Sources of Spatial Variation in Herbivory and Performance of

an Invasive Non-native Plant, Common Burdock (Arctium minus)

By

Yoonsoo Lee

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Ecology and Evolutionary Biology

University of Toronto

© Copyright by Yoonsoo Lee 2013

ii

Sources of Spatial Variation in Herbivory and Performance of an

Invasive Non-native Plant, Common Burdock (Arctium minus)

Yoonsoo Lee

Master of Science

Ecology and Evolutionary Biology

University of Toronto

2013

Abstract

The herbivory experienced by non-native invasive plants may depend on their local

environments, such as herbivore abundance. In this study, I performed a common garden

experiment with plants sampled from 11 populations of Arctium minus, from southern Ontario to

near its northern range limit. I also compared performance and herbivory of burdock in open and

understory habitats. Finally, I conducted freezing tolerance experiments with the lepidopteran

seed predator Metzneria lapella, and palatability tests with plants from different populations.

Results suggested that the previously described latitudinal trends in herbivore damage among

populations are due to environmental differences rather than genotypic differences among

populations. At a local scale, plants of open habitat were less damaged and had better

performance than understory plants. Burdock has not escaped damage by herbivores in its

invaded range; instead variation among sites in herbivore populations and impacts may

significantly affect the invasiveness of this species.

iii

Acknowledgments

I would like to first thank my supervisor, Dr. Peter Kotanen. I feel very lucky that he graciously

accepted me to work in his lab. Under his guidance, I was able to learn so much and got even

more interested in plant ecology. His enthusiasms in science always encouraged me to challenge

myself and become better scientist. I am also thankful for his patience to put up with my slow

progress in my study. I would also like to thank my committee members, Dr. Megan

Frederickson and Dr. Marc Johnson, for all their help and advice throughout my research. I

would also like to thank Dr. Ivana Stehlik and her summer field course during summer 2010

which got me interested in plant-herbivore interaction. I also thank Dr. Arthur Weis for lending

me a space at Koffler Scientific Reserve for my common garden experiment.

I would like to thank Dasvinder Kambo for being an excellent example as a senior graduate

student and sharing his knowledge and experiences. I like to also thank Johanna Perz for being

the best assistant a graduate student can ask for. Without her help, the common garden would not

have been successful.

I would also like to give special thanks to my girlfriend Christine Yoo for voluntarily helping me

with the common garden and forest samplings even though she is not a biologist and scared of

insects. Her bravery is much appreciated and cannot be matched.

Finally, I would like to thank my parents who were not able to be with me most of the time, but

always found a way to show their support and love across the sea. Their encouragements and

trusts never failed to give me strength during my difficult times. Last but not least, I would like

to thank my sister for always being supportive, empathetic, and the best sister I can ever hope for.

iv

Table of Contents

Abstract ....................................................................................................................................................... ii

Acknowledgments ...................................................................................................................................... iii

Table of Contents ....................................................................................................................................... iv

List of Tables .............................................................................................................................................. vi

List of Figures ........................................................................................................................................... vii

List of Appendices ..................................................................................................................................... ix

Chapter 1: General Introduction ............................................................................................................... 1

Intercontinental Variation - Invasive Species ................................................................................... 2

Intracontinental Variation - Differences in Marginal Populations ................................................. 3

Local Variation - Population Differences among Microhabitats .................................................... 4

My Research......................................................................................................................................... 6

References ............................................................................................................................................ 8

Chapter 2: Differences in herbivore damage and performance for plants from a latitudinal range of

populations ................................................................................................................................................. 13

Introduction ....................................................................................................................................... 14

Methods .............................................................................................................................................. 18

Study Species ............................................................................................................................... 18

Common Garden Transplant Experiment .................................................................................... 19

Palatability Experiment ............................................................................................................... 21

Freezing Tolerance Experiment .................................................................................................. 22

Statistical Analysis ....................................................................................................................... 23

Results ................................................................................................................................................. 24

Common Garden Experiments..................................................................................................... 24

Palatability Experiment ............................................................................................................... 26

Freezing Tolerance Experiment .................................................................................................. 27

Discussion ........................................................................................................................................... 27

v

Herbivory Differences among Populations ................................................................................. 28

Performance Differences among Populations ............................................................................. 31

Resistance Differences among Populations ................................................................................ 32

Figures ................................................................................................................................................ 36

References .......................................................................................................................................... 52

Chapter 3: Differences in herbivore damage to Arctium minus in open and forest habitat ............... 57

Introduction ....................................................................................................................................... 58

Methods .............................................................................................................................................. 62

Study Species ............................................................................................................................... 62

Study Sites .................................................................................................................................... 63

Statistical Analysis ....................................................................................................................... 64

Results ................................................................................................................................................. 65

Discussion ........................................................................................................................................... 66

Tables .................................................................................................................................................. 71

Figures ................................................................................................................................................ 76

References .......................................................................................................................................... 84

Chapter 4: General Conclusion ................................................................................................................ 89

Does latitudinal variation in attack reflect underlying variation in resistance to herbivores? .. 90

Do local differences in environmental conditions affect herbivory? ............................................. 91

Implications ........................................................................................................................................ 91

Future Work ...................................................................................................................................... 93

References .......................................................................................................................................... 95

Appendix 2-I: Distribution of Data .......................................................................................................... 97

Appendix 2-II: Unequal Variance of Data Test .................................................................................... 103

vi

List of Tables

Table 3-1A: Analysis of variance of the leaf damage ................................................................................. 71

Table 3-1B: Analysis of variance of the average number of serpentine miners .......................................... 71

Table 3-1C: Analysis of variance of the average number of blotch miners ................................................ 71

Table 3-2A: Analysis of variance of the number of M. lapella larvae in a capitulum ................................ 72

Table 3-2B: Analysis of variance of the proportion of damaged seeds per capitulum ................................ 72

Table 3-3A: Analysis of variance of the average height ............................................................................. 73

Table 3-3B: Analysis of variance of the average circumference ................................................................. 73

Table 3-4A: Analysis of variance of the number capitulae per plant .......................................................... 74

Table 3-4B: Analysis of variance of the number of seeds per capitulum .................................................... 74

Table 3-5: Analysis of variance of the trichome density ............................................................................. 75

vii

List of Figures

Chapter 2

Figure 2-1: Map of source populations........................................................................................................ 36

Figure 2-2: Chi-square analysis of survival rate vs. population .................................................................. 37

Figure 2-3A: ANCOVA model, % leaf area damage vs. latitude in August 2011 ...................................... 38

Figure 2-3B: ANCOVA model, % leaf area damage vs. latitude in August 2012 .................................... 38

Figure 2-3C: ANCOVA model, number of larvae per capitulum vs. latitude ............................................. 38

Figure 2-4: ANCOVA model, height vs. latitude ........................................................................................ 39

Figure 2-5: ANCOVA model, stem circumference vs. latitude .................................................................. 40

Figure 2-6: Regression model, survival rate during summer vs. latitude .................................................... 41

Figure 2-7A: ANCOVA model, total capitulum mass vs. latitude .............................................................. 42

Figure 2-7B: ANCOVA model, total number of seedheads per individual vs. latitude .............................. 42

Figure 2-7C: ANCOVA model, individual capitulum mass vs. latitude ..................................................... 42

Figure 2-7D: ANCOVA model, number of seeds per capitulum vs. latitude .............................................. 42

Figure 2-8: ANOVA model, leaf toughness vs. latitude ............................................................................. 43

Figure 2-9: ANCOVA model, leaf trichome density vs. latitude ................................................................ 44

Figure 2-10A: Palatability test (T. ni) