Different initial responses of the canop

Oikos 000: 001–007, 2014
doi: 10.1111/oik.01940
 2014 he Authors. Oikos  2014 Nordic Society Oikos
Subject Editor: Chistopher Lortie. Editor-in-Chief: Dustin Marshall. Accepted 29 October 2014

Different initial responses of the canopy herbivory rate in mature
oak trees to experimental soil and branch warming in
a soil-freezing area
Masahiro Nakamura, Tatsuro Nakaji, Onno Muller and Tsutom Hiura
M. Nakamura (masahiro@fsc.hokudai.ac.jp), T. Nakaji, O. Muller and T. Hiura, Tomakomai Research Station, Field Science Center
for Northern Biosphere, Hokkaido Univ., JP-053-0035 Tomakomai, Japan. MN also at: Nakagawa Experimental Forest, Field Science Center
for Northern Biosphere, Hokkaido Univ., Otoineppu, JP-098-2501 Nakagawa, Japan. Present address for OM: Inst. for Bio- and Geosciences,
IBG-2: Plant Sciences, Forschungszentrum Jülich GmbH, DE-5425 Jülich, Germany.

Plants and insects comprise more than 50% of known species on earth, and their interactions are of major importance
in most natural ecosystems. To understand the mechanisms by which global warming affects plant–insect interactions in
the canopy of mature cool-temperate forests with a freeze–thaw cycle, we examined changes in the herbivory rate and leaf
traits in oak Quercus crispula. From 2007 to 2009, we experimentally increased the temperature of the surrounding soil and
canopy branches of mature oak trees by approximately 5°C using electric heating cables. Soil warming decreased the rate
of herbivory in the canopy, whereas branch warming had no effect. he magnitude of the effect of soil warming on canopy
herbivory varied. For the first year, the decrease was 32%, but this doubled (63%) in the third year. Branch warming did

not affect canopy leaf traits; however, soil warming decreased the leaf nutritional quality by decreasing N and increasing the
carbon:nitrogen (CN) ratio for three years. Additionally, soil warming increased total phenolics in the third year. Stepwise
multiple regression models showed that among the leaf traits that were changed by soil warming, N explained the variation
in herbivory for the first and second years, whereas total phenolics explained it for the third year. Our experimental results
demonstrate that soil warming drives the rate of herbivory in the canopy of mature oak trees, and the magnitude of the
soil warming effect was gradually enhanced during the initial three years. his suggests the importance of belowground
temperature elevation in predicting the effect of global warming on plant–insect interactions in a forest canopy.

he mean global temperature is predicted to increase by 1.8–
4.0°C by 2100 under various climate scenarios (IPCC 2007).
Temperature is a key factor that impacts numerous ecological
processes such as soil respiration, soil nitrogen (N) mineralization, and aboveground plant growth (Rustad et al. 2001,
Chung et al. 2013). Experimental warming is an effective
approach to determine the effect of increasing temperature
on ecological processes, with few confounding factors (e.g.
other variables that covary spatially and temporally with temperature). herefore, a number of field experiments have been
initiated worldwide to study the effects of simulated global
warming (Shaver et al. 2000, Rustad et al. 2001). A wide
range of techniques (e.g. greenhouses, open-top chambers
and electric infrared heaters) have been developed to experimentally warm a variety of small plants, including those of

the tundra, grasslands, and sapling trees (reviewed by Rustad
et al. 2001). Within forests, most insect species diversity and
plant–insect interactions are concentrated in the canopy of
mature trees, rather than in the understory, because of higher
plant productivity (Basset 2003). However, few studies have
examined the responses of mature trees to experimental
warming in natural forests (Nakamura et al. 2010b).

