vs. sieved samples. We tested the following

two main hypotheses: (1) previous experience of 3 years

of soil warming would reduce the respiration rates of

intact cores when incubated at a common temperature,

and (2) this response would be mediated through

changes in substrate availability within the soil and so

differences between warmed and ambient plot samples

would be lost when sieving made additional substrates

available (alternatively, if differences were maintained,

this would be consistent with the response being

mediated by thermal acclimation of the microbial com-

munity).

Materials and methods

Field experimental design

The soil incubated in this experiment was a sandy loam

(pH 6.5) which had been taken from the soil-warming

and shading experiment established in the experimen-

tal garden at the University of York. This experiment

1762 I . P. H A R T L E Y et al.

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consisted of twelve main 2 m2 plots which were seeded

with a mixture of northern grassland species in July

1998 (see Edwards et al., 2004), with Holcus lanatus L.

later becoming the dominant species. Each main plot

contained two subplots, one ambient temperature

subplot and a subplot in which the soil was warmed

to 3 1C above ambient (Fig. 1) using low-voltage heating

cables connected to a 1 m� 0.5 m steel grid with a

25mm� 25 mm mesh size (Ineson et al., 1998). The steel

grid resulted in the heat being spread evenly through-

out the plot and so minimized lateral thermal gradients.

Controllers, connected to arrays of thermistor probes

placed 2 cm below the heating meshes, maintained a

3 1C difference between warmed and ambient subplots.

Warming was switched on continuously from mid

January 2000 until the soils were sampled on 30th

January 2003.

Of the 12 pairs of plots, four were left unshaded and

the other eight plots were exposed to two levels of

shading (‘half shade’ and ‘full shade’; Fig. 1). The half

shade treatment blocked 70% of ambient photosynthetic

photon density and the full shade treatment blocked out

86%. These shading values were measured using PAR

sensors placed 75 cm above the ground, but shading

at leaf level may have been lower (Heinemeyer et al.,

2004).

Soil sampling

On 30th January 2003 the heating cables were removed

and four soil cores (68 mm in diameter and 100 mm in

depth) were taken from each plot. On the following day,

to avoid between-plot differences in soil moisture con-

tent, the cores were adjusted to water holding capacity

by adding deionized water. Three of the cores were left

intact and placed into 2 L jars and then incubated at

three different temperatures.

Two days after the soils were adjusted to water

holding capacity, the final core was dry-sieved first

through a 2 mm mesh and then through a 500 mm mesh

with roots being removed at each step. Approximately

10 g of sieved soil was added to each of three 125 cm3

bottles and these were then incubated at the three

different temperatures.

Soil incubation

The cores and sieved samples were incubated at 4, 8

and 12 1C for 4 weeks. The 4 1C treatment reflected the

mean soil temperature that the control plots had ex-

perienced over the preharvest month while 12 1C was

the maximum soil temperature recorded in the warmed

subplots over the preharvest month. During incubation,

the intact cores and sieved samples were maintained at

water holding capacity by weighing the bottles and jars

every 2 days and then adding the correct amount of

deionized water. Deionized water reservoirs were

maintained in each incubator to allow the water addi-

tions to be made at the correct temperature.

Respiration measurement

After 3 days of incubation at each temperature, the

respiration rates of the intact cores were measured.

The jars containing the intact cores were closed and

over-pressured with 50 cm3 of CO2-free air. The head-

space was initially sampled (t0) and again after 3 h of

incubation by removing 10 cm3 of headspace gas into

stoppered syringes. The jars were then opened until the

next set of measurements.

Two days after sieving, measurements of the respira-

tion rates in the sieved samples were made. As for the

measurements made on the intact cores, the serum

bottles were sealed and then over-pressured, but this

time using 25 cm3 of CO2-free air. Again, the headspace

was sampled at t0 and then after 3 h of incubation. The

serum bottles were then reopened until the next set of

measurements. Soil respiration rates were measured on

a further three occasions with a sampling interval of 1

week for both the intact cores and the sieved samples.

