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 J ü lich GmbH, DE-5425 J ü lich, 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 aff ects 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 eff ect. h e magnitude of the eff ect of soil warming on canopy herbivory varied. For the fi rst year, the decrease was 32%, but this doubled (63%) in the third year. Branch warming did not aff ect 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 fi rst 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 eff ect was gradually enhanced during the initial three years. h is suggests the importance of belowground temperature elevation in predicting the eff ect of global warming on plant – insect interactions in a forest canopy.
h e 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) mineral-ization, and aboveground plant growth (Rustad et al. 2001, Chung et al. 2013). Experimental warming is an eff ective approach to determine the eff ect of increasing temperature on ecological processes, with few confounding factors (e.g. other variables that covary spatially and temporally with tem-perature). h erefore, a number of fi eld experiments have been initiated worldwide to study the eff ects 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 experi-mentally 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 aff ecting 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 eff ects also have the potential to signifi cantly infl uence herbivore performance. However, data addressing the indirect eff ects of global warming on plant – insect interactions in mature forests remain scarce.
Previous studies have identifi ed mechanisms that explain indirect (plant-mediated) eff ects of global warm-ing on herbivorous insects (Dury et al. 1998, Veteli et al. 2002, Zvereva and Kozlov 2006, Bidart-Bouzat and Imeh-Nathaniel 2008, DeLucia et al. 2012). h ese studies focused
on plant phenotypic plasticity under global warming con-ditions (Nicotra et al. 2010). Temperature elevation may aff ect 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). h e 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 tempera-ture elevation due to the dilution eff ects of plant growth; this might, in turn, increase insect performance (Zvereva and Kozlov 2006).
Latitudinal gradient studies also provide useful pre-dictions of global warming eff ects (Andrew and Hughes 2005, Kozlov 2008, Adams and Zhang 2009, Hiura and Nakamura 2013). Forests in warmer climates reportedly suff er 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 con-trast, a large-scale study of four late-successional tree species ( Quercus alba , Acer rubrum , Fagus grandifolia and
Liquidambar styracifl ua ) reported a signifi cant latitudi-nal 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 aff ect herbivory on late-successional tree species, which dominate mature forests. However, the mechanism by which global warming aff ects 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 spe-cies. To better understand the mechanism by which global warming aff ects plant – insect interactions in the canopy of mature oak trees, fi eld experiments must warm above-ground and belowabove-ground regions separately (Weih and Karlsson 2001). h us, we conducted experimental soil and branch warming using electric heating cables to examine how global warming aff ects plant – insect interactions in the canopy of a cool-temperate forest. In general, soil warm-ing causes no change in leaf phenology (Bronson et al. 2009) and we showed that the time of leaf emergence was unaff ected 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 varia-tion 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 eff ect of experimental warming on canopy herbivory vary temporally?
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). h e 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). h e soil parent material is clastic pumice and sand that was deposited by eruptions of Mt. Tarumae in 1667 and 1739 (Shibata et al. 1998). h e mean monthly temperatures range from – 3.2 to 19.1 ° C. h e 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
Artifi cial 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 fi ve 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 dis-turbance. To determine the eff ect 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 fi ve 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 warm-ing increased acorn production and extended the length of the growing season of canopy leaves by inducing later leaf fall. To determine the eff ect 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 analy-ses that sites with little or no snow cover showed changes in soil temperature were more synchronous with air tem-perature. In our experimental forest, snow cover is relatively little (20 – 50 cm in snow depth). h us, both of soil and branch temperatures were set to be approximately 5 ° C
(K cables) coupled with a controller to govern the power supply. When the diff erence 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 diff erence 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 Para-bapta 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). h us, 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). h e median of each class was used for the sta-tistical 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. h e mean leaf mass per area (LMA) was calculated for each sample. h e leaves were ground into a fi ne powder using an analytical mill for chemical analysis. h eir 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 environ-mental changes (Bryant et al. 1983), were then calculated. h e concentrations of condensed tannin and total phenolics in the leaves were determined according to the methods of Julkunen-Titto (1985). h ese analyses were conducted from 2007 to 2009.
Statistical analyses
To determine the eff ects of soil warming and year on her-bivory 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 ANO-VAs. Individual trees were replicated in the analysis. h ere were some diff erences 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) diff ered. h e data in Table 2 are based on fi ve replications, and those in Table 3 on three. However, the diff erences were not statistically sig-nifi cant. To determine the leaf traits that contributed most to the observed variation in the rate of herbivory each year,
Table 1. Two-way ANOVA for the effects of soil warming, year, and their interaction on leaf traits.