Plant–insect interactions are of major importance
in most natural ecosystems because the two groups are
extremely diverse and comprise almost 50% of all known
species on Earth (Price 1997). Previous studies have
predicted that climatic changes could impact the species
composition and ecosystem function of forests through
changes in populations and the distribution of herbivorous
insects (Ayres and Lombardero 2000, Logan et al. 2003).
Temperature is a very important factor directly affecting
insect population dynamics through the modulation of
survival, development rates, and dispersal (reviewed by
Bale 2002, Robinet and Roques 2010). However, Shaver

et al. (2000) suggested that indirect temperature effects
also have the potential to significantly influence herbivore
performance. However, data addressing the indirect effects
of global warming on plant–insect interactions in mature
forests remain scarce.
Previous studies have identified mechanisms that
explain indirect (plant-mediated) effects of global warming on herbivorous insects (Dury et al. 1998, Veteli et al.
2002, Zvereva and Kozlov 2006, Bidart-Bouzat and ImehNathaniel 2008, DeLucia et al. 2012). hese studies focused
EV-1

on plant phenotypic plasticity under global warming conditions (Nicotra et al. 2010). Temperature elevation may
affect the primary and secondary metabolism of plants
(Veteli et al. 2002), in which the role of plant secondary
metabolites is well established in terms of defense against
insect herbivores (Bidart-Bouzat and Imeh-Nathaniel
2008, DeLucia et al. 2012). he co-evolution theory
suggests that plant secondary metabolites are likely to be
the most important mediators of plant–insect interactions
(Ehrlich and Raven 1964, Cornell and Hawkins 2003).
A recent meta-analysis of laboratory experimental results

using only sapling and seedling trees predicted decreased
production of plant secondary metabolites under temperature elevation due to the dilution effects of plant growth;
this might, in turn, increase insect performance (Zvereva
and Kozlov 2006).
Latitudinal gradient studies also provide useful predictions of global warming effects (Andrew and Hughes
2005, Kozlov 2008, Adams and Zhang 2009, Hiura and
Nakamura 2013). Forests in warmer climates reportedly
suffer more herbivory than do forests in cooler climates
(Coley and Aide 1991, Coley and Barone 1996). For
example, Kozlov (2008) showed that the rate of herbivory
on white birch Betula pubescens, a pioneer tree species,
decreased at high latitudes in northern Europe. In contrast, a large-scale study of four late-successional tree
species (Quercus alba, Acer rubrum, Fagus grandifolia and
Liquidambar styraciflua) reported a significant latitudinal gradient in the rate of herbivory, with less damage
in lower-latitude areas of eastern North America (Adams
and Zhang 2009). From this result, it is expected that
global warming would adversely affect herbivory on latesuccessional tree species, which dominate mature forests.
However, the mechanism by which global warming affects
plant–insect interactions on late-successional tree species
is poorly understood.

Here, we report the initial three-year (2007–2009)
results of an experimental warming of mature Quercus
crispula (18–20 m in height), a late-successional tree species. To better understand the mechanism by which global
warming affects plant–insect interactions in the canopy
of mature oak trees, field experiments must warm aboveground and belowground regions separately (Weih and
Karlsson 2001). hus, we conducted experimental soil and
branch warming using electric heating cables to examine
how global warming affects plant–insect interactions in the
canopy of a cool-temperate forest. In general, soil warming causes no change in leaf phenology (Bronson et al.
2009) and we showed that the time of leaf emergence was
unaffected by branch warming (Nakamura et al. 2010b).
In this experiment, instead of focusing on phenological
asynchrony between egg hatching and budbreak
(Harrington et al. 1999), we focused on leaf traits
(bottom–up factors) as the main variable explaining variation in herbivory (Strong et al. 1984). We addressed the
following questions: 1) do canopy leaf traits and herbivory
respond to soil or branch experimental warming? 2) Which
canopy leaf traits are responsible for the observed variation
in canopy herbivory? 3) How does the magnitude of the
effect of experimental warming on canopy herbivory vary

temporally?
EV-2

Material and methods
Site description
We performed a warming experiment in the Tomakomai
Experimental Forest (TOEF; 42°40′N, 141°36′E) of the
Field Science Center for Northern Biosphere, Hokkaido
University (detailed description in Hiura 2001). he site is
a mature cool-temperate forest with Quercus crispula trees as
the dominant species. Access to the tree canopy was provided
by a construction crane that covers approximately 0.5 ha of
the forest canopy (jib length, 41 m at a height of 25 m).
he soil parent material is clastic pumice and sand that was
deposited by eruptions of Mt. Tarumae in 1667 and 1739
(Shibata et al. 1998). he mean monthly temperatures range
from –3.2 to 19.1°C. he annual precipitation is 1450 mm,
and snow cover reaches a depth of 20–50 cm. Soil freezing
occurs from late December to early May, and a freeze-thaw
cycle is observed during winter (Ueda et al. 2013).