To measure the change in the headspace CO2 con-

centration over the period of incubation, gas samples

were injected into a gas sampling valve (Rheodyne Inc.,

Bensheim, Germany) connected to a 1 cm3 gas sampling

loop. The gas was then injected into a CO2-free air

Unshaded

UnshadedUnshaded

Unshaded

Full shade Full shade

Full shade

Full shade

Half shade

Half shade

Half shade

Half shade

Warmed

Ambient

1 : 1 1 : 2 1 : 3 1 : 4 1 : 5 1 : 6 1 : 7 1 : 8

2 : 1 2 : 2 2 : 3 2 : 4 2 : 5 2 : 6 2 : 7 2 : 8

3 : 1 3 : 2 3 : 3 3 : 4 3 : 5 3 : 6 3 : 7 3 : 8

1 m

Fig. 1 Diagram of the experimental set-up of the soil-warming

meshes and shading treatments in the experimental garden at

the University of York.

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stream flowing through an infra-red gas analyser (model:

ADC-225 MK3, ADC Bioscientific Ltd, Herts, UK) and

peak heights were recorded on a chart recorder (model:

SE 120 BBC Goerz Metrawatt, Austria). Standards of 377

and 512 ppm were run between every set of 10 samples.

Root biomass and soil dry-weight analysis

At the end of the incubation, the intact cores were

broken up and sieved. Stones, fine and coarse roots

were removed and weighed, with fine roots being

defined as roots with a diameter o1 mm. The roots

and stones were oven-dried at 80 1C and weighed to

produce a measurement of root and stone content for

each of the incubated cores. Subsamples of soil from

each intact core and sieved sample were removed and

oven-dried at 105 1C to obtain a measure of soil moist-

ure content which was in turn used to calculate the dry

weight of soil in each sample.

C content and d13C ratio analysis

Root-free soil samples and H. lanatus leaves removed

from each plot were oven-dried at 60 and 105 1C

respectively, and then ground in a custom-built end-

over-end shaker, before being weighed into 6 mm� 4 mm

tin cups (Elemental Microanalysis Ltd, Okehampton,

UK). These samples were then run, with sugar stan-

dards, through a elemental analyser combustion unit

(Flash EA1112, ThermoFinnigan, CA, USA) coupled to

a isotope ratio mass spectrometer (Provac Services,

Cheshire, UK) to allow their d13C ratios and C contents

to be measured.

Data analysis

Root biomasses were expressed in terms of milligram

dry weight of root per gram dry weight of soil

(mg g dw�1). Headspace volumes, in the jars and Whea-

ton bottles, were measured allowing CO2 fluxes to be

expressed in microgram of C per gram dry weight of

soil per hour (mg C g dw�1 h�1). The sieved samples

taken from the first unshaded plot (subplots 1 : 1 and

1 : 2) respired at a rate well over twice as high as the

samples taken from all other plots. This was linked to

an ant colony which had developed in this specific plot,

and therefore, these samples were considered as out-

liers and were not included in the final analysis.

Statistical analyses were carried out using SPSS (SPSS

Science, Birmingham, UK). Repeated-measures ANOVAs,

using warmed and ambient subplots as the within-

subject variables and shading as the between-subject

factor, were used to investigate whether there were

significant effects of the warming and shading treat-

ments on (1) the respiration rates of the intact cores

and restructured samples, (2) root biomass, and (3) the

d13C ratio and C content of the H. lanatus leaves and

soil samples taken from the plots. Repeated-measures

ANOVAs were also used to investigate whether the

differences between warmed and ambient subplot soils

changed through time and, when significant differences

were observed, paired t-tests were used to investigate

which weeks differed significantly. Linear regressions

were used to investigate the relationship between root

biomass and the respiration rates of the intact cores.

