CLIMATE WARMING EFFECTS ON LEAF HERBIVORY AND LEAF QUALITY IN
EASTERN TEMPERATE FOREST SPECIES
by
FERN RAFFELA LEHMAN
(Under the Direction of Jacqueline E. Mohan)
ABSTRACT
Herbivory may be indirectly affected by warming via warming-induced phytochemical and structural changes. The goal of the following two studies was to examine leaf herbivory on tree species growing under elevated soil and air temperature. In the first study, I examined chewing leaf herbivory on red maple (Acer rubrum L.) and white oak (Quercus alba L.) seedlings in a natural forest setting. In the second study, I fed leaves from the warmed white oaks to black-dotted brown moths (Cissusa spadix C.) in laboratory feeding preference trials.
The effect of warming on chewing leaf herbivory depended on leaf quality (e.g. C:N) as well as the phenology of leaves. Overall, however, warming led to reduced herbivory in the field for both species. The feeding study results complimented the field study results for white oak in that the larvae fed less on warmed white oak leaves.
INDEX WORDS: Climate change, Global warming, Soil warming, Herbivory, Nitrogen,
Carbon:nitrogen ratio, Red maple, Acer rubrum, White oak, Quercus alba,
Open-top chamber, Temperate forest, Seedling, Cissusa spadix, Black-
dotted brown moth, Choice feeding, No choice feeding
CLIMATE WARMING EFFECTS ON LEAF HERBIVORY AND LEAF QUALITY IN
EASTERN TEMPERATE FOREST SPECIES
By
FERN RAFFELA LEHMAN
B.A., Goshen College, 2008
A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment
of the Requirements for the Degree
MASTER OF SCIENCE
ATHENS, GEORGIA
2012
© 2012
Fern Raffela Lehman
All Rights Reserved
CLIMATE WARMING EFFECTS ON LEAF HERBIVORY AND LEAF QUALITY IN
EASTERN TEMPERATE FOREST SPECIES
by
FERN RAFFELA LEHMAN
Major Professor: Jacqueline E. Mohan
Committee: Kamal J. Gandhi
Paul E. Hendrix
Electronic Version Approved:
Maureen Grasso
Dean of the Graduate School
The University of Georgia
May 2012
ACKNOWLEDGEMENTS
I’d first like to thank my advisor, Jacqueline Mohan, who has been a source of great enthusiasm and support throughout my project. She has also helped immensely with advice on data analyses and writing. I am also very grateful for writing feedback from Shafkat Khan and Paul Frankson.
Many thanks to my lab mates Katy Bridges, Peter Baas, Kaitlin Maclean, and Megan
Machmuller for their field help and writing feedback. Thank you to the field techs at Duke
Forest and Harvard Forest for collecting the monthly herbivory data and to all the PIs on the “hot plants” project for allowing me to conduct research in the chambers!
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS………………………………………………………………………iv
LIST OF FIGURES……………………………………………………………………………....vi
CHAPTER
1 INTRODUCTION…………………………………………………………………...…1
2 LEAF HERBIVORY AND LEAF QUALITY RESPONSES IN RED MAPLE (ACER
RUBRUM) AND WHITE OAK (QUERCUS ALBA) GROWN IN ELEVATED SOIL
AND AIR TEMPERATURES..……………………………………………………...…4
3 FEEDING PREFERENCE OF BLACK-DOTTED BROWN MOTH (CISSUSA
SPADIX) ON WHITE OAK (QUERCUS ALBA) GROWN IN ELEVATED SOIL
AND AIR TEMPERATURES..……..…………………………………..…………….31
4 CONCLUSIONS…………………………………………………………………...…59
REFERENCES…………………………………………………………………………………..62
v
LIST OF FIGURES
Page
Figure 2.1: Warming effect on leaf mass herbivory in red maple……………………….…...... 26
Figure 2.2: Warming effect on red maple phytochemistry (N, C:N, δ15N)…………....………...27
Figure 2.3: Warming effect on red maple specific leaf area...…………………………………...28
Figure 2.4: Warming effect on red maple herbivory rates in two forest settings………...... 29
Figure 2.5: Warming effect on white oak herbivory rates in a forest setting……….…………...30
Figure 3.1: Warming effect on leaf mass herbivory on white oak during choice feeding
preference trials…………………………….………...... 52
Figure 3.2: Warming effect on herbivory rates on white oak during choice feeding preference
trials….…...... 53
Figure 3.3: Warming effect on herbivory rates on white oak during no choice feeding
preference trials…………………………………………………………………...54
Figure 3.4: Warming effect on leaf quality of choice preference leaves....…………………...... 55
Figure 3.5: Warming effect on leaf quality of no choice preference leaves...…………………...57
Figure 3.6: Tree species host feeding preferences of Cissusa spadix larvae…………………….58
vi
CHAPTER 1
INTRODUCTION
Global mean surface temperatures are projected to increase 1.4-5.8°C by 2100 AD as a consequence of rising green-house gases concentrations (IPCC, 2007). Mean surface temperatures in the USA alone are predicted to increase 3-4°C within the next 50 years (Karl et al., 2009). How terrestrial ecosystems will respond is a great deal of interest with our rapidly changing climate. A meta-analysis using 32 ecosystem warming studies show increases in plant primary productivity by 19%, soil respiration by 20%, and net nitrogen mineralization rates by
46% across a variety of forest and non-forest ecosystems and geographic coordinates (Rustad et al., 2001). Herbivore responses were not included in this analyses as previous warming experiments have generally not tracked herbivory. Warming may indirectly increase plant productivity by increasing soil nutrient availability due to higher litter decomposition and nitrogen (N) mineralization rates. Leaf N concentrations increased (Butler et al. 2011) in conjunction with greater N mineralization rates in a temperate forest warming study (Melillo et al., 2011). Changes in the phytochemical constituents of plants could have cascading effects on higher trophic levels. Herbivory may be indirectly affected by warming via warming-induced phytochemical changes associated with greater soil N bioavailability. Insect herbivores can play an important role in forest nutrient cycling and primary productivity (Stadler et al., 2001; Cronin et al., 2010; Ritchie et al., 1998; Chapman et al., 2003; Frost and Hunter, 2004; Throop et al.,
2004; Meehan and Lindroth, 2007) and numerous global change studies have examined the indirect effects of CO2 fertilization on plants and herbivory. Few, however, have examined the
1
indirect effects of warming on plants and herbivory (Cornelissen, 2011; Zvereva et al., 2006).
The goal of the following two studies was to examine leaf herbivory on tree species growing under elevated temperatures in natural forest settings. It is the only study to my knowledge that examined naturally occurring herbivory on temperate species growing in both elevated soil and air temperatures.
In the first study (Chapter 2), I examined leaf herbivory on red maple (Acer rubrum L.) and white oak (Quercus alba L.) juvenile trees in a natural forest setting. These are common eastern temperate forest species that exhibit contrasting responses to soil warming. Red maple juvenile growth responds positively to warming, whereas white oak shows a neutral growth response (Mohan, unpublished data). I predicted that soil warming would indirectly affect herbivory via changes in leaf chemistry (phytochemistry). Insect herbivores, often N limited, frequently respond to increases in foliar nitrogen by feeding more (Mattson, 1980). Soil warming led to higher maximum rates of photosynthesis and higher green leaf N rates in juvenile tree species (red maple, sugar maple, and black cherry) growing faster with warming (Mohan et al., 2010). To test my predictions, I measured %leaf mass eaten, leaf quality (carbon:nitrogen,
N, δ 15N, SLA), and %total plant leaf herbivory in red maple. I also measured %total plant leaf herbivory in white oak. Measurements were taken in soil and air warming experiments at
Harvard Forest (MA) and Duke Forest (NC).
In the second study (Chapter 3), I fed white oak (Q. alba) leaves from juvenile trees growing in the warming experiments to black-dotted brown moths (Cissusa spadix C.) in laboratory feeding preference trials. Warming led to reduced herbivory on white oak in the field
(Lehman, unpublished, Chpt 2), though it was not determined whether the reductions were due to changes in leaf chemistry or other biotic factors (e.g. parasitoids, herbivore population levels).
2
Leaf nitrogen decreased and condensed tannins increased in another oak species (Quercus robur
L.) exposed to a +3C rise in temperature (Dury et al., 1998). The black-dotted brown moth has been outbreaking in Athens, Georgia for the past three years and is known to defoliate white oak, red oak (Quercus rubra), and post oak (Quercus stellata), making it an appropriate model organism for the feeding trials. To test the effects of warming on larvae feeding preferences, I conducted choice and no-choice feeding trials over two different spring outbreaks. I quantified and compared %leaf mass eaten and leaf quality (C:N, N, δ 15N, and SLA).
3
CHAPTER 2
LEAF HERBIVORY AND LEAF QUALITY RESPONSES IN RED MAPLE (ACER
RUBRUM) AND WHITE OAK (QUERCUS ALBA) GROWN IN ELEVATED SOIL AND AIR
TEMPERATURES1
1 Lehman, Fern R., Shafkatul I. Khan, Jerry M. Melillo, James S. Clark, and Jacqueline E. Mohan. To be submitted to Global Change Biology. 4
Abstract
Climate warming has the potential to affect herbivory by changes in leaf chemistry (nitrogen, carbon:nitrogen ratio). Invertebrate herbivores often respond to increased nitrogen availability in leaves, which can occur in direct nitrogen fertilization studies or indirectly through soil warming induced nitrogen enhancements. Soil warming increases soil N mineralization and increases plant available N. Leaf herbivory data (% leaf mass eaten, % total plant leaf herbivory) were collected for red maple (Acer rubrum) and white oak (Quercus alba) growing under three temperature conditions (ambient temperature, +3°C, +5°C) and two light conditions (gap, shade) in two temperate deciduous forests (Harvard Forest, Duke Forest). Leaf quality data
(phytochemistry, specific leaf area) and % leaf-mass-eaten data for leaves with signs of herbivory were collected for red maple at Harvard Forest, while % total plant leaf herbivory data were collected for red maple and white oak at Harvard Forest and Duke Forest. Percent leaf mass eaten in red maple with signs of herbivory was neither affected by warming (p=0.2239) nor habitat. Correlations between leaf quality and herbivory could only be performed for trees from the gap habitat due to insufficient leaf material in the shade. Leaf chemistry (N, C:N, δ15N) did not explain percent leaf mass eaten. However, specific leaf area was positively related to % leaf mass eaten (p=0.023), meaning that thinner leaves exhibited elevated herbivory. Contrary to our hypothesis, warming had a significant negative effect on leaf N (p=0.0218), particularly in the shade. This is likely due to earlier spring bud-break and advanced phenological development expressed by trees in the warmed plots. There was a habitat effect on C:N (p<0.0001), with lower C:N values in the Gap, but there was no habitat*temperature interaction effect (p=0.4162).