Leaf traits Source of variation F-value p - value
LMA (g m -2 ) Year 0.31 0.72
Soil warming 0.00 0.96
Year ⫻ Soil warming 0.02 0.98
N (mg g -1 ) Year 5.28 ⬍ 0.01
Soil warming 6.53 0.01
Year ⫻ Soil warming 6.26 0.77
CN Year 13.56 ⬍ 0.01
Soil warming 6.95 0.01
Year ⫻ Soil warming 0.78 0.47
Condensed tannin (mg g ⫺1 )
Year 0.5 0.59
Soil warming 0.21 0.64
Year ⫻ Soil warming 0.16 0.86
Total phenolics (mg g ⫺1 )
Year 11.74 ⬍ 0.01
Soil warming 6.04 0.02
Year ⫻ Soil warming 0.55 0.58
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 Leaf traits Control Soil warming F-value
p-value (one-side-test)
2007 LMA (g m ⫺2 ) 99.16 ⫾ 3.77 98.27 ⫾ 6.12 0.02 0.45
(1st year) N (mg g ⫺1 ) 23.40 ⫾ 1.14 20.93 ⫾ 1.10 2.46 0.08
CN 22.69 ⫾ 1.11 25.99 ⫾ 1.50 3.13 0.06
condensed tannin (mg g ⫺1 ) 17.12 ⫾ 1.30 17.82 ⫾ 2.24 0.74 0.4
total phenolics (mg g⫺1 ) 178.32 ⫾ 7.22 188.04 ⫾ 7.65 0.85 0.19
2008 LMA (g m ⫺2 ) 99.39 ⫾ 4.78 100.19 ⫾ 5.89 0.01 0.46
(2nd year) N (mg g ⫺1 ) 25.07 ⫾ 0.29 23.84 ⫾ 0.85 1.9 0.10
CN 19.10 ⫾ 0.17 20.29 ⫾ 0.77 2.28 0.08
condensed tannin (mg g ⫺1 ) 19.24 ⫾ 1.62 18.88 ⫾ 2.69 0.01 0.46
total phenolics (mg g⫺1 ) 184.80 ⫾ 10.24 204.69 ⫾ 10.53 1.83 0.11
2009 LMA (g m ⫺2 ) 95.46 ⫹ 3.93 96.16 ⫹ 6.37 0.09 0.46
(3rd year) N (mg g ⫺1 ) 25.55 ⫹ 0.86 23.87 ⫹ 0.69 2.46 0.08
CN 20.28 ⫹ 0.70 21.69 ⫹ 0.75 1.89 0.10
condensed tannin (mg g ⫺1 ) 10.61 ⫹ 0.70 11.76 ⫹ 1.41 0.53 0.24
warming did not aff ect 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 signifi cantly aff ected 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 fi rst (2007) and second years (2008), soil warming decreased the concentration of N and increased the CN ratio in canopy leaves, although the signifi cance 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 signifi cantly (p ⫽ 0.05), although the eff ects on the N concentration and CN ratio were only marginally signifi cant (N, p ⫽ 0.08; CN, p ⫽ 0.10). In contrast, branch warming did not aff ect 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. h e model indicated that the N and condensed tannin in canopy leaves could explain the variation in can-opy herbivory in the fi rst year (2007) (Table 4), although only the eff ect of N was signifi cant (p ⬍ 0.01). However, in the second year (2008), N, condensed tannin, and total 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 feed-ing activity of insect herbivores (Strong et al. 1984). We removed leaf traits for which the variance infl ation factor (VIF) values exceeded 10 in the stepwise model because VIF provides a measure of the extent to which the vari-ance of an estimated regression coeffi cient is increased by multicollinearity.
Results
Response of canopy herbivory rate
h e response of the canopy herbivory rate in mature trees diff ered between experimental soil- and branch-warming conditions. Before soil warming, there was no diff erence in herbivory rate between control and soil-warmed trees (p ⫽ 0.23; Fig. 1). After the commencement of soil warming, however, the herbivory rate signifi cantly decreased in June and August of the fi rst year (2007) (June, p ⫽ 0.03; August, p ⫽ 0.02). In the second (2008) and third years (2009), soil-warmed trees also experienced a lower herbivory rate, but only in August (2008, p ⬍ 0.01; 2009, p ⬍ 0.01). h e mag-nitude of the infl uence of soil warming on herbivory rate was gradually enhanced during the initial thrtee years. In the fi rst year (2007), the decrease was 32% in soil-warmed trees com-pared with control trees (Fig. 2). In the third year (2009), the decrease was twice as high (63%). In contrast, branch
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 Control
Branch
warming F-value
p-value (one side test)
LMA (g m ⫺2 ) 99.77 ⫾ 11.06 91.28 ⫾ 8.19 ⫺0.55 0.31
N (mg g ⫺1 ) 23.63 ⫾ 1.01 24.96 ⫾ 1.37 0.79 0.24
CN 21.84 ⫾ 1.67 19.92 ⫾ 1.27 ⫺1.11 0.16
Condensed tannin (mg g ⫺1 ) 11.55 ⫾ 2.26 10.28 ⫾ 1.36 ⫺0.48 0.33
Total phenolics (mg g⫺1 ) 156 ⫾ 27.01 147.39 ⫾ 20.62 ⫺0.27 0.40
Figure 1. Herbivory rate of soil-warmed trees (fi lled bars, n ⫽ 5) and control trees (open bars, n ⫽ 5) from 2006 to 2009. Values are mean ⫾ SE. Asterisk shows a signifi cant diff erence ( * p ⬍ 0.05, * * p ⬍ 0.10). We measured the canopy herbivory rate twice a year (June and August). Soil warming occurred from 2007 – 2009.