Experimental soil and branch warming
Artificial warming was applied to branches and the soil
surrounding the roots of mature Q. crispula (18–20 m
in height), a late-successional tree species. Five mature
Q. crispula trees whose canopy was accessible by a gondola
hanging from a construction crane were selected. In spring
2007, we established four 5 ⫻ 5 m plots with one or two
oak trees in the center of each plot. An electric heating cable
120 m in length (copper resistance wire) was inserted into
the soil at 20-cm intervals at a depth of 5–10 cm in each plot
(similar to Peterjohn et al. 1993, Melillo et al. 2002). Due
to soil warming, the snow cover in the experimental plots
was less than that in the control plots during winter. We also
selected five oak trees around the canopy construction crane
as control plots, into which shovels were inserted to the same
depth as in the warmed plots to ensure a similar level of disturbance. To determine the effect of soil warming on canopy
herbivory and leaf traits, we randomly selected ten branches
in the canopy of each tree.
For branch warming, we selected three trees from the
five that were experiencing soil warming and warmed a part

of each tree’s canopy region. Electric heating cables were
attached with adhesive tape to upper canopy branches and
continuous (yearly) heating began in the spring of 2008.
On each tree, 1–3 thick branches were selected, and on each
branch, ⬎ 30 current year shoots were wired. Additional
details on the methods of branch warming were published by
Nakamura et al. (2010b), who reported that branch warming increased acorn production and extended the length of
the growing season of canopy leaves by inducing later leaf
fall. To determine the effect of branch warming on the rate
of herbivory in the canopy and on leaf traits, we randomly
selected ten warmed and ten control branches on each tree.
Jungqvist et al. (2014) predicted using modeling analyses that sites with little or no snow cover showed changes
in soil temperature were more synchronous with air temperature. In our experimental forest, snow cover is relatively
little (20–50 cm in snow depth). hus, both of soil and
branch temperatures were set to be approximately 5°C
higher than ambient temperatures using thermal sensors

Table 1. Two-way ANOVA for the effects of soil warming, year, and
their interaction on leaf traits.
Leaf traits

LMA (g

m-2)

N (mg g-1)

CN

Condensed tannin
(mg g⫺1)
Total phenolics
(mg g⫺1)

Source of variation

F-value

p-value

Year

Soil warming
Year ⫻ Soil warming
Year
Soil warming
Year ⫻ Soil warming
Year
Soil warming
Year ⫻ Soil warming
Year
Soil warming
Year ⫻ Soil warming
Year
Soil warming
Year ⫻ Soil warming

0.31
0.00
0.02
5.28
6.53

6.26
13.56
6.95
0.78
0.5
0.21
0.16
11.74
6.04
0.55

0.72
0.96
0.98
⬍ 0.01
0.01
0.77
⬍ 0.01
0.01
0.47
0.59
0.64
0.86
⬍ 0.01
0.02
0.58

(K cables) coupled with a controller to govern the power
supply. When the difference in temperature between the
control and treatments was ⬍ 5°C, the controller relay
switch opened and power was supplied to the electric cable.
When the difference was ⱖ 5°C, the relay closed. Due to
the temperature controller, branch warming increased the
branch temperatures by ∼5°C and leaf temperatures by
∼1.5°C.
Response variables
In the cool-temperate zone of Japan, the seasonal trend of
macro-lepidopteran larvae (dominant chewing herbivores)
on oaks displays two peaks, in June and August (Yoshida
1985). Inurois punctigera and Cosmia exgua were abundant
in June, whereas Pseudoips fagna, Moma alpium and Parabapta clarissa were abundant in August. Such two peaks in
herbivore activity are probably explained by seasonal changes
in nutrients and chemical defense (e.g. tannins) in oak leaves
(Feeny 1970). hus, we observed canopy herbivory twice a
year (June and August). We measured the rate of herbivory
on canopy leaves from ten current-year shoots from each