Warming led to reduced % total leaf herbivory in shade red maples at Harvard Forest and shade white oaks at Duke Forest. There was no effect of warming on red maples at Duke Forest,
5
however. In conclusion, warming at Harvard Forest did not have an indirect effect on leaf-level herbivory in red maple exhibiting signs of herbivory via changes in leaf chemistry. However, herbivores were discriminating against warmed red maple plants at Harvard Forest. Herbivores were also discriminating against warmed white oaks at Duke Forest. We suggest that the reduction in herbivory had to do with the advanced phenology and thus reduced palatability of older warmed leaves. However, further studies linking herbivory, phenology, and leaf quality would are warranted.
Keywords: Herbivory, soil warming, nitrogen, red maple, Acer rubrum, white oak, Quercus alba, open-top chamber, C:N ratio
Introduction
Global mean surface temperatures are projected to increase 1.4-5.8°C by 2100 AD as a consequence of rising green-house gases concentrations (IPCC, 2007). Mean surface temperatures in the USA alone are predicted to increase 3-4°C within the next 50 years (Karl et al., 2009). How terrestrial ecosystems will respond is a great deal of interest in our rapidly changing climate. A meta-analysis using 32 ecosystem warming studies show increases in plant primary productivity by 19%, soil respiration by 20%, and net N mineralization rates by 46% across a variety of forest and non-forest ecosystems and geographic coordinates (Rustad et al.,
2001). Warming may indirectly increase plant productivity by increasing nutrient availability due to higher litter decomposition and N mineralization rates. Leaf N concentrations increased
(Butler et al. 2011) in conjunction with greater N minerlization rates in a temperate forest warming study (Melillo et al., 2011). Changes in the phytochemical constituents of plants could have cascading effects on higher trophic levels. Insect herbivory may be indirectly affected by warming via warming-induced phytochemical changes associated with greater soil N
6
bioavailability. We know that insect herbivores can play an important role in forest nutrient cycling and primary productivity (Stadler et al., 2001; Cronin et al., 2010; Ritchie et al., 1998;
Chapman et al., 2003; Frost and Hunter, 2004; Throop et al., 2004; Meehan and Lindroth, 2007) and numerous global change studies have looked at the indirect effects of CO2 fertilization on plants and herbivory. Few, however, have examined the indirect effects of warming on plants and herbivory (Cornelissen, 2011; Zvereva et al., 2006).
Warming can affect plant susceptibility to herbivory in direct and indirect ways. Plant susceptibility to herbivory is mediated by plant nutritional quality or the phytochemical constituents of the plant. Phytochemical constituents that influence herbivory include leaf nitrogen, carbon, phenolics, water content, toughness, and thickness or specific leaf area (SLA)
(Mattson, 1980; Shure et al., 1998; Dury et al., 1998; Kudo, 2003). Variations in these constituents define the quality of food for herbivores. Nitrogen is often a limiting nutrient for herbivore growth, reproduction, survivorship, body size, and population size particularly in the temperate zone (Mattson, 1980). Chewing damage on red oak saplings increased by 13% in one nitrogen fertilization experiment (Cha et al. 2010). Warming indirectly affects plant growth rates and phytochemical constituents via changes in soil nutrient availability and warming may directly affect plant photosynthetic and growth rates. Soil warming led to higher maximum rates of photosynthesis and higher green leaf N rates in juvenile tree species (red maple, sugar maple, and black cherry) growing faster with warming (Mohan et al., unpublished data). Increased net nitrogen mineralization and soil inorganic N availability led to increased leaf N concentrations in trees under warming treatment (Butler et al., 2011; Schmidt et al., 2002). Increased leaf N may make certain warmed plant species more susceptible to herbivory. Warming-induced leaf N enrichment (73%) led to a six-fold increase in herbivory on the dwarf shrub Vaccinium myrtillus
7
(Richardson et al., 2002). Defense compounds may also exhibit strong response to warming.
Overall, phenolic responses to warming have been species-specific and range from negative to positive increases in plant secondary chemistry (Bidart-Bouzat and Imeh-Nathaniel, 2008).
Condensed tannins increased in Quercus robur (Dury et al., 1998) and phenolics decreased in
Salix myrsinifolia (Veteli et al., 2002). Warming also affects plant phenology, leading to earlier leaf-out (Liu et al., 2001) and faster growth rates (Melillo et al., 2011; Mohan, unpublished).
Warming can indirectly affect herbivory by accelerating plant growth and changing plant phytochemistry, as the example above with V. myrtillus supports. Warming effectively speeds up nutrient cycling rates and makes plant-available nitrogen increasingly available.
There were three main objectives in this study. First, to test the impact that warming has on herbivory and leaf phyotochemistry using juvenile red maple and white oak as model organisms. Soil warming over 6 years led to a reduction in leaf C:N ratios in canopy red maple at Harvard Forest (Butler et al., 2011). Juvenile red maple is also one of four species that consistently benefits from soil warming by growing faster under warmer conditions (Mohan, unpublished data). Over the last several decades this species has become increasingly dominant in eastern temperate forests (Abrams, 1998). White oak is a common co-dominant in eastern forests, and in contrast to red maple, shows a neutral growth response to warming (Butler et al.,
2011). The second objective was to test how phytochemical quality affects rates and amounts herbivory, specifically C:N. Herbivory tends to respond positively to increases in leaf nitrogen, or reduced C:N ratios. The final objective was to determine what temporal trends herbivory showed under warming as leaves aged. The main hypotheses were:
H1: Warmed plants exhibit decreased leaf C:N, due to soil warming induced increases
in soil and leaf N.
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H2a: Decreased leaf herbivory on warmed trees when herbivores get the same amount of
N per biomass consumed.
H2b: Increased leaf herbivory on warmed leaves when there is more nutritious N to gain.
H3: Herbivory responses to warming will vary as leaves age.
The warming experiment was duplicated at two locations along the eastern U.S.: Harvard Forest
(MA) and Duke Forest (NC). Herbivory and phytochemical observations were taken at Harvard
Forest, whereas only herbivory observations were taken at Duke Forest. Observations were made during the growing season of 2010. This is the first study to examine herbivory in a natural forest setting using an experiment that directly warms soils in addition to warming the air within open-top chambers. We predicted that warming would reduce leaf C:N ratios and that leaf herbivory would respond by either either increasing (more nutritious leaves) or decreasing
(less leaf biomass required to gain requisite N). Our predictions are based on recent data showing positive growth in red maple seedlings under warming and past data showing lower C:N ratios in canopy red maples. The inverse relationship between herbivory and C:N ratios, where herbivory decreases with higher C:N ratios, has been called compensatory feeding (Docherty et al., 1996; Schadler et al., 2007). CO2 fertilization studies in particular lead to increases in leaf
C:N, making foliage less nutritious for herbivores, and leading to increased feeding (Bezemer et al., 1998). Perhaps the opposite of compensatory feeding will occur when C:N ratios decrease under warming, since the foliage quality is increasing, and herbivores are gaining more nitrogen per unit carbon. Conversely, increasing leaf quality may make leaves more advantageous to consume and lead to increased feeding as has been seen during fertilization studies. Changes in leaf nitrogen content, rather than more complex chemical constituents, were the focus of this study, since biomass of leaf samples was limited. Juvenile red maple and white oak (one-year-
9
old seedlings in this study) leaves were small and didn’t yield enough sample to do additional chemical analyses like secondary compounds. However, phenolics did not change in Acer rubrum with a +3°C change in temperature (Williams et al., 2003). Herbivory rates will likely interact with plant development and leaf age. Most herbivory occurs within the first month of leaf development when leaf nitrogen content is highest and phenolic content is lowest (Aide,
1993).
Few global change studies have examined indirect effects of warming on herbivory. This study is the only one of which we are aware that examines herbivory in response to both soil and air warming. Including impacts of warmer soils and increased N bioavailabilities on leaf-and plant-scale herbivory and phytochemistry provides a more realistic view of how forest ecosystems will respond to potentially altered herbivory with climate change.
Materials and methods
Plant growth conditions
First-year-old seedlings of Acer rubrum (red maple) and Quercus alba (white oak) were planted in spring 2010 as seeds into open-top chambers. The chamber design is replicated (n=3) with three temperature treatments and two light treatments. The open-top chambers (18.3m2 ) maintained soil and air at these temperatures, ambient, +3°C, and +5°C, and were located in two habitat/light conditions (closed forest canopy shade and canopy gap conditions). The experiment is further replicated by geographic location; experimental infrastructure is duplicated at Harvard
Forest (MA) and at Duke Forest (NC). At each location, common gardens of tree species native to the eastern temperate biome were planted as seeds (n=10-20 individuals per species) into the chambers. Tree gardens represented twenty-one species common to eastern deciduous forests, ranging from species near their southern or northern limits, and species with more extensive
10
distributions. Metallic mesh was buried around the perimeter of each chamber to exclude non- invertebrate herbivores.
We measured herbivory at both locations, focusing exclusively on the two most dominant species at Duke Forest and the most common species (red maple) at Harvard Forest. At Harvard
Forest, we quantified % herbivory of Acer rubrum and at Duke Forest we did so for both Acer rubrum and Quercus alba. These species were the most abundant and readily measurable species at their respective locations. Notably, these species displayed contrasting growth responses to warming, from a positive response in A. rubrum to a neutral response in Q. alba over eight years of soil warming (Mohan, unpublished). Inferences about the effect of warming on herbivory are drawn at the individual, plant level.
Harvard Forest is a mixed-deciduous forest in north-central Massachusetts (42°N, 72°W).
The dominant tree species include Quercus rubra, Acer rubrum, Betula lenta, Pinus strobus, and
Tsuga canadensis. It is an old-field forest, established after the abandonment from agriculture at the turn of the 20th century (Melillo, 2002). Overall, it has a cool and moist temperate climate, with average annual temperatures that vary from -7°C in January to 20°C in July. The average annual precipitation is 110 cm and is distributed fairly evenly throughout the year. The dominant soil type is characterized as mainly sandy loam glacial till, with some alluvial and colluvial deposits, moderately well drained, and acidic. It is an upland region dominated by granite, gneiss, and schist bedrock.