Results

Intact cores

Respiration rate. Overall, significant differences were

observed between the respiration rates of the intact

cores taken from the warmed and ambient subplots,

and also between intact cores taken from the three

different shading treatments (Fig. 2). At 4 1C, signi-

ficant differences were only observed between

shading treatments, with samples taken from the full

shade treatment respiring at a rate significantly lower

than both unshaded (P 5 0.020) and half shade

(P 5 0.019) samples. For the samples incubated at 8 1C,

there was also no significant warming effect despite

cores taken from ambient plots respiring at on average

a 29% higher rate. However, the respiration rate of

samples taken from the unshaded plots was

significantly higher than that of samples taken from

the half shade (P 5 0.003) and full shade treatments

(P 5 0.001). However, at 12 1C a significant difference

Soi

l res

pira

tion

rate

(µg

C g

dw

−1 h

−1)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

8°C12°C

Half shade Full shadeUnshadedAmbient Warmed Ambient Warmed Ambient Warmed

4°C

Fig. 2 The respiration rate measured in the intact cores taken

from the different warming and shading treatments and incu-

bated at the three different temperatures. The rates represent

the mean of the four measurement weeks. Error bars represent

11 SE (n 5 4).

1764 I . P. H A R T L E Y et al.

r 2007 The AuthorsJournal compilation r 2007 Blackwell Publishing Ltd, Global Change Biology, 13, 1761–1770

was observed between the respiration rates of samples

taken from the ambient and warmed subplots

(P 5 0.017) with the ambient subplot rate being on

average 26% higher (Fig. 2). In addition, the difference

between unshaded and half shade (P 5 0.001) and

unshaded and full shade (Po0.001) plots was

maintained.

The differences between warmed and ambient

subplot cores were not constant over time, especially

at 12 1C. Soil respiration rates did not differ significantly

between cores taken from the warmed and ambient

subplots in the first week of sampling (Fig. 3a).

However, for the unshaded and half shade treatments

there was a higher respiration rate in the ambient

subplot samples. One week later, as respiration rates

in the cores taken from the warmed subplots had

declined relative to the respiration rates of the

ambient subplot cores, a significant difference was

observed between subplots (Fig. 3, P 5 0.027) and this

difference was maintained in week 3 (P 5 0.008) and

week 4 (P 5 0.004). The change over time in the relative

rates of respiration in the warmed and ambient subplots

was statistically significant (Fig. 3b, P 5 0.015).

Interestingly, significant warming effects were only

observed in cores taken from the unshaded and half

shade treatments (weeks 2–4: Po0.012) but not in the

full shade cores (week 1–4: P40.568). However, no

significant interaction between warming and shading

treatments was identified.

Differences between shading treatments depended

on incubation temperature and sampling time. At 12 1C,

cores from the unshaded plots respired at a significantly

higher rate than cores from the two shading treatments

throughout (Po0.001), and from week 2 onwards,

significant differences were also observed at 8 1C

(Po0.001). At 4 1C, during week 3 the respiration rates

of unshaded plot cores were significantly higher than

the full shade cores (P 5 0.008).

Root biomass. Soil warming reduced root biomass

significantly in cores taken from warmed subplots

with fine (P 5 0.022), coarse (P 5 0.019) and total root

biomass (P 5 0.012, Fig. 4) all being affected. Shading

also reduced root biomass significantly (Fig. 4), with

fine root biomass in the unshaded plots being

significantly higher than fine root biomass in the half

shade (Po0.001) and full shade plots (P 5 0.002).

However, there was no significant difference between

full and half shade plot samples and no significant

interaction between shading and warming treatments.

In addition, coarse root biomass was not affected by

shading (P 5 0.115).

Regression analyses were carried out between the

respiration rates of the intact cores and the root

biomasses removed through destructively sampling

cores at the end of the experiment. A significant

positive correlation was only observed for the ambient

cores incubated at 12 1C (Fig. 5, Po0.001).