canopy branch according to the following six classes: 1) no
damage, 2) ⬍ 10% of leaf area lost, 3) 11–25% loss, 4) 26–
50% loss, 5) 51–75% loss, and 6) ⬎ 75% loss (Nakamura
et al. 2010a). he median of each class was used for the statistical analysis of chewing herbivory rate (i.e. 0, 5, 18, 38,
63 and 88% loss, respectively). To identify leaf traits that
could be responsible for the variation in herbivory rate, one
or two leaves that were sampled from each branch in August
were freeze-dried for 12 h and their dry mass was measured.
he mean leaf mass per area (LMA) was calculated for each
sample. he leaves were ground into a fine powder using an
analytical mill for chemical analysis. heir N and carbon (C)
contents (percentage of dry mass) were measured with a CN
analyzer. CN ratios, which are often used as the basis for
predicting the responses of trees and herbivores to environmental changes (Bryant et al. 1983), were then calculated.
he concentrations of condensed tannin and total phenolics
in the leaves were determined according to the methods of
Julkunen-Titto (1985). hese analyses were conducted from
2007 to 2009.
Statistical analyses
To determine the effects of soil warming and year on herbivory rate and leaf traits in the canopy, we used two-way
repeated measures ANOVAs. To compare the herbivory
rates and leaf traits between soil-warmed and control trees
in each year from 2006 (before soil warming) to 2009, we
used one-way ANOVAs. To compare the herbivory rates
and leaf traits between warmed and control canopy regions
on each soil-warmed tree, we also used one-way ANOVAs. Individual trees were replicated in the analysis. here
were some differences in leaf trait values for soil-warmed
trees in 2009; these were between soil-warmed trees (Table
2) and control branches on soil-warmed trees used for
branch-warming studies (Table 3) because the number of
replications (individual trees) differed. he data in Table
2 are based on five replications, and those in Table 3 on
three. However, the differences were not statistically significant. To determine the leaf traits that contributed most
to the observed variation in the rate of herbivory each year,

Table 2. Leaf traits of soil-warmed (n ⫽ 5) and control trees (n ⫽ 5) in August from 2007 to 2009. Values
are mean ⫹ SE.
Year
2007
(1st year)

Leaf traits

LMA (g m⫺2)
N (mg g⫺1)
CN
condensed tannin (mg g⫺1)
total phenolics (mg g⫺1)
2008
LMA (g m⫺2)
(2nd year) N (mg g⫺1)
CN
condensed tannin (mg g⫺1)
total phenolics (mg g⫺1)
2009
LMA (g m⫺2)
(3rd year) N (mg g⫺1)
CN
condensed tannin (mg g⫺1)
total phenolics (mg g⫺1)

Control

Soil warming

99.16 ⫾ 3.77
23.40 ⫾ 1.14
22.69 ⫾ 1.11
17.12 ⫾ 1.30
178.32 ⫾ 7.22
99.39 ⫾ 4.78
25.07 ⫾ 0.29
19.10 ⫾ 0.17
19.24 ⫾ 1.62
184.80 ⫾ 10.24
95.46 ⫹ 3.93
25.55 ⫹ 0.86
20.28 ⫹ 0.70
10.61 ⫹ 0.70
132.73 ⫹ 5.21

98.27 ⫾ 6.12
20.93 ⫾ 1.10
25.99 ⫾ 1.50
17.82 ⫾ 2.24
188.04 ⫾ 7.65
100.19 ⫾ 5.89
23.84 ⫾ 0.85
20.29 ⫾ 0.77
18.88 ⫾ 2.69
204.69 ⫾ 10.53
96.16 ⫹ 6.37
23.87 ⫹ 0.69
21.69 ⫹ 0.75
11.76 ⫹ 1.41
163.48 ⫹ 15.79

p-value
F-value (one-side-test)
0.02
2.46
3.13
0.74
0.85
0.01
1.9
2.28
0.01
1.83
0.09
2.46
1.89
0.53
3.42

0.45
0.08
0.06
0.4
0.19
0.46
0.10
0.08
0.46
0.11
0.46
0.08
0.10
0.24
0.05

EV-3

Table 3. Leaf traits of warmed (n ⫽ 3) and control canopy regions (n ⫽ 3) on each soil-warmed tree in
August 2009. Values are mean ⫹ SE.
Leaf traits
LMA (g m⫺2)
N (mg g⫺1)
CN
Condensed tannin (mg g⫺1)
Total phenolics (mg g⫺1)

Control

Branch
warming

F-value

p-value
(one side test)