Duke Forest lies in the eastern edge of the North Carolina Piedmont Plateau (35°N,
79°W) and is also a post-agricultural forest dominated by Quercus alba and Acer rubrum. The warming experiment is located in an upland forest in the Eno Division of Duke Forest which, like Harvard Forest, was abandoned from agriculture early in the last century.
11
Measurements
Herbivory. Two types of leaf chewing herbivory measurements were taken at Harvard Forest and one at Duke Forest. At Harvard Forest, leaves were photographed in the field for later quantification of leaf mass eaten. In addition, individual plants were surveyed for total plant percent herbivory regularly throughout the 2010 growing season. At Duke Forest, individual plants were similarly surveyed for total plant herbivory but not photographed.
At Harvard Forest, non-destructive photographic leaf sampling was done over five days in the second week of August 2010. The photographic work documented accumulated summer herbivory. Individual leaves with signs of herbivory were digitally photographed (Olympus
Evolt 10 MP) in situ against 5 quadrille graphing paper (5.08 mm2). Digital photographs were later analyzed in the lab to quantify the average leaf area missing and the average leaf biomass missing for individual tree seedlings.
Separate estimates of total plant percent herbivory were visually assessed and recorded monthly or bi-monthly on individual plants at Harvard Forest and Duke Forest during 2010.
The digital photographs were uploaded into a computer program (Image J) that analyzes area.
Each image was converted into a binary image so that the photosynthetic leaf area stood in contrast to the background. Herbivory was defined as missing tissue from chewing activity on a pixel-by-pixel basis. No other signs of herbivory (e.g. galls, mining) were present on leaves
(personal observation). After the binary conversion, total leaf area (mm2), excluding any holes or missing tissue, could then be quantified. This total leaf area was then subtracted from the original leaf area (before damage from chewing) to determine the amount of missing leaf tissue.
The original leaf area was quantified by retracing any missing leaf perimeters and digitally filling
12
in damaged areas, then reanalyzing them. Leaf biomass was calculated by dividing leaf area by specific leaf area (SLA, mm2mg-1 d.w.), which was calculated for each leaf: mm2 / mm2mg-1= mg leaf biomass
Phytochemistry and SLA. Fully expanded leaves were sampled from individual plants.
Depending on the size of the plant, 1-2 two whole leaves were collected. A cork borer was used to take punch samples from the leaves. The leaves and leaf punches were kept cool (4oC) ~48 hours then put in a drying oven at 40°C for at least 48 hours. After drying, both leaves and leaf punches were weighed. The area of the leaf punch and its weight were used to calculate SLA.
Dried leaves and leaf punches were ground and analyzed for percent C, N, and δ15N via microdumas combustion. The ratio of carbon:nitrogen (C:N) was later calculated for individual plants.
Statistical analyses
Data were organized by habitat, temperature, and herbivory date for survey data. Leaf biomass missing and total plant percent herbivory were quantified per individual. The ratio of carbon:nitrogen and SLA were also calculated for individuals. Herbivory variables were arcsine- transformed and leaf quality variables (N, C:N, and SLA) were log10-transformed. A general linear model (Proc GLM SAS) was used to determine the relationships between herbivory, habitat, temperature, and the different leaf quality measurements (C, N, and SLA). SLA sampling size was not sufficient for the +3 °C treatment. The effect of habitat and temperature on leaf quality and δ15N were also analyzed using a general linear model. Tukey’s least square means was used to determine habitat*temperature differences.
Repeated measure PROC GLM (SAS) was used to determine patterns of herbivory for pre-treatment and experimentation during 2010 summer at Harvard Forest and 2010 at Duke
13
Forest. The herbivory data was arcsine-transformed and plotted by Julian date. Julian date is based on the day of the year out of 365 days.
Results
Leaf Mass and leaf mass versus phytochemistry. Leaf mass eaten on any given leaf appeared higher at elevated temperatures in the shade, however, the trend was not significantly linear
(Figure 2.1; p=0.2239). There was no significant difference between herbivory in the shade and gap, nor between treatments in the gap. Leaf mass eaten in the gap was not explained by differences in leaf C:N (p=0.4467), however, there was an important contribution from gap leaf
N (p=0.0928). There was a strong positive correlation between leaf mass eaten and SLA in gap leaves (p=0.023).
Phytochemical and SLA data is presented for red maple leaves from the Harvard Forest warming experiment. There was a habitat effect on C:N (Figure 2.2; GLM p<0.0001), with lower C:N values in the Gap, but there was no habitat*temperature interaction effect (p=0.4162).
In the shade, the average leaf C:N ranged from 27.56±0.63 to 30.91±1.52 mgg-1, highest in leaves grown in the +5°C treatment. In the gap, the average leaf C:N ranged from 21.22±1.29 to
22.79±1.42 mgg-1. Leaf N also depended on habitat and displayed higher levels in the gap
(Figure 2.2; p<0.0001). Temperature had a significant effect on leaf N (p=0.0218), which differed depending on the habitat. In the shade, leaf N was reduced at +5C (15.93±0.80 mgg-1) in comparison to the +3 and ambient treatments. In the gap, leaf N showed a unimodal response to warming, but showed the highest average overall in the +5C treatment and second highest in the ambient treatment (24.9±1.24 mgg-1). The average leaf N content in the shade ranged from
15.93±0.80 to 17.02±0.52 mgg-1. The average leaf N content in the gap ranged from 19.49±0.92 to 24.9±1.24 mgg-1. Gap leaves were 15N enriched (Figure 2.2; GLM p<0.0001) compared to
14
shade leaves. There was a positive relationship (increasing enrichment) between heating and
δ15N in the Gap and a negative relationship (increasing depletion) in the Shade (p=0.0087).
Shade leaves showed δ15N values ranging from -3.01±0.29 to -2.19± 0.30 mgg-1 and gap leaves showed values ranging from -0.38±0.70-2.32±0.69 mgg-1. The SLA did not respond significantly to either habitat or temperature (Figure 2.3; GLM p=0.0985), however, there tended to be an overall unimodal response to temperature. Specific leaf area data was not presented for
+3°C shade, because of insufficient leaf material for punch sampling. The average SLA in the shade ranged from 25.69±1.09 to 27.51±2.38 mgg-1, while the average SLA in the gap ranged from 21.54±1.11 to 29.05±5.16 mgg-1.
Total plant herbivory over time.
Herbivory on red maple at Harvard Forest and Duke Forest. The rate of total plant herbivory at
Harvard Forest was greater in the shade versus the gap (Figure 2.4; Repeated Measures GLM p=0.0101 for date*habitat interaction), particularly from June 16-September 1. The effect of temperature depended on the habitat and date (p=0.0370). Shade herbivory was reduced in both heated treatments compared to the ambient treatment during May and from July to September 1st.
In contrast, gap herbivory showed no strong temperature responses, regardless of the date. The rate of leaf herbivory at Duke Forest was not explained by habitat (p=0.2990 Date*habitat) nor by temperature (p=0.1289 date*habitat*treat).
Herbivory on white oak at Duke Forest. Overall, the rate of herbivory was greater in the shade versus the gap (Figure 2.5; Repeated measures p=0.0039 for date*habitat) and the effect of temperature depended on the habitat and date (p=0.0208 habitat*date*treat). In the shade, herbivory was significantly higher in the +5C treatment starting in May, then both +5C and +3C treatments were significantly higher in June. In July, +3C and ambient herbivory rose higher
15
than +5C herbivory. From August-November, herbivory rate was reduced in both heated treatments compared to ambient. In the gap, the herbivory rate also tended to be higher in the
+5C treatment at the beginning of the summer. Both gap heated treatments were reduced in comparison with ambient herbivory from September-November.
Discussion
Herbivory is predicted to be impacted directly and indirectly by climate warming (Bale et al.,
2002; Zvereva, 2006; Cornelissen, 2011). Warming studies have documented faster development among invertebrate herbivores (Veteli et al., 2002; Williams et al., 2003) and faster growth rate in plants (Melillo et al., 2011, Butler et al., 2011, Mohan, unpublished). Less well known are the indirect effects of warming on herbivory via changes in plant chemistry or phytochemical makeup (Cornelissen, 2006; Zvereva et al., 2006). We predicted that soil warming would lower leaf C:N in red maple and lead to either greater herbivory, due to increased leaf palatability, or reduced herbivory, since herbivores gain a higher proportion of leaf nitrogen per unit mass consumed. In this study, while leaf level herbivory in red maple did not respond to temperature or phytochemical makeup, whole plant level herbivory in red maple did respond negatively to elevated temperature. Similarly, this study showed that whole plant herbivory in white oak responds negatively to increased temperatures in a natural forest understory environment. Leaf level herbivory on red maple in this study was not related to phytochemical makeup, but was positively related to specific leaf area, or leaf thickness in gap leaves (a lower SLA means thicker leaves). Leaf quality (chemistry and thickness) was affected by warming to some extent. Leaf N and δ 15N showed strong habitat dependent temperature responses, whereas leaf C:N and SLA showed no temperature responses.
16
Warming by +3C and +5C negatively affected leaf herbivory in both red maple and white oak, though it depended on light availability and the time of year. While warmed red maples in the shade were discriminated against, when herbivory did occur on a leaf, it did not matter what temperature or habitat treatment the leaf occurred in or what its phytochemistry was. Herbivory did respond, however, to leaf thickness (SLA) in the gap habitat. Red maple SLA is positively correlated to leaf N (Shahba et al., 2009). In other words, thicker leaves have less nitrogen.
However, SLA or leaf thickness did not depend on the habitat or temperature in our study. Our findings support previous studies showing that herbivory and phytochemistry can be strongly light dependent (Dudt and Shure 1994; Agrell et al., 2000; Muth et al., 2008). Whole plant herbivory was greater overall in the shade than in the gap on both red maple and white oak seedlings. Not surprisingly, sunlight availability led to greater levels of leaf N and lower levels of C:N in gap red maple leaves. Red maple saplings had greater leaf N levels in a light environment versus a shade environment (Osier and Jennings, 2007). It is difficult, however, to tease apart the effect that light availability and leaf age had on phytochemistry, since leaves were only sampled once for phytochemistry. Data on these plants showed that warmed red maples and white oaks grew faster than their ambient counterparts and that gap plants grew faster than shade plants (Mohan, unpublished). Warmed red maple were breaking bud about 9 days earlier in 2010 than ambient red maple in the soil warming experiment at Duke Forest in 2010 (Salk et al., unpublished). Considering these observations, we can assume that warmed plants were older, which may have had implications for their phytochemistry. Leaf nitrogen concentration is greatest at the beginning of bud development and declines over time. In contrast, leaf phenolics and toughness tend to increase over time (Kudo, 2003). Our red maple phytochemical results, which provide a snapshot of leaves at the end summer, suggest the importance of habitat and
17
light for leaf phytochemistry at summer’s end. In our study, while leaf N was reduced in +5C shade leaves, it was elevated in +5C gap leaves. Furthermore, C:N values did not show a temperature response. Therefore, based on these results, warming does not appear to have an indirect affect on leaf level herbivory in red maple via changes in phytochemistry. Leaf N was marginally reduced and leaf water content significantly reduced in mature red maple leaves grown under +3.5C (air temperature), but this did not affect gypsy moth larvae performance
(Williams et al., 2003). Leaf N and water content decreased in red maple leaves as they aged
(Williams et al., 2003). Despite their findings, it should be reiterated that soil warming, which makes our study unique, can have important implications for soil nitrogen cycling and plant nutrient uptake. Warming in our study did have some important effects on phytochemistry that should not be overlooked and could help explain the effects that warming had on whole plant herbivory.