Sieved samples

During the first measurement, the gas samples taken

from two of the soils from the shaded plots were

contaminated (due to leaky septa), which prevented a

comparison being made between shading treatments. In

addition, respiration rates were high and declined over

the first week by �40%, thereafter remaining fairly

stable (Fig. 6a). To avoid results being dominated by

the pulse of CO2 release observed during the first

measurement date, only weeks 2–4 were included in

the analysis of the mean respiration rates (Fig. 7). No

significant difference in the respiration rates of the

Res

pira

tion

rate

(µg

C g

−1so

il dw

h−1

)

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Ambient Warmed

*

** **

(a)

Week 1 Week 2 Week 3 Week 4

Res

pira

tion

rate

of t

he a

mbi

ent s

ubpl

ot c

ores

(pro

port

ion

of w

arm

ed s

ubpl

ot r

ate)

1.0

1.2

1.4

1.6

1.8(b)

b

b

a a

Fig. 3 The changes in the respiration rates in the warmed and

ambient subplot intact cores incubated at 12 1C over the 4 week

incubation period. Graph (a) shows the mean respiration rates

�1 SE (n 5 12). When significant differences between warmed

and ambient subplot cores were identified they are indicated

with *(Po0.05) and **(Po0.01). Graph (b) shows the respiration

rates in the ambient subplot cores expressed as a proportion of

the rate in the warmed cores. Scale bars labelled with different

letters differ significantly (i.e. between week differences are

identified). Error bars represent 11 SE (n 5 12).

WA R M I N G E F F E C T S O N S O I L R E S P I R AT I O N 1765

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samples taken from the warmed and ambient subplots

was observed at any of the three incubation tempera-

tures, even when excluding the full shade plots. At

12 1C the respiration measured in samples taken from

unshaded plots was significantly higher than in the half

shade (P 5 0.022) and full shade samples (P 5 0.009).

However, as with the intact cores, between weeks the

respiration rates of the warmed subplot samples de-

clined as a proportion of the respiration rates in the

ambient subplot samples but only when incubated at

12 1C (Fig. 6b, P 5 0.008). By week 3, there was a

significant difference between the rate of respiration in

the warmed and ambient subplot samples (Fig. 6a,

P 5 0.045) and this difference was maintained in week

4 (P 5 0.048).

Throughout weeks 2–4, the samples taken from the

unshaded plots respired at a significantly higher rate

than soil from the half and full shade plots but only

when incubated at 12 1C (Fig. 7).

C content and d13C ratios

The H. lanatus leaf samples taken from the warmed

subplots had a significantly more negative d13C ratio

than the samples from the ambient subplots (Table 1)

but no further significant warming or shading effects on

the C content or d13C ratio of the leaves or soil were

observed (Table 1).

Discussion

The effect of soil warming

Respiration rates. We hypothesized that prior experience

of three years of soil warming would reduce the

respiration rate of intact cores when incubated at a

common temperature. This hypothesis was supported

by the fact that warmed subplot cores respired at a

significantly lower rate than ambient subplot cores

when incubated at 12 1C (Fig. 2). The reduction in the

respiration rate of the warmed subplots samples was

on average 26%. This result is in broad agreement with

the findings of other long-term field experiments in

which soil warming has only resulted in a transient

stimulation of soil respiration (Luo et al., 2001; Rustad

et al., 2001; Eliasson et al., 2005). However, it should be

mentioned that during the incubation of the intact

cores, root death probably occurred which could have

affected substrate availability and microbial activity.

Therefore, the rates observed in the intact cores may

not have been completely indicative of the rates of

respiration occurring under undisturbed field conditions.