eff ects of plant growth. Previous studies have largely focused on plant photosynthetic responses to aboveground (air) temperature elevation and the accumulation of carbohy-drates. Such diff erential responses of plant traits between the meta-analysis and our warming experiment may be due to size-dependent tree responses (Nabeshima et al. 2010). h is 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 buff er-ing 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 indi-rect (plant-mediated) eff ects 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 aff ect
canopy leaf traits and herbivory. Experiments at higher tem-peratures are necessary to make further conclusions.
Responses to experimental soil warming
In contrast to branch warming, soil warming decreased the nutritional quality of canopy leaves (N, CN ratio, and total phenolics). Belowground increases in temperature infl uence various ecological processes that aff ect canopy leaf traits. Previous laboratory and snow manipulation studies have suggested that soil freezing drives soil N mineralization dur-ing winter because the increased mortality of microbes and fi ne roots results in a release of labile organic N to the soil (Schimel and Clein 1996, Groff man 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 win-ter, 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). h ese results suggest that elevation of the belowground temperature and the associated changes in soil N mineralization have a more pronounced infl uence 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 aff ect plant – insect interactions (Dury et al. 1998, Veteli et al. 2002, Zvereva and Kozlov 2006, Bidart-Bouzat and Imeh-Nathaniel 2008). h e 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. styracifl ua ). Both studies observed mature trees of late-successional spe-cies in cool-temperate forests. h ese results indicate that negative indirect (plant-mediated) eff ects of global warming phenolics explained the variation in canopy herbivory, and all
were signifi cant (p ⬍ 0.01). Among these selected leaf traits, soil warming only decreased the concentration of N in the fi rst and second years. In the third year (2009), LMA, con-densed tannin, and total phenolics explained the variation in canopy herbivory. h e eff ects of condensed tannin and total phenolics were signifi cant (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
h e 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 seed-ling 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 Variable b SE F-value p-value
2007 (intercept) ⫺5.36 4.98 ⫺1.07 0.29
(1st year) N 0.56 0.17 3.23 ⬍ 0.01
condensed tannin 0.19 0.12 1.51 0.14
2008 (intercept) ⫺4.75 7.14 ⫺0.67 0.51
(2nd year) N 0.82 0.22 3.57 ⬍ 0.01
condensed tannin 0.51 0.12 4.24 ⬍ 0.01
total phenolics ⫺0.07 0.02 ⫺3.28 ⬍ 0.01
2009 (intercept) 15.95 2.77 5.76 ⬍ 0.01
(3rd year) LMA ⫺0.04 0.03 ⫺13.5 0.18
condensed tannin 0.56 0.23 2.42 0.02
total phenolics ⫺0.07 0.02 ⫺4.49 ⬍ 0.01
aff ects plant – insect interactions in mature cool-temperate forests, this warming experiment should be continued using mature oak trees because indirect eff ects of temperature are likely more pronounced in the long- than in the short-term (Shaver et al. 2000).
Acknowledgements – h is 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 Environ-ment (no. D-0909 to TH) and the EnvironEnviron-ment Research and Technology Development Fund (S-9-3).
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Annual variation in the effect of soil warming on canopy herbivory
Our experiment revealed annual variation in indirect (plant-mediated) eff ects of soil warming on the rate of herbivory in the canopy. h e magnitude of the soil warming eff ect on canopy herbivory was gradually enhanced during the initial three years of the study. h e magnitude of the infl uence of global warming may vary temporally (Shaver et al. 2000). Melillo et al. (2002) reported that an average 28% increase in CO 2 fl ux due to soil warming was stabilized during the fi rst six years. h e eff ect of soil warming on soil respiration then substantially decreased. h ere are two possible causes of the annual variation in the eff ect 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 fi rst 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 nutri-tional 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 artifi cially high densities, or, con-versely, 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 landscape-level 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. h us, the eff ects of herbivore con-gregation 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). h erefore, our results can provide meaningful predic-tions of landscape-level responses to global warming.
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