The observation that herbivores were discriminating against warmed red maple and white oak could be related to the advanced phenology (decreased palatability) of the warmed plants as well as the effects of light availability on phytochemistry. It is interesting to note that initial herbivory rates were much higher on warmed white oaks in the shade at the beginning of the summer (Figure 2.4). It wasn’t until August that herbivory on oaks at both +3C and +5C declined and was much less than herbivory on ambient oaks. We know that warmed plants were breaking bud sooner than ambient plants (Salk et al., unpublished), which means that warmed oaks leaves may have been available sooner for herbivory than ambient plants. Most herbivory occurs within first month of leaf development, when leaf palatability is greatest (Aide, 1993). It appears that the proportion of herbivory rose, stabilized, then declined as ambient white oaks aged (Figure 2.5). Herbivory in the warmed oaks began earlier, rose fast, and then declined
18
sharply. A sample of the treated white oak leaves taken at the end of April showed that warming led to significantly less leaf N and marginally higher leaf C:N (Lehman et al., unpublished), providing evidence for advanced phenology in warmed oak leaves. The geographic location of red maple plants had an effect on herbivory rates, as temperature and habitat did not explain herbivory on red maples at Duke Forest. It is unclear whether this had to do with a differing assemblage and abundance of herbivores at each site, since herbivores were not measured. Air warming led to significant changes in an insect community composition (Villalpando et al.,
2009). Red maple is known to be predated by insect herbivores such as the gypsy moth (Lymantria dispar), the linden looper (Erannis tiliaria), the elm spanworm (Ennomos subsignaria), the red maple spanworm (Itame pustularia), and the polyphemus moth (Anthera polyphemus) (Hutnick and Yawney, 1961; Wagner, 2005). Common leaf herbivores of white oak include the gypsy moth (Lymantria dispar), orange-striped oakworm (Anisota senatoria), variable oakleaf caterpillar (Heterocampa manteo), several oak leaf tiers (Psilocorsis spp.), and walkingstick (Diapheromera femorata) (Minkler, 1965). While the species of herbivores were not observed for this study, the photographs of herbivory and observations in the field showed that herbivory was predominantly done by leaf chewers.
Warming appeared to have an indirect N-fertilization effect that led to changes in phytochemistry, particularly in the gap where warming lead to high leaf N levels at +5 C.
Furthermore, the source of N became increasingly 15N enriched in gap leaves, indicating that gap red maples were gaining recalcitrant sources of nitrogen. Warming soils to +5C lead to increased decay of the soil labile carbon pool, resulting in increased availability of inorganic nitrogen to plants, and further to increased carbon storage in canopy trees (Melillo et al., 2011).
Data from the current chamber experiment is showing that warmed red maple plants grow bigger
19
in the gap and grow bigger at warmer temperatures (Mohan, unpublished data). Warmed plants are growing and taking up more nitrogen. The original Prospect Hill warming experiment at
Harvard Forest begun in 1991 showed that warming the soils up to +5C increased the availability of inorganic nitrogen to plants (Peterjohn et al., 1994; Melillo et al., 2002). A related soil warming study, located at Harvard Forest, concluded that soil warming stimulates microbial activity by increasing nitrogen mineralization and plant available N (Melillo et al., 2011). The increasing 15N enrichment of gap leaves could be due to the increased availability of recalcitrant sources of nitrogen mined by soil microbes for plant uptake.
Conclusions and future work
Climate change is expected to have multiple direct and indirect effects on plant-herbivore interactions. Warming has accelerated phenology in a variety of species (Salk et al., unpublished; Buse et al., 1999; Williams et al., 2003; van Asch et al., 2007; Liu et al., 2001), which can have important implications for the synchronicity between plants and their invertebrate hosts (Visser et al., 2001). Warming may indirectly affect herbivory via phytochemical changes. In our study, warming did not have an indirect effect on red maple herbivory via phytochemical changes as predicted. While warming did reduce leaf N shade +5 leaves and increase leaf N in +5 gap leaves, this did not have a significant effect on leaf palatability (C:N). It may be that warmed red maple and white oak plants were discriminated against due to advanced phenology and decreasing palatability of aged leaves. However, further research must be done that relates temporal changes in leaf phytochemistry with herbivory measurements.
20
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25
Shade Gap
12 12
10 10
8 8
6 6
% Leaf Mass Eaten 4
% Leaf Mass Eaten Mass % Leaf 4
2 2
0 0 Ambient +3 C +5 C Ambient +3 C +5 C
Figure 2.1: Warming effect on leaf mass herbivory in red maple. The proportion of leaf mass eaten from Acer rubrum (red maple) seedlings in three soil/air temperature treatments (ambient, +3°C, +5°C) and two habitat treatments (shade understory, gap). Leaf samples were collected in the first week of August, 2010. Leaf mass data were arcsine-transformed and phytochemical data were log10-transformed and analyzed using general linear models (n=128). Leaf mass eaten did not vary significantly with temperature nor habitat type (GLM p=0.2239). Leaf mass eaten was not explained by C:N (p=0.4467), but was weakly explained by N (p=0.0928). There was a correlation between leaf mass eaten and SLA (p=0.0118).
26
Shade Gap
35 35
30 30
25 25
20 20
C:N
C:N 15 15
10 10
5 5
0 0 Ambient + 3 C + 5 C Ambient + 3 C + 5 C
30 30
25 25
20 20
d.w.)
-1
d.w.) 15 -1 15
N (mgg
N (mgg 10 10
5 5
0 0 Ambient + 3 C + 5 C Ambient + 3 C + 5 C
4 4
2 2
N
15 0
N
15 0
-2 -2
-4 -4 Ambient + 3 C + 5 C Ambient + 3 C + 5 C
Figure 2.2: Warming effect on red maple phytochemistry (N, C:N, δ15N). Phytochemical results (C:N, N, δ15N) for Acer rubrum (red maple) leaves growing in three temperature and two habitat treatments. All the values, except for the δ15N values, were log10-transformed and all data were analyzed using a general linear model (n=87). There was a habitat effect on C:N (p<0.0001), lower C:N values in the Gap, but no habitat*temperature effect (p=0.4162). Leaf N also depended on habitat and displayed higher levels in the gap (p<0.0001). Leaf N was affected also by temperature (p=0.0218), though the response depended on the habitat. In the shade, leaf N was slightly reduced at +5. In contrast, gap leaf N showed the greatest overall increase in the +5 treatment. Gap leaves were 15N enriched (p<0.0001) compared to shade leaves. There was a positive relationship between heating and δ15N in the gap and a negative relationship in the shade (p=0.0087).
27
Shade Gap
40 40
30 30
d.w.)
d.w.)
-1
-1
mg
mg
2 2 20 20
SLA (mm
SLA (mm SLA 10 10
0 0 Ambient + 5 C Ambient + 3 C + 5 C
Figure 2.3: Warming effect on red maple specific leaf area. Punch samples were taken from leaves of seedlings growing in the two habitat and three temperature treatments and specific leaf area was calculated for each seedling. There was not enough leaf sample from +3, shade treatment, so that SLA average is not available. The values were log10-transformed and analyzed using a general linear model (n=34). Leaf SLA was not explained by temperature or habitat (GLM p=0.0985). However, +3 Gap was lower than both Gap Ambient and Gap +5 treatments (T-test p=0.0787; p=0.0146 respectively).
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Shade HF Gap HF
25 25
20 20
15 15
10 10
5 5
% Total Plant Leaf Herbivory
% Total Plant Leaf Herbivory Leaf Plant % Total 0 0
-5 -5 80 100 120 140 160 180 200 220 240 260 80 100 120 140 160 180 200 220 240 260 Julian Day Julian Day
Shade DF Gap DF
14 14
12 12
10 10
8 8
6 6
4 4
% Total Plant Herbivory % Total
% Total Plant Herbivory 2 2
0 0
50 100 150 200 250 300 350 50 100 150 200 250 300 350 Julian Day Julian Day
Shade Ambient Gap Ambient Shade +3 Gap +3 Shade +5 Gap +5
Figure 2.4: Warming effect on red maple herbivory rates in two forest settings. Visual percentage estimates of total plant leaf herbivory for Acer rubrum (red maple) at Harvard Forest and Duke Forest in 2010. Data were arcsine-transformed and analyzed using a repeated measures general linear model. A) The rate of leaf herbivory at HF (n=3275) was greater in the shade versus the gap (p=0.0101 date*habitat), particularly from June 16- September 1. The effect of temperature depended on the habitat and date (type I p=0.0370). Shade herbivory was reduced in both heated treatments compared to the ambient treatment during May and from July to September 1st. In contrast, gap herbivory showed no strong temperature responses, regardless of the date. B) The rate of leaf herbivory at DF (n=3814) was not explained by habitat (p=0.2990 Date*habitat) nor by temperature (p=0.1289 date*hab*treat).
29
Shade DF Gap DF
35 35
30 30
25 25
20 20
15 15
10 10
% Total Plant Herbivory 5 5
% Total Plant Leaf Herbivory Plant Leaf % Total
0 0
50 100 150 200 250 300 350 50 100 150 200 250 300 350 Julian Day Julian Day
Shade Ambient Gap Ambient Shade +3 Gap +3 Shade +5 Gap +5
Figure 2.5: Warming effect on white oak herbivory rates in a forest setting. Visual percentage estimates of total plant leaf herbivory on Quercus alba (white oak) at Duke Forest in 2010 (n=3814). Overall, the rate of herbivory was greater in the shade versus the gap (p=0.0039 date*habitat) and the effect of temperature depended on the habitat and date (type I p=0.0208 habitat*date*treat). In the shade, herbivory was significantly higher in the +5 treatment starting in May, then both +5 and +3 treatments were significantly higher in June. In July, +3 and ambient herbivory rose higher than +5 herbivory. From August-November, herbivory rate was reduced in both heated treatments compared to ambient. In the gap, the herbivory rate also tended to be higher in the +5 treatment at the beginning of the summer. Both gap heated treatments were reduced in comparison with ambient herbivory from September- November.