99.77 ⫾ 11.06
23.63 ⫾ 1.01
21.84 ⫾ 1.67
11.55 ⫾ 2.26
156 ⫾ 27.01

91.28 ⫾ 8.19
24.96 ⫾ 1.37
19.92 ⫾ 1.27
10.28 ⫾ 1.36
147.39 ⫾ 20.62

⫺0.55
0.79
⫺1.11
⫺0.48
⫺0.27

0.31
0.24
0.16
0.33
0.40

we used stepwise multiple regression models. Several leaf
traits (e.g. tannin, phenolics, N and leaf toughness) are
reported to be proximate factors that explain the feeding activity of insect herbivores (Strong et al. 1984). We
removed leaf traits for which the variance inflation factor
(VIF) values exceeded 10 in the stepwise model because
VIF provides a measure of the extent to which the variance of an estimated regression coefficient is increased by
multicollinearity.

Results
Response of canopy herbivory rate
he response of the canopy herbivory rate in mature trees
differed between experimental soil- and branch-warming
conditions. Before soil warming, there was no difference
in herbivory rate between control and soil-warmed trees
(p ⫽ 0.23; Fig. 1). After the commencement of soil warming,
however, the herbivory rate significantly decreased in June
and August of the first year (2007) (June, p ⫽ 0.03; August,
p ⫽ 0.02). In the second (2008) and third years (2009), soilwarmed trees also experienced a lower herbivory rate, but
only in August (2008, p ⬍ 0.01; 2009, p ⬍ 0.01). he magnitude of the influence of soil warming on herbivory rate was
gradually enhanced during the initial thrtee years. In the first
year (2007), the decrease was 32% in soil-warmed trees compared with control trees (Fig. 2). In the third year (2009),
the decrease was twice as high (63%). In contrast, branch

Figure 1. Herbivory rate of soil-warmed trees (filled bars, n ⫽ 5)
and control trees (open bars, n ⫽ 5) from 2006 to 2009. Values are
mean ⫾ SE. Asterisk shows a significant difference (* p ⬍ 0.05, **
p ⬍ 0.10). We measured the canopy herbivory rate twice a year
(June and August). Soil warming occurred from 2007–2009.

EV-4

warming did not affect the herbivory rate in 2008 and 2009
(June 2008, p ⫽ 0.73; August 2008, p ⫽ 0.51; June 2009,
p ⫽ 0.54; August 2009, p ⫽ 0.52; Fig. 3).
Response of canopy leaf traits
Soil warming significantly affected the N, CN ratio, and
total phenolics of canopy leaves (p ⬍ 0.05, Table 1). However,
the canopy leaf traits induced by soil warming all changed
during the three study years. In the first (2007) and second
years (2008), soil warming decreased the concentration of
N and increased the CN ratio in canopy leaves, although
the significance levels were marginal (N, p ⬍ 0.10; CN,
p ⬍ 0.10: Table 2). In the third year (2009), soil warming
increased the total phenolics concentration significantly
(p ⫽ 0.05), although the effects on the N concentration and
CN ratio were only marginally significant (N, p ⫽ 0.08; CN,
p ⫽ 0.10). In contrast, branch warming did not affect any
canopy leaf traits (all leaf traits in 2009, p ⬎ 0.10; Table 3).
Relationships between canopy leaf traits and
herbivory rate
In the stepwise regression model using data for 2007
and 2009, we removed the CN ratio when the VIF value
exceeded 10 because the CN ratio was highly correlated
with N. he model indicated that the N and condensed
tannin in canopy leaves could explain the variation in canopy herbivory in the first year (2007) (Table 4), although
only the effect of N was significant (p ⬍ 0.01). However, in
the second year (2008), N, condensed tannin, and total

Figure 2. Yearly variation in decrease percentage (control trees – soil
warming trees) / control trees ⫻ 100% in canopy herbivory rate due
to soil warming in August from 2007 to 2009.