We hypothesized that, by breaking up soil

aggregates and making previously protected SOM

available, sieving would reduce the differences in

respiration rates between warmed and ambient

subplot soils if substrate availability rather than

thermal acclimation was responsible for the observed

reduction in the respiration rates of the warmed subplot

intact cores. In support of this hypothesis, overall the

respiration rates in the sieved samples taken from

the warmed subplots were only 6% lower than those

in ambient subplot samples when incubated at 12 1C

(Fig. 7), and significant differences were not observed

until after 3 weeks of incubation (Fig. 6a). Further, the

near identical rates of respiration early in the incubation

(Fig. 6b) suggest that microbial respiration had not

acclimated to the soil warming treatment. Over time,

the decline in the rate of respiration in both the intact

Unshaded Half shade Full shade

Tot

al r

oot b

iom

ass

(mg

g dw

−1)

0.0

0.2

0.4

0.6

0.8

1.6

1.4

1.2

1.0

AmbientWarmed

Fig. 4 The mean total root biomass extracted from the intact

cores taken from the different warming and shading treatments.

Root biomasses are expressed as mg dry weight of roots per

gram dry weight of soil (mg g dw�1). Error bars represent

11 SE (n 5 4).

Total root biomass (mg g dw−1)

0.0 0.5 1.0 1.5 2.0 2.5

Res

pira

tion

rate

(µg

C g

dw

−1 h

−1)

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

y = 0.099x + 0.129

R2 = 0.83

Fig. 5 The relationship between the total dry-weight of roots

taken from the ambient cores, incubated at 12 1C, and the mean

respiration rate of each core over the 4 week period. A linear

regression is plotted.

1766 I . P. H A R T L E Y et al.

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cores and sieved samples taken from the warmed

subplots, relative to those taken from the ambient

subplots, provides further evidence of substrate

availability becoming increasingly limiting in these

soils (Figs 3 and 6b). Had thermal acclimation been

important then respiration rates may have been

expected to have become more similar over time as

microbes adapted to the constant incubation conditions,

although the duration of this experiment may have

been to short for such changes to have occurred.

In summary, the results of this empirical

investigation agree with the conclusions of recent

modelling studies suggesting that the reduction in the

positive response of soil respiration to warming can be

explained by changes in substrate availability rather

thermal acclimation/adaptation of microbial activity

(Kirschbaum, 2004; Eliasson et al., 2005; Knorr et al.,

2005). However, one main caveat should be mentioned;

sieving may have affected the microbial community

structure. It is known that sieving can affect

particularly sensitive microbes, such as methano-

trophs (Roslev et al., 1997). Yet it seems unlikely that

sieving would reverse the thermal acclimation of the

respiration arising from the entire microbial

community. Given that the hypothesized thermal

acclimation is implicated in the long-term response of

soil respiration to temperature, and therefore can be

expected to take a number of years, it seems especially

unlikely that physical disturbance would reverse this

entire process.

Root responses and labile C. The cores taken from the

warmed subplots had a significantly and markedly

lower root biomass than the ambient subplot cores

(Fig. 4). Previous work in this ecosystem has

demonstrated that root growth was controlled mainly

by radiation flux, whereas root death in autumn and

winter was enhanced by soil warming (Edwards et al.,

2004). Sampling for this current experiment took place

in January, and therefore the root biomasses measured

in the warmed subplots may not be indicative of the

amount of C available in the soil which may explain the

lack of correlation between root biomass and respiration

rate in the warmed subplot cores. It appears that an

increased rate of decomposition in the warmed

subplots, rather than a change in C input, is the most

likely explanation for the reduction in substrate

availability within the warmed subplot soil samples.

No significant effect of warming on the respiration

rates of the intact cores or sieved samples taken from

the full shade plots was observed (Fig. 2). The low rate

of C input into the soil in this treatment may have

reduced the effect of soil warming on substrate

availability suggesting that warming had the greatest

effect on the turnover of recently fixed C and agrees

with the studies of Eliasson et al. (2005) and Knorr et al.

(2005) which linked the declining warming response of

soil respiration to depletion of labile SOM. As root

biomass in the full shade plots was low, labile C

inputs may have been small and, hence, no warming

effect was observed. As the recalcitrant SOM pool tends

to be much larger, it has been suggested that

decomposition activities associated with this pool are

less likely to become substrate limited (Eliasson et al.,

2005).