30
CHAPTER 3
FEEDING PREFERENCE OF BLACK-DOTTED BROWN MOTH (CISSUSA SPADIX) ON
WHITE OAK (QUERCUS ALBA) GROWN IN ELEVATED SOIL AND AIR
TEMEPRATURES2
2 Lehman, Fern R., Kamal J. Gandhi, Shafkatul I. Khan, Paul T. Frankson, David R. Coyle, Jerry M. Melillo, James S. Clark, and Jacqueline E. Mohan. To be submitted to Global Change Biology. 31
Abstract
Insect herbivory is largely mediated by leaf palatability, defined mainly by the variation in plant nitrogen content and carbon:nitrogen ratio. Herbivores often respond to increased nitrogen content by consuming more plant matter (Mattson, 1980). Climate warming may affect herbivory indirectly via changes in plant chemistry and palatability (Cornelissen, 2011; Zvereva et al., 2006). We predicted that soil and air warming would lead to increased leaf N and increased herbivory as leaves become more palatable. This study utilized choice and no choice feeding preference trials during April 2010 and 2011 to observe differences in cumulative leaf mass eaten and herbivory rates on leaves from juvenile trees grown in four temperature conditions (ambient Whitehall Forest, ambient Duke Forest, +3°C Duke Forest, and +5°C Duke
Forest). Leaves were fed to black-dotted brown moth (Cissusa spadix) larvae that were collected during outbreaks of April 2010 and 2011 in Athens, GA. There were five total feeding trials:
Tree species no choice in 2010 using several tree species (NCSP), Choice 2010 using white oak leaves produced under different temperatures (CH 2010), a similar choice experiment in 2011
(CH 2011), a 36-hour duration No Choice using white oak leaves using warmed white oaks in
2011 (NC 36), and a similar 12-hour No Choice feeding trial in 2011 (NC 12). The NCSP trial confirmed that larvae fed on white oak (6.875±3.125%), red oak (9.63±4.97%), and red maple
(1.25±0.82%). The relationship between warming, phytochemistry (C:N, %N, and ∂15N), specific leaf are (SLA), and percent leaf mass eaten was analyzed for the CH 2010 and CH 2011 trials.
The effect of warming on phytochemistry and SLA was also analyzed in leaves from the NC 36 hour trial. Percentage leaf mass loss was reduced for leaves produced under warmer conditions compared to ambient leaves for both Choice trials (2010 p=0.2960; 2011 p=0.0839). During the
CH 2010 trial, mass loss was negatively correlated to C:N (p=0.0003) and positively correlated
32
to N (p=0.0003). During the CH 2011 trial, mass loss showed a positive correlation with SLA or leaf thickness (p=0.0761). Herbivory rates were consistently reduced in the warmed leaves in all but one of the feeding trials (CH 2010 p=0.0432; CH 2011 p=0.3477; NC 36 p=0.1296; NC 12 p=0.0037). Contrary to our hypothesis, warming led to consistently elevated C:N (CH 2011 p=0.0006; CH 2010 p<0.0001; NC 36 p=0.0943), reduced N (CH 2010 p=0.0828; CH 2011 p<0.0001; NC 36 p=0.1027), and reduced SLA or thicker leaves (CH 2010 p=0.1437; CH 2011 p<0.0001; NC 36 p=0.0266). Our observations of reduced herbivory on white oak leaves in the lab support field observations of reduced herbivory on warmed white oaks. The reduced palatability of warmed leaves suggest warming-induced advances in leaf phenology, which further supports field observations of advanced phenology of warmed trees. In conclusion, elevated soil and air temperatures do have an indirect effect on herbivory via changes in leaf palatability that may be explained by warming-induced aging of leaves due to earlier spring bud break.
Keywords: Herbivory, warming, nitrogen, C:N ratio, white oak, Quercus alba, black-dotted brown moth, Cissusa spadix, choice feeding
Introduction
Rising global temperatures and levels of CO2 are both well documented components of anthropogenic global change. Studies have utilized both elements of change to determine their direct and indirect effects on plants, animals, and plant-animal interactions (Post et al., 1999;
Tylianakis et al., 2008). The indirect effects of CO2 on herbivory have been studied extensively
(Kinney et al., 1997; Agrell et al., 2000; Stiling and Cornelisson, 2007; Stiling et al., 2009;
Knepp et al., 2005; Hall et al., 2005; Knepp et al., 2007; Wang et al., 2009; Massad and Dyer,
2010); less well known are the indirect effects of warming on herbivory (Cornelissen, 2011;
33
Zvereva et al., 2006). Climate warming is expected to have an indirect effect on herbivory by changing phytochemical constituents of the herbivore host plant. Phytochemical constituents that herbivores respond to such as nitrogen (N), water content, lignin, and defense compounds
(Mattson, 1980; Shure et al., 1998; Dury et al., 1998; Awmack and Leather, 2002; Kudo, 2003;
Massad and Dyer, 2010), can shift with warming-induced changes in plant nutrient uptake and growth. Soil warming leads to a nutrient fertilization effect, where warming increases net nitrogen mineralization and soil inorganic N availability that lead to increased leaf N concentrations in plants (Butler et al. 2011; Schmidt et al., 2002). This can lead to accelerated plant growth and species-specific changes in leaf nitrogen (N) concentrations (e.g. Vaccinium myrtillus Richardson et al,. 2002; Acer rubrum Mohan, unpublished; Quercus robur Dury et al.,
1998). Phytophagous insect herbivores, often limited by nitrogen, may respond by increasing their consumption if leaves become more palatable (greater leaf N) (Mattson, 1980). Chewing damage on red oak saplings increased by 13% in one nitrogen fertilization experiment (Cha et al., 2010). The importance of the ratio of carbon:nitrogen for herbivory is highlighted in CO2 fertilization studies, where herbivores increase their feeding when leaves become less palatable
(higher C:N, increased secondary compounds), which is termed ‘compensatory feeding’
(Doucherty et al., 1996; Bezemer, 1998; Knepp et al., 2007; Schadler et al., 2007). This is explained by increased allocation of carbon to secondary compounds. In contrast, herbivores have responded to lower C:N values, or higher nitrogen content, by increasing their feeding
(Mattson, 1980). Warming effects on plant growth could have important implications for nutrient flows from primary producers to primary consumers. Numerous studies have examined the responses of soils and primary producers to warming (Bonan et al., 1992; Rustad et al., 2001;
34
Schmidt et al., 2002; Rinnan et al., 2009). However, more needs to be known about the cascading effects that warming has on higher trophic levels.
Climate warming may indirectly affect insect herbivore feeding via changes in plant phytochemical constituents. The effects of soil warming on soil nitrogen availability are similar to N fertilization studies which have lead to greater rates of herbivory. Therefore, we may predict that insect herbivores will increase their feeding if the plant material becomes more palatable (lower C:N due to increases in leaf N) or reduce their feeding if plant material becomes less palatable (higher C:N due to decreases in leaf N). There are many factors that affect herbivores in nature, however, and field experiments make it difficult to narrow down the main factors of interest. Lab studies can help focus on a few factors that affect herbivory. Choice feeding experiments are often used to determine insect herbivore preferences among a variety of feeding material (Wang et al., 2008).
The purpose of this study was to determine whether leaves from white oaks (Quercus alba) grown in warmed conditions would affect herbivory preferences of black-dotted brown moth, Cissusa spadix, (Lepidoptera) larvae and whether those changes would correspond to warming induced changes in leaf palatability. The black-dotted brown moth is native to the study location (Athens, GA) and is known to feed on white oak and other species from the white oak group. Outbreaks of C. spadix were previously un-documented until April 2010, when they began to outbreak in Athens, GA and throughout the deep southeastern US (GA, AL, SC, and southern NC). They have since gone through outbreaks in April, 2011, and again this April,
2012. A soil and air warming experiment set up at Duke Forest (NC) is being used to determine the effects of warming on tree demographics and soil biogeochemistry as well as plant-level herbivory on multiple eastern temperate species. Field observations of plant level leaf herbivory
35
have shown reduced herbivory on white oaks at the Duke warming experiment (Chapter 2).
Actual herbivory in nature, however, can depend on many factors such as predation, herbivore populations, and trophic interactions (Rosenheim, 1998; Barton, 2010). Whether reduced levels of white oak herbivory in at Duke Forest were due to changes in leaf palatability is of yet unknown. Therefore, a lab study was designed to ascertain if reductions in white oak herbivory observed in the field corresponded to changes in leaf palatability. The following hypotheses were formed:
H1: Leaf nitrogen (N) will increase and leaf carbon:nitrogen ratio (C:N) will decrease with soil and air warming.
H2: Herbivory will increase due to soil and air warming induced increases in leaf N and
reductions in leaf C:N.
Materials and methods
Choice and No Choice feeding trials took place during two years. The first Choice trial took place on April 29, 2010 and the second trial took place on April 28-29, 2011. Also, two No
Choice feeding trials took place on April 28-29, 2011. In the feeding trials, black-dotted brown moth larvae (Cissusa spadix) were given white oak (Quercus alba) leaves and herbivory levels were monitored over time. The larvae were collected from outbreaks occurring in Athens, GA during late April of both years. 2010 was the first known outbreak of this moth species in the south. The white oak leaves were collected from seedlings growing in a soil and air warming experiment at Duke Forest (NC) as well as from ambient-temperature white oak seedlings at
Whitehall Forest (Athens, GA). In the Choice trials, single larvae were given a choice between leaves grown at four different temperature treatments (ambient Whitehall Forest, ambient Duke
36
Forest, +3°C Duke, and +5°C Duke). In the No Choice trials, single larvae were given one leaf, from one of the four temperature conditions.
Plant growth conditions
Leaves used in the feeding experiments were collected from naturally recruited first-year
Quercus alba seedlings growing in open-top warming chambers at the Duke Forest experiment.