Figure 3. Herbivory rate of warmed canopy regions (filled bars,
n ⫽ 3) and control canopy regions (open bars, n ⫽ 3) on an individual soil-warmed tree in 2008 and 2009. Values are mean ⫾ SE.
Asterisk shows a significant difference (* p ⬍ 0.05, ** p ⬍ 0.10).
We measured the canopy herbivory rate twice a year (June and
August). Branch warming occurred from 2008–2009.

effects of plant growth. Previous studies have largely focused
on plant photosynthetic responses to aboveground (air)
temperature elevation and the accumulation of carbohydrates. Such differential responses of plant traits between the
meta-analysis and our warming experiment may be due to
size-dependent tree responses (Nabeshima et al. 2010). his
implies that the response of sapling trees to aboveground
temperature elevation is plastic, whereas mature trees may
lose this capability or their response may include a buffering capacity. Previous studies have reported that many of the
physiological and morphological characteristics of woody
plants vary depending on tree size (Bond 2000, Delagrange
et al. 2004, Nabeshima et al. 2010). Our results suggest that
an aboveground rise in temperature is not likely to have indirect (plant-mediated) effects on the rate of herbivory in the
canopy of mature oak trees. Alternatively, it is possible that
a 1.5°C increase in leaf temperature is not enough to affect
canopy leaf traits and herbivory. Experiments at higher temperatures are necessary to make further conclusions.
Responses to experimental soil warming

phenolics explained the variation in canopy herbivory, and all
were significant (p ⬍ 0.01). Among these selected leaf traits,
soil warming only decreased the concentration of N in the
first and second years. In the third year (2009), LMA, condensed tannin, and total phenolics explained the variation in
canopy herbivory. he effects of condensed tannin and total
phenolics were significant (condensed tannin, p ⫽ 0.02; total
phenolics, p ⬍ 0.01). Among these selected leaf traits, soil
warming increased the concentration of total phenolics in
the third year only.

Discussion
Responses to experimental branch warming
he absence of any response in the rate of herbivory and
leaf traits to branch warming in mature Quercus crispula
trees (Fig. 3) contradicts the results of a recent meta-analysis
of experimental warming studies using sapling and seedling trees (Zvereva and Kozlov 2006). In the meta-analysis
by Zvereva and Kozlov (2006), temperature elevation was
suggested to decrease the concentrations of plant secondary
metabolites (e.g. phenolics and tannins) due to the dilution

Table 4. Stepwise multiple regression models for leaf traits related
with herbivory rate in August from 2007 to 2009.
Year
2007
(1st year)

Variable

(intercept)
N
condensed tannin
2008
(intercept)
(2nd year) N
condensed tannin
total phenolics
2009
(intercept)
(3rd year) LMA
condensed tannin
total phenolics

b
⫺5.36
0.56
0.19
⫺4.75
0.82
0.51
⫺0.07
15.95
⫺0.04
0.56
⫺0.07

F-value

p-value

4.98 ⫺1.07
0.17
3.23
0.12
1.51
7.14 ⫺0.67
0.22
3.57
0.12
4.24
0.02 ⫺3.28
2.77
5.76
0.03 ⫺13.5
0.23
2.42
0.02 ⫺4.49

SE

0.29
⬍ 0.01
0.14
0.51
⬍ 0.01
⬍ 0.01
⬍ 0.01
⬍ 0.01
0.18
0.02
⬍ 0.01

In contrast to branch warming, soil warming decreased the
nutritional quality of canopy leaves (N, CN ratio, and total
phenolics). Belowground increases in temperature influence
various ecological processes that affect canopy leaf traits.
Previous laboratory and snow manipulation studies have
suggested that soil freezing drives soil N mineralization during winter because the increased mortality of microbes and
fine roots results in a release of labile organic N to the soil
(Schimel and Clein 1996, Groffman et al. 2001, Tierney
et al. 2001). Our experiment shows that soil warming in a
cool-temperate forest with a mild freeze–thaw cycle resulted
in no soil freezing during winter, and that this decreased
the inorganic and dissolved organic N pool to 17–25% of
control levels (Ueda et al. 2013). Because plants absorb N
not only during the growing season but also during winter, even when leaves are abscised (Andersen and Michelsen
2005, Ueda et al. 2011), the reduction in the available N
pool in soil should reduce plant N uptake. Accordingly, our
experiment shows that soil warming decreased the N content
while increasing the CN ratio and total phenolics in canopy
leaves (Table 2). hese results suggest that elevation of the
belowground temperature and the associated changes in soil
N mineralization have a more pronounced influence on the
canopy leaf traits of mature oak trees than does elevation of
the aboveground temperature.
We showed that soil warming decreased the rate of
canopy herbivory in mature oak trees, probably due to a
decrease in leaf nutritional quality (N, CN ratio and total
phenolics). Changes in leaf traits induced by global warming
are expected to affect plant–insect interactions (Dury et al.
1998, Veteli et al. 2002, Zvereva and Kozlov 2006, BidartBouzat and Imeh-Nathaniel 2008). he results from our
experimental study are consistent with those of a large-scale
latitudinal study by Adams and Zhang (2009), who reported
a decrease in herbivory rate at lower latitudes on four tree
species (Q. alba, A. rubrum, F. grandifolia and L. styraciflua).
Both studies observed mature trees of late-successional species in cool-temperate forests. hese results indicate that
negative indirect (plant-mediated) effects of global warming
EV-5