The effect of ecosystem shading

The shading treatments also appeared to have caused a

reduction in substrate availability in the soil. The rates

of respiration in the intact cores taken from the shaded

plots were lower then those from the unshaded plots

Res

pira

tion

rate

(µg

C g

−1 s

oil d

w h

−1)

0.10

0.15

0.20

0.25

0.30AmbientWarmed

**

Week 1 Week 2 Week 3 Week 4

Res

pira

tion

rate

of a

mbi

ent s

ubpl

ots

sam

ples

(pro

port

ion

of w

arm

ed s

ubpl

ot r

ate)

0.90

0.95

1.00

1.05

1.10

1.15

1.20 (b)

(a)

b

ab

a

a

Fig. 6 The changes in the rates of respiration in the warmed

and ambient subplot sieved samples incubated at 12 1C over the

4 week incubation period. Graph (a) shows the mean respiration

rates �1 SE (week 1: n 5 9, weeks 2–4: n 5 11). When significant

differences between warmed and ambient subplot cores were

identified they are indicated with *(Po0.05). Graph (b) shows

the rate of respiration in the ambient subplot samples expressed

as a proportion of the rate in the warmed cores. Scale bars

labelled with different letters differ significantly (i.e. between

week differences are identified). Error bars represent 11 SE

(week 1: n 5 9, weeks 2–4: n 5 11).

WA R M I N G E F F E C T S O N S O I L R E S P I R AT I O N 1767

r 2007 The AuthorsJournal compilation r 2007 Blackwell Publishing Ltd, Global Change Biology, 13, 1761–1770

when incubated both at 8 and 12 1C (Fig. 2). It appears

that shading, through effects on plant productivity and

root biomass (Edwards et al., 2004), reduced substrate

availability in the soil, consequently reducing hetero-

trophic soil respiration especially at higher incubation

temperatures. This result is in agreement with the study

of Bond-Lamberty et al. (2004) that identified a strong

link between autotrophic and heterotrophic soil respira-

tion across forest sites and linked this to variations in

primary productivity. In addition, in situ, the impor-

tance of photosynthetic substrate availability in regulat-

ing belowground respiration is being increasingly

recognized (Craine et al., 1999; Hogberg et al., 2001;

Janssens et al., 2001; Hartley et al., 2006).

In the sieved samples incubated at 12 1C, respiration

rates in the shaded plot samples were significantly

lower than those in the unshaded plot samples

(Fig. 7). However, at 12 1C the magnitude of the differ-

ence between shaded and unshaded intact cores was

�45% and sieving reduced this to �15%. Again, it

appears that sieving reduced differences in substrate

availability between treatments.

C content and d13C ratios

The d13C ratio of H. lanatus leaves taken from the

warmed subplots was significantly more negative than

the d13C ratio of leaves taken from the ambient subplots

(Table 1). If soil-warming had resulted in an increase in

water-stress in the warmed subplot plants, then it

would have been expected that stomatal apertures

would have been decreased causing a reduction in

C-isotope discrimination and a consequent increase in

the d13C ratio (Smedley et al., 1991). Thus, no evidence

for soil warming resulting in water-stressed plants was

produced. Therefore, in agreement with Edwards et al.

(2004), there was little isotopic evidence of soil warming

affecting plant growth in this system.

Despite respiration measurements identifying a re-

duction in substrate availability in the soils taken from

the shaded plots and warmed subplots, no effect was

observed on the bulk C content of the soils (Table 1). This

result demonstrates how difficult it is to measure a

‘small change in a big number’ such as a treatment

effect on the amount of C stored in soils, as such

changes are likely to be within the error term of the

measurement techniques used (Valentini et al., 2000).