The chambers control both air and soil temperatures (ambient, +3°C, and +5°C) and are located in intact forest understory low-light conditions. Whole seedlings were collected from chambers, bagged with paper towels moistened with deionized (DI) water, and shipped overnight to Athens,
GA. The seedlings were immediately placed in DI water until the feeding trials began that evening. Naturally recruited white oak seedlings were also collected from the understory of
Whitehall Forest and were immediately placed in water until the feeding trials began.
Duke Forest lies in the eastern edge of the North Carolina Piedmont Plateau (35°N,
79°W) and is a post-agricultural forest dominated The warming experiment is located in an upland forest in the Eno Division of Duke Forest which is dominated by white oak and red maple. Whitehall Forest is similarly a naturally recruited upland oak-maple forest that is regenerating after agricultural land abandonment early in the last century.
Larvae sampling
In both years of the experiment, outbreaking caterpillars were collected from white oak tree trunks during late April at Whitehall Forest (33o53'05'' N 083o21'28'' W). They were immediately placed in jars and fed young Quercus alba leaves for one day. They were then without food for 48 hours before the experiment started.
Based on observations of caterpillar morphology and pupal rearing, the 2010 outbreaking caterpillar was identified as Cissusa spadix (David Wagner, unpublished data; John Pickering,
37
unpublished data). However, DNA barcoding of a population of the 2011 outbreaking caterpillars identified them as a mixture of C. spadix and common white oak moth (Phoberia atomaris) (Gandhi et al., unpublished data). Based on larvae behavior and morphology, we believe the larvae used in our feeding trials wereC. spadix. However, there was a chance that the populations we used contained common white oak moth. The caterpillars are out-breaking again this spring 2012, and we are collecting larvae for DNA barcoding in order to verify the species, which we are also using again for a third year of feeding trials.
There were outbreaks of C. spadix during April 2010 and 2011 in Athens, Clarke County,
GA and throughout the deep southeastern US (GA, AL, SC, southern NC). The larvae were defoliating oak trees, particularly white oak and post oak (Quercus stellata). The larvae spent the day in the leaf litter, crawled up the tree after sunset, and consumed canopy leaves at night.
Larvae feeding preferences were confirmed by personal observations in the field as well as a no choice feeding preference trial using six tree species commonly occurring at Whitehall Forest: white oak (Q. alba), red oak (Q. rubra), red maple (Acer rubrum), tulip poplar (Liriodendron tulipifera), sweetgum (Liquidambar styraciflua), and loblolly pine (Pinus taeda). In the lab, larvae fed exclusively on white oak (6.875±3.125%), red oak (9.63±4.97%), and red maple
(1.25±0.82%) leaves and did not consume tulip poplar, sweetgum, or loblolly pine (Figure 3.6).
Experimental design
Choice feeding trial 2010. Eighteen large (150 mm diameter) petri dishes were randomly laid out for Choice 2010 (CH 2010) feeding trial using whole Quercus alba leaves from seedlings grown at the Duke Forest soil and air warming experiment and from the ambient Whitehall
Forest. Eighteen larvae were randomly selected and assigned to three treatment leaves per dish: an ambient leaf from Whitehall Forest (Amb WH, n=18), an ambient leaf from Duke Forest
38
(Amb DF, n=18), and one heated leaf either from the +3oC treatment (n=9) or the +5oC treatment
(n=9) from Duke Forest (+3 DF or +5 DF). Treatment leaves were randomly assigned to dishes.
Moist cotton was wrapped on the tips of leaf stems to avoid desiccation.
Choice feeding trial 2011. The experimental design for the Choice 2011 (CH 2011) feeding trial was the same, except that the number of replicates for each treatment was n=32 for ambient leaves and n=16 for warmed leaves. Thirty-two petri dishes were laid out for choice feeding trials using Quercus alba leaves from the Duke warming experiment and from Whitehall
Forest. Thirty-two larvae were then randomly selected and assigned to three treatments per dish: an ambient leaf from Whitehall Forest (n=32), an ambient leaf from Duke Forest (n=32) and one warmed leaf from Duke Forest (+3°C or +5°C, n=16).
Before either choice experiment began, Cissusa spadix larvae were starved for 48 hours.
The feeding trials began at sundown (around 8:00 pm), when Cissusa spadix caterpillars are known to actively feed, and lasted for 24 hours for the 2010 experiment and 36 hours for the
2011 experiment. Feeding position and percent herbivory were recorded at hours 0, 1, 2, 4, 8,
12, 18, 20, and 24 for the 2010 trial. Feeding position and percent herbivory was recorded at hours 0, 2, 4, 8, 12, 24, 36 for the 2011 trial. If larvae were not actively feeding or immobile within the first hour, they were exchanged for randomly selected new larvae.
No choice species preference 2010. Thirty medium-sized (100 mm diameter) petri dishes were randomly laid out for No Choice host species preference feeding trials using whole leaves from six different mature tree species: white oak (Q. alba), red oak (Q. rubra), red maple (Acer rubrum), tulip poplar (Liriodendron tulipifera), sweetgum (Liquidambar styraciflua), and loblolly pine (Pinus taeda). Two leaves from each species was placed randomly placed into five dishes (n=10). Single larvae were randomly placed into dishes. The feeding trial began at 8:00
39
p.m. on May 5, 2010 and lasted 12 hours, with observations of herbivory at hours 0.5, 1, 6, and
12.
No choice feeding trials 2011. A total of 30 petri dishes (n=10; ambient Duke Forest,
+3°C DF, and +5°C DF) were used for two separate no choice feeding trials. The first no choice feeding trial ran for 36 hours (NC 36) and the second no choice feeding trial ran for 12 hours
(NC 12). Larvae were without food for 48 hour before the NC 36 trial, whereas larvae used in the NC 12 trial were collected and used on the same evening as the feeding trial. Single larvae were randomly assigned to one treatment/leaf per dish. Both feeding trials began at 8:00 pm on the evenings of April 28 and April 29, 2011. Visual estimates of herbivory were taken at hr 0, 1,
4, 12, 24, and 36 during the NC 36 trial. Estimates for the NC 12 feeding trial were at hours 1 and 12.
Larvae were weighed (n=18) before and after the CH 2010 trial. A sub-set of larvae
(n=8) were weighed before the CH 2011 trial. Treatment leaves were photographed before and after choice feeding trials in order to estimate biomass removed. Photographs were analyzed using Image J software to determine total leaf area. Leaf perimeters were reconstructed in order to determine leaf area removed. Leaf biomass (mg) removed was calculated by dividing leaf area removed by specific leaf area (SLA, mm2mg-1), calculated for each leaf. Leaf mass removed was not calculated for leaves used in the no choice feeding experiments. Instead, visual estimates of herbivory were used for analyses in the no choice feeding trials.
Phytochemical sampling and analyses
Two fully expanded leaves were removed from each plant from adjacent buds,, one for the feeding trial and one for phytochemical and SLA analysis. Leaves were analyzed for% C, %N, and natural isotope δ 15N. Using a cork borer, a leaf punch sample was taken from each leaf and
40
the whole leaves and punch samples were either dried for 48hrs at 60°C (CH 2010 leaves) or freeze-dried (CH 2011, NC 36 and 12 leaves). Punched samples were weighed and SLA was calculated for each sample leaf. Whole leaf samples were manually ground to a fine powder consistency, then prepared for combustion analysis. An Elemental Analyzer-Isotope Ratio Mass
Spectrometer (EA-IRMS) was used to determine leaf C, N, and natural isotope δ15N and δ13C.
Leaf samples were taken for analyses for 2010 and 2011 choice feeding trials and for the 36 hrs no choice feeding trial done in 2011.
Statistical analyses
Data were organized by dish number, hour, and temperature condition. Herbivory was quantified as leaf area consumed and leaf mass consumed. The values for leaf mass eaten and herbivory rates were logit-transformed for the choice trials. The herbivory rates were also logit- transformed for the No Choice 2011 trials. Leaf chemistry (C:N, N), except for δ 15N, and SLA values were log10-transformed. A general linear model (proc GLM SAS) was used to determine the relationships between leaf mass loss, temperature treatment, leaf C:N, N, δ15N, and SLA. A repeated measures general linear model (proc GLM SAS) was used to determine herbivory rates progressed over time in ambient versus warmed leaves.
Results
Choice feeding trials herbivory 2010 & 2011. The effect of temperature on cumulative percent leaf mass eaten in the 2010 feeding experiment was not significantly different among leaf types
(n=50; p=0.2960), though there was a significant relationship with temperature in the 2011 experiment (n=96; p=0.0839) (Figure 3.1). The effects of warming on Duke Forest leaves alone are presented as well, since Duke Forest leaves represent a homogenous set of plants. In 2010, warming marginally reduced herbivory (p=0.1198) when only Duke Forest leaf herbivory was
41
analyzed and Whitehall leaf herbivory was excluded. In contrast, warming in 2011 had a very significant negative effect on leaf mass eaten (p=0.0064) when only Duke Forest leaf herbivory was analyzed and Whitehall leaf herbivory was excluded. In the 2010 Choice trial (CH 2010), percentage leaf mass eaten ranged from 20.43±11.29% to 48.80±10.65%, with the lowest
o occurring on +5 C treated leaves and the highest mass eaten on Ambient Duke Forest (Amb DF) leaves. Mass eaten in CH 2010 was most similar between leaves grown in ambient conditions at
Whitehall Forest (Amb WH) (26.21±9.18%) and +5°C treatments. In the 2011 Choice trial (CH
2011), percentage leaf mass eaten ranged from 1.87±1.36 to 10.94±4.6%, again with the highest
o mass eaten on Amb DF leaves and the lowest eaten on +5 C leaves. In 2011, mass eaten was similar between leaves grown at Amb WH and Amb DF conditions.
In 2010, herbivory rates were greatest on Amb DF leaves (type I p=0.0432, type III p=0.0130; Figure 3.2). In 2011, herbivory rates did not differ significantly between warmed and ambient leaves (type I p=0.3477, type II p=0.3439; Figure 3.2).
No choice species preference 2010. Larvae fed exclusively on white oak
(6.875±3.125%), red oak (9.63±4.97%), and red maple (1.25±0.82%) leaves (Figure 3.6).
No choice feeding trials herbivory 2011. During the 36 hr trial, herbivory rates were greatest in ambient DF leaves (type I p=0.1296, type III p=0.4932; Figure 3.3). There was no hour*treat interaction (type I p=0.6093). During the 12 hr trial, herbivory rates were greatest in ambient DF leaves (hour*treat n=60; type I p=0.0037, type III p=0.0037; Figure 3.3).