on the canopy herbivory rate may be characteristic of latesuccessional tree species in mature cool-temperate forests.
Annual variation in the effect of soil warming on
canopy herbivory
Our experiment revealed annual variation in indirect (plantmediated) effects of soil warming on the rate of herbivory
in the canopy. he magnitude of the soil warming effect on
canopy herbivory was gradually enhanced during the initial
three years of the study. he magnitude of the influence of
global warming may vary temporally (Shaver et al. 2000).
Melillo et al. (2002) reported that an average 28% increase
in CO2 flux due to soil warming was stabilized during the
first six years. he effect of soil warming on soil respiration
then substantially decreased. here are two possible causes of
the annual variation in the effect of soil warming on canopy
herbivory. First, a gradual change in nutritional leaf quality
is induced by soil warming. Our experiment showed that for
the first year, soil warming decreased N and increased the
CN ratio, and that the total phenolics increased due to soil
warming in the third year. Such leaf nutritional changes (total
phenolics) induced by soil warming are likely to strengthen
plant defenses against herbivorous insects. Second, there may
have been a gradual change in species composition and/or a
decrease in insect abundance due to changes in the nutritional quality of the leaves in the previous year.
Our warming experiment was conducted at the plot level.
Herbivorous insects that prefer local conditions in warmed
plots may congregate at artificially high densities, or, conversely, those that are repelled by the treatments may choose
to avoid them (Englund and Cooper 2003, Moise and
Henry 2010). Small-scale studies that attempt to extrapolate
the results of plot-level warming experiments to landscapelevel responses may thus result in inaccurate interpretations.
However, because we applied a soil warming treatment to
mature oak trees (18–20 m in height), the canopy area of
each soil-warmed individual tree was larger than in other
previous experiments. hus, the effects of herbivore congregation and avoidance may have been relatively small in
our experiment. Additionally, our results are consistent with
those of a large-scale latitudinal study (Adams and Zhang
2009). Such large-scale studies can reinforce plot-level results
(Shaver et al. 2000, Rustad et al. 2001, Moise and Henry
2010). herefore, our results can provide meaningful predictions of landscape-level responses to global warming.
In conclusion, our warming experiment clearly demonstrates that plant–insect interactions in the canopy responded
differently to soil and branch warming of mature oak trees
(a late-successional tree species). Soil warming in a mature
cool-temperate forest with a freeze-thaw cycle decreased
the nutritional quality of leaves and the rate of herbivory
in the canopy, whereas branch warming had no effect on
canopy leaf traits or the herbivory rate. he magnitude of the
indirect (plant-mediated) effects of belowground temperature elevation on canopy herbivory was gradually enhanced
during the initial three years of the study. hese results suggest that belowground temperature elevation due to global
warming in a soil freezing area is an important driving
force of plant–insect interactions in the canopy. For a better
understanding of the mechanism by which global warming
EV-6

affects plant–insect interactions in mature cool-temperate
forests, this warming experiment should be continued using
mature oak trees because indirect effects of temperature are
likely more pronounced in the long- than in the short-term
(Shaver et al. 2000).
Acknowledgements – his work was supported by grants from the
Japan Society for the Promotion of Science (no. 26450188 to MN,
no. 19657007 and 21248017 to TH), the Ministry of Environment (no. D-0909 to TH) and the Environment Research and
Technology Development Fund (S-9-3).

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