Further work is required that investigates the effect of

soil warming on the different SOM pools. Again, in

support of previous modelling exercises (Eliasson et al.,

2005; Knorr et al., 2005), a possible explanation for the

patterns observed in the study presented here, is that

changes in the size of the relatively small labile C pool

are responsible for the changes in respiration rates

and that these changes cannot be detected in the

bulk C content of the soil. Isotopic (Heath et al., 2005;

Trueman & Gonzalez-Meler, 2005; Trumbore, 2006)

and fractionation approaches (Cardon et al., 2001; Chris-

tensen, 2001) may be necessary to identify direct effects

of treatments on SOM turnover and storage.

Conclusions

Intact soil cores taken from plots exposed to three-years

of warming and shading, respired at lower rates than

cores taken from ambient subplots and unshaded plots.

Table 1 The effect of shading and warming on the d13C ratio and carbon (C) content (%C) of the soil and Holcus lanatus leaves

Unshaded Half shade Full shade Ambient Warmed

Leaf d13C �33.5 � 0.1 �33.8 � 0.2 �33.7 � 0.2 �33.4 � 0.1a �33.9 � 0.2b

Leaf %C 41.0 � 0.6 41.9 � 0.2 41.6 � 0.4 41.4 � 0.4 41.5 � 0.3

Soil d13C �25.5 � 0.1 �25.6 � 0.1 �25.5 � 0.1 �25.6 � 0.1 �25.5 � 0.1

Soil %C 4.6 � 0.1 4.4 � 0.1 4.5 � 0.1 4.4 � 0.0 4.5 � 0.1

The mean C content and d13C ratios are shown as well as �1 SE (n 5 4). Within a d13C ratio or C content measurement, warming

treatments labelled with different letters differ significantly.

Soi

l res

pira

tion

rate

(µg

C g

dw

−1 h

−1)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.204°C 8°C 12°C

Half shade Full shadeUnshadedAmbient Warmed Ambient Warmed Ambient Warmed

Fig. 7 The respiration rate measured in the sieved samples

from the different warming and shading treatments incubated

at the three different temperatures. The rates represent the mean

of the latter three measurement weeks, i.e. rates after the initial

pulse of respiration had declined. Error bars represent 11 SE

(n 5 3 unshaded, n 5 4 half and full shade).

1768 I . P. H A R T L E Y et al.

r 2007 The AuthorsJournal compilation r 2007 Blackwell Publishing Ltd, Global Change Biology, 13, 1761–1770

However, sieving substantially reduced the differences

between warming and shading treatments. These

results suggest that different substrate availabilities

caused the differences observed in the intact core

respiration rates. Therefore, this response to warming,

mediated through changes in substrate availability and

not through active adaptation of the microbial commu-

nity, should not be referred to as ‘acclimation’. The

results presented here were also consistent with re-

sponses being largely due to changes in the substrate

availability in the recently fixed, labile SOM pool and

not due to changes in CO2 production associated with

the decomposition of the recalcitrant soil C pool. While

studies have demonstrated that microbial community

structure may be altered by soil warming, no study as

yet appears to have formally demonstrated thermal

acclimation of heterotrophic respiration. It must, there-

fore, be assumed that temperature does indeed increase

heterotrophic activity and that the main limiting factor

for a sustained response is the amount of substrate

available for decomposition. The magnitude of C losses

induced by warming a particular soil may be depen-

dent on the organic content and the chemical and

physical properties of the soil which regulate substrate

availability. Understanding how different soil types will

respond to warming remains of key importance in the

modelling of global C-cycle feedbacks.

Acknowledgements

We are extremely grateful for the opportunity to sample the soilsfrom the long-term experiment which was set up and run by E. J.Edwards and A. H. Fitter. During the main experimental periodthe plots were maintained by C. P. Lancaster and P. Scott. Thetemperature control system was designed and built by D. Ben-ham. The members of the Ineson, Fitter, Atkin and Hodgelaboratory groups helped during harvesting of the plots. I. P.H.’s PhD Studentship was funded by NERC and Forest Researchthrough the UK Centre for Terrestrial Carbon Dynamics (CTCD).

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