Herbivory vs. leaf quality Choice 2010. Results are presented for correlations that first include, and then exclude, ambient Whitehall leaf herbivory and leaf quality. In 2010, mass
Eaten (%) was positively correlated with leaf N (n=54; p=0.0003) and negatively correlated with
C:N (n=54; p=0.0003). Mass eaten was not explained by SLA (n= 54; p=0.79) nor by δ15N
42
(n=54; p=0.9050). When ambient Whitehall leaves were excluded, mass eaten was still positively correlated with leaf N (n=34; p=0.0032) and C:N (n=34; p=0.0004). There was still no impact of warming on SLA (n=34; p=0.6962) and δ15N (n=34; 0.4549).
Herbivory vs. leaf quality Choice 2011. Results are presented for correlations that first include, and then exclude, ambient Whitehall leaf herbivory and leaf quality. In 2011, mass eaten (%) was not correlated to either N (n=41; p=0.9088) nor C:N (n=41; p=0.8403). Mass eaten was not explained by SLA (n=41; p=0.80) nor by δ 15N (n=41; p=0.916). When ambient
Whitehall leaves were excluded, mass eaten showed a nearly significant positive correlation with leaf N (n=31; p=0.1328) and nearly significant negative correlation with C:N (n=31; p=0.1054).
There was a nearly significant positive correlation between mass eaten and SLA (n=31; p=0.0761). There was still no correlation with N15 (n=31; p=0.3620), even when ambient
Whitehall leaves were excluded.
Phytochemistry and SLA Choice 2010. Elevated temperature lead to significant increases in C:N, with the greatest increase at +5°C (p=0.0006; Figure 3.4). Leaf C:N ranged from
17.24±0.71 to 24.03±1.39 mgg-1, with the highest ratio in leaves from the +5 °C treatment and the lowest in leaves from Amb DF condition. Leaf C:N was similar between leaves from the
Amb WH condition (20.73±0.94mgg-1) and leaves from the +3oC treatment at Duke (19.05±1.48 mgg-1). Leaf N showed a response to warming (p=0.0828), appearing most reduced at +5oC
(Figure 3.4). The amount of N was lowest at +5oC (21.06±1.54) and highest at Amb DF
(26.21±1.47mgg-1). There was similar %N in the +5oC leaves and Amb WH leaves
(23.01±1.10mgg-1). Specific leaf area (p=0.1437) and δ15N (p=0.1453) showed nearly significant responses to temperature (Figure 3.4). The SLA values were lowest at +5oC
(37.48±1.73 mm2mg-1) and highest at Amb DF (43.92±1.76 mm2mg-1). The values for δ15N
43
were lowest in leaves grown in the Amb WH condition (-5.13±0.19) and highest in leaves grown in the +5oC treatment (-4.64±0.24).
Phytochemistry and SLA Choice 2011. In 2011, leaf C:N increased with warming
(p<0.0001; Figure 3.4). Leaf C:N ranged from 20.76±1.03 to 32.31±2.18 mgg-1, with the highest ratio in leaves from the Amb WH condition and the lowest in leaves from Amb DF condition.
These different C:N ratoios from ambient-temperature leaves at the two sites likely results from fundamental differences in soil type and chemistry. While Whitehall Forest occurs on highly- weathered Ultisol soils, upland soil at the Eno Division at Duke Forest where this experiment takes place are comprised of 85% Ultisol and 15% Alfisol soils with inherently higher fertility.
Leaf C:N was similar between leaves from the Amb WH condition and leaves from the +3oC treatment at Duke (30.02±1.70 mgg-1). In general leaf N decreased with warming (p<0.0001).
The amount of N was highest at Amb DF (23.26±1.05 mgg-1). There was similar %N in the
+3oC leaves (16.48±1.01 mgg-1) and the Amb WH leaves (15.16±0.95 mgg-1). Leaf δ15N
(n=41; p=0.0771) did not respond to warming, with values ranging from -4.49±0.45 to -
2.85±0.19 mm2mg-1. Leaf SLA decreased with warming thus warming produced thicker leaves at the date of harvesting (n=93; p<0.0001) and ranged from 37.63±1.28 to 53.39±1.37 mm2mg-1.
There was a similar SLA in +3°C leaves (37.63±1.28 mm2mg-1) and Amb WH leaves
(40.38±1.26 mm2mg-1).
Phytochemistry and SLA No Choice 36 hr 2011.
Warming had a positive effect on C:N ratios and foliar %N (p=0.09 and 0.10, respectively;
Figure 3.5. The mean leaf C:N ratio ranged from 21.60±1.03 to 25.61±1.72, while the average leaf N ranged from 19.23±1.14 mgg-1 to 22.60-1.15 mgg-1. Warming had no effect on δ 15N
(p=0.2118 Figure 3.5), which ranged from -5.02±0.42 to -4.20±0.42. Warming significantly
44
reduced SLA (p=0.0266; Figure 3.5), which ranged from 41.78±2.04 mm2mg-1 to 48.36±1.39 mm2mg-1.
Discussion
Insect herbivory on leaves often depends on leaf palatability, phytochemistry, and structural characteristis. Hebivores often respond to increases in leaf N by consuming more, such as in herbivory responses to plant nitrogen fertilization studies (Mattson, 1980; Cha et al., 2010). Soil and air warming were predicted to have an indirect effect on herbivory via changes in phytochemistry, particularly increases in leaf N. Soil warming led to an indirect N fertilization effect, increasing soil N mineralization rates and inorganic N availabilities to plants (Melillo et al., 2002, 2011) and leading to decreases in leaf C:N in mid-summer canopy Acer rubrum trees
(Butler et al., 2011). Our findings support the prediction that warming would affect phytochemistry, however warming lead to increased C:N levels, rather than decreased C:N levels as we expected. There was a decrease in leaf mass herbivory on warmed leaves in 2010, especially on plants grown at +5oC (Figure 3.1), and again a decrease in 2011 on warmed leaves
(3.1). Leaf mass eaten corresponded to changes in leaf N and leaf C:N in 2010, but did not correspond to N and C:N in 2011. This result in 2011 was mainly explained by the fact that ambient Whitehall leaves had similar phytochemistry to warmed (+3oC) leaves, even though ambient Whitehall leaves were eaten as much as ambient Duke Forest leaves (Figure 3.4). This suggests that in 2011 Whitehall leaves were still palatable, despite having high C:N and low N content compared to warmed Duke Forest leaves that had very low herbivory levels, high C:N ratios, and low N content. When Whitehall leaf herbivory and leaf chemistry were not considered in the analysis, then reduced mass eaten corresponded to higher C:N ratios and lower
N content. Furthermore, increasing leaf thickness in Duke Forest leaves led to reduced
45
herbivory. Warmed leaves consistently had lower herbivory rates during the choice and no choice feeding trials (Figure 3.2 and 3.3). The leaf quality (phytochemistry and SLA) results were fairly consistent across the Choice 2010, 2011, and the No Choice 36 hr leaves. Warming led to reduced leaf quality; they had increased C:N ratios, reduced N, and reduced SLA (thicker leaves) (Figure 3.4 and 3.5). The results of our lab-based study are similar to a concurrent study examining naturally occurring herbivory in the field, where plants are susceptible to a variety of invertebrate herbivore populations and herbivore interactions with predators or parasites.
Herbivory was reduced on the warmed Quercus alba (white oak) seedlings growing in the natural forest setting (Lehman et al., in prep Chpt 2). Previous studies using another oak species found similar phytochemical responses to air warming alone (Dury et al., 1998; Buse et al.,
1998). A +3°C increase in temperature led to reduced leaf palatability, by reducing leaf nitrogen concentration and increasing condensed tannins in Pedunculate oak (Quercus robur L.) (Dury et al., 1998). The reduction of leaf nitrogen concentration corresponded to feeding by early instar larvae, whereas the increase of condensed tannins corresponded to the end of the larval feeding stage, leading the authors to conclude that adult fecundity would be adversely affected by increased temperatures. In a related study, increased fiber in air warmed Pedunculate oak led to reduced Winter Moth (Operophtera brumata L.) pupal mass and fecundity (Buse et al., 1998).
Phytochemistry changes over the lifespan of a leaf. Leaf nitrogen concentration is greatest at the beginning of bud development and declines over time. In contrast, leaf phenolics and toughness tend to increase over time (Kudo, 2003). The negative response in leaf C:N with elevated temperature may be explained by advanced leaf phenology of warmed plants. Warmed red maples were breaking bud about 9 days earlier than ambient plants at Duke Forest (Salk et al., unpublished). The observations of increased levels of leaf C:N, reduced leaf N, and thicker
46
leaves (lower SLA) with increasing temperature provides evidence that warmed oak leaves were older than ambient leaves and thus less palatable to herbivores (Figure 3.4 and 3.5).
Conclusion and future direction
Climate change is expected to affect herbivory several direct and indirect ways. Warming is expected to directly affect the synchronicity between hosts and their herbivores; studies show that warming accelerates phenology in many plant species (van Asch et al., 2007). Warming is also expected to indirectly affect herbivores via changes in phytochemistry. While a few studies have looked at the indirect effects of warming on herbivory, our study is the first to use plants that have undergone soil and air warming, which has been known to change leaf chemical properties. Warming led to reduced leaf palatability, which led to reduced herbivory. Therefore, we may conclude that warming does have an indirect affect on herbivory via changes in phytochemstry in white oak. This work highlights the role of earlier spring bud-break for impacting spring herbivory rates via reductions in leaf tissue quality. Future warming work that considers herbivory and phytochemical changes as leaves develop as well as measures plant nitrogen uptake could help determine the contribution of each to changes in phytochemistry and corresponding herbivory.
47
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2010
70
60
50
40
30
% Leaf Mass Eaten Mass % Leaf 20
10
0 Amb WH Amb DF +3 DF +5 DF
2011
18
16
14
12
10
8
% Leaf Mass Eaten 6
4
2
0 Amb WH Amb DF +3 DF +5 DF
Ambient Whitehall Forest Ambient Duke Forest +3 C Duke Forest +5 C Duke Forest
Figure 3.1: Warming effect on leaf mass herbivory on white oak during choice feeding preference trials. The percentage of leaf mass eaten from white oak leaves during choice feeding trials with Cissusa spadix larvae in 2010 and 2011. The effect of temperature on leaf mass eaten in the 2010 feeding experiment was not significant (n=50; p=0.2960), though there was a nearly significant relationship with temperature in the 2011 experiment (n=96; p=0.0839). Error bars represent standard error.
52
2010
1.0
0.8
0.6
0.4
0.2
Proportion Leaf Herbivory
0.0
0 5 10 15 20 25 30 Hour
2011
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
Proportion Leaf Herbivory 0.02
0.00
0 10 20 30 40 Hour
Amb WH Amb DF 3 DF 5 DF
Figure 3.2: Warming effect on herbivory rates on white oak during choice feeding preference trials. The proportion of herbivory in two choice feeding trials with white oak leaves grown at four temperature conditions (ambient Whitehall, ambient Duke Forest, +3°C, +5°C). The 2010 choice trial lasted for 24 hours and the 2011 choice trial lasted for 36 hours. 2010 Choice Feeding: Herbivory rates were greatest on Amb DF leaves (hour*treat n=576; type I p=0.0432, type III p=0.0130). 2011 Choice Feeding: Herbivory rates did not differ significantly between warmed and ambient leaves (hour*treat n=672; type I p=0.3477, type III p=0.3439). Error bars represent standard error.
53
No Choice 36 Hours
0.30
0.25
0.20
0.15
0.10
0.05
Proportion Leaf Herbivory
0.00
0 10 20 30 40 Hour
No Choice 12 Hours
0.7
0.6
0.5
0.4
0.3
0.2
Proportion Leaf Herbivory 0.1
0.0
0 2 4 6 8 10 12 14 Hour
Amb DF 3 DF 5 DF
Figure 3.3: Warming effect on herbivory rates on white oak during no choice feeding preference trials The proportion of herbivory in two no choice feeding trials with white oak leaves grown in three temperature conditions (ambient Duke Forest, +3°C, +5°C) that lasted 36 hours (top) and 12 hours (bottom). 36 Hr: Herbivory rates were greatest in ambient DF leaves (n=120; type I p=0.1296, type III p=0.4932). There was no hour*treat interaction (type I p=0.6093). 12 Hr: Herbivory rates were greatest in ambient DF leaves (hour*treat n=60; type I p=0.0037, type III p=0.0037). Error bars represent standard error.
54
2010 2011
40 40
30 30
20 20
C:N
C:N C:N
10 10
0 0 Amb WH Amb DF +3 DF +5 DF Amb WH Amb DF +3 +5
35 35
30 30
25 25
20 20
d.w.)
d.w.)
-1
-1
15 15
N (mgg
N (mgg
10 10
5 5
0 0 Amb WH Amb DF +3 DF +5 DF Amb WH Amb DF +3 DF +5 DF
0 0
-1 -1
-2 -2
N
N
15 -3 -3
-4 -4
-5 -5
-6 -6 Amb WH Amb DF +3 DF +5 DF Amb WH Amb DF +3 DF +5 DF
60 60
50 50
40 40
d.w.)
d.w.)
-1
-1
mg
mg
2
30 2 30
20 20
SLA (mm SLA
SLA (mm
10 10
0 0 Amb WH Amb DF +3 DF +5 DF Amb WH Amb DF +3 DF +5 DF 55
Figure 3.4: Warming effect on leaf quality of choice preference leaves. Phytochemical and specific leaf area results for white oak (Quercus alba) leaves collected from seedlings growing at two ambient and two heated conditions (Amb WH, Amb DF, +3°C, +5°C) during the April 2010 and 2011 choice feeding trials. The Amb WH leaves were from seedlings growing in ambient conditions in a shaded forest understory at Whitehall Forest (Athens, GA). The other leaves were collected from seedlings growing in ambient and heated conditions in a shaded understory at Duke Forest (Durham, NC). Phytochemical data, except for δ15N, and SLA were log10-transformed and all data were analyzed using a general linear model. Choice 2010: Leaf C:N increased with warming (n=54; p=0.0006), especially at +5. In contrast, leaf N tended to decrease with warming, especially at +5 (n=54; p=0.0828). Leaf δ15N (n=54; p=0.1453) and SLA (n=54; p=0.1437) did not respond to warming. Choice 2011: Leaf C:N increased with warming (n=41; p<0.0001). In contrast, leaf N decreased (n=41; p<0.0001). Leaf δ15N (n=41; p=0.0771) did not respond to warming. Leaf SLA decreased with warming (n=93; p<0.0001). Error bars represent standard error.
56
30 25
25 20
20 15
d.w.)
15 -1
C:N
10
N (mgg 10
5 5
0 0 Amb DF 3 DF 5 DF Amb DF 3 DF 5 DF
60 0
50 -1
40 -2
d.w.)
-1
mm
2 30
N
15 -3
20 -4 SLA (mm
10 -5
0 -6 Amb DF 3 DF 5 DF Amb DF 3 DF 5 DF
Amb DF 3 DF 5 DF
Figure 3.5: Warming effect on leaf quality of no choice preference leaves. Phytochemistry (C:N, N, δ 15N) and SLA in white oak leaves (Q. alba) grown in three temperature conditions (ambient Duke Forest, +3°C, +5°C). The leaves were used in the No Choice 36 hour feeding trial on April 28, 2011. All of the values, except for δ 15N, were log10-transformed and analyzed using a general linear model (proc GLM SAS). Warming had a nearly significant positive effect on C:N ratios (n=29; p=0.0943), but no effect on N (n=29; p=0.1027). Warming had no effect on δ 15N (n=29; p=0.2118). Warming significantly reduced SLA (n=30; p=0.0266). Error bars represent standard error.
57
No Choice 2010 Host Preference
16
14
12
10
8
% Herbivory 6
4
2
0 PITA LIST LITU ACRU QUAL QURU
Figure 3.6: Tree species host feeding preferences of Cissusa spadix larvae. The % herbivory on leaves used in no choice host species preference feeding trials. Leaves were collected from six species: Pinus taeda (PITA), Liquidambar styraciflua (LIST), Acer rubrum (ACRU),
Quercus alba (QUAL), and Quercus rubrum (QURU). The feeding trial began at 8:00 p.m. May 5, 2010 and lasted 12 hours. Larvae fed exclusively on white oak (6.875±3.125%), red oak (9.63±4.97%), and red maple (1.25±0.82%) leaves. Error bars represent standard error.
58
CHAPTER 4
CONCLUSIONS
Global mean surface temperatures are projected to increase 1.4-5.8°C by 2100 AD as a consequence of rising green-house gases concentrations (IPCC, 2007). Mean surface temperatures in the USA alone are predicted to increase 3-4°C within the next 50 years (Karl et al., 2009). How terrestrial ecosystems will respond is a great deal of interest in our rapidly changing climate. A meta-analysis using 32 ecosystem warming studies show increases in plant primary productivity by 19%, soil respiration by 20%, and net nitrogen mineralization rates by
46% across a variety of forest and non-forest ecosystems and geographic coordinates (Rustad et al., 2001). Warming may indirectly increase plant productivity by increasing nutrient availability due to higher litter decomposition and N mineralization rates. Leaf N concentrations increased (Butler et al. 2011) in conjunction with greater N minerlization rates in a temperate forest warming study (Melillo et al., 2011). Changes in the phytochemical constituents of plants could have cascading effects on higher trophic levels. Insect herbivory may be indirectly affected by warming via warming-induced phytochemical changes associated with greater soil N bioavailability. We know that insect herbivores can play an important role in forest nutrient cycling and primary productivity (Stadler et al., 2001; Cronin et al., 2010; Ritchie et al., 1998;
Chapman et al., 2003; Frost and Hunter, 2004; Throop et al., 2004; Meehan and Lindroth, 2007) and numerous global change studies have looked at the indirect effects of CO2 fertilization on plants and herbivory. Few, however, have examined the indirect effects of warming on plants and herbivory (Cornelissen, 2011; Zvereva et al., 2006). The goal of the following two studies
59
was to examine leaf herbivory on tree species growing under elevated temperatures in a natural forest setting. It is the only study to my knowledge that examined naturally occurring herbivory on temperate species growing in both elevated soil and air temperatures.
The field studies of herbivory complimented the lab studies of herbivory well. Warming consistently lead to reduced herbivory, both on red maples in the field, and on white oaks both in the field and in laboratory feeding trials. Warming also lead to reduced leaf palatability in both red maple leaves (lower SLA or increased leaf thickness) and white oaks (lower N, higher C:N, and lower SLA). However, phytochemistry changes over the lifespan of a leaf. Leaf nitrogen concentration is greatest at the beginning of bud development and declines over time. In contrast, leaf phenolics and toughness tend to increase over time (Kudo, 2003). The negative responses in leaf C:N with elevated temperature may be explained by advanced leaf phenology of warmed plants. Warmed red maples were breaking bud about 9 days earlier than ambient plants at Duke Forest (Salk et al., unpublished). The observations of increased levels of leaf
C:N, reduced leaf N, and thicker leaves (lower SLA) with increasing temperature provides evidence that warmed oak leaves were older than ambient leaves and thus less palatable to herbivores.
Climate change is expected to have multiple direct and indirect effects on plant-herbivore interactions. Warming has accelerated phenology in a variety of species (Salk et al., unpublished; Buse et al., 1999; Williams et al., 2003; van Asch et al., 2007; Liu et al., 2001), which can have important implications for the synchronicity between plants and their invertebrate hosts (Visser et al., 2001). Warming may indirectly affect herbivory via phytochemical changes. While a few studies have looked at the indirect effects of warming on herbivory, our study is the first to use plants that have undergone soil and air warming, which
60
has been known to change leaf chemical properties. In our study, warming did not have an indirect effect on red maple herbivory via phytochemical changes as predicted. Unfortunately, data were not available to see whether this was still the case for shade red maple leaves, which were obviously negatively affected by warming on a whole plant level. While warming did reduce leaf N shade +5 leaves and increase leaf N in +5 gap leaves, this did not have a significant effect on leaf palatability (C:N). In the choice and no choice feeding trials, warming led to reduced leaf palatability which led to reduced herbivory. We may conclude that warming can have an indirect affect on herbivory via changes in phytochemistry in white oak and potentially in red maple as well. Furthermore, it may be that warmed red maple and white oak plants were discriminated against due to advanced phenology and decreasing palatability of aged leaves. However, further research must be done that relates temporal changes in leaf phytochemistry with herbivory measurements. This work highlights the role of earlier spring budbreak for impacting spring herbivory rates via reductions in leaf tissue quality. Future warming work that considers herbivory and phytochemical changes as leaves develop as well as measures plant nitrogen uptake could help determine the contribution of each to changes in phytochemistry and corresponding herbivory. The effect of climate warming on these predominant eastern species has the potential to scale up to higher trophic levels, especially through changes in leaf phenology and chemistry.
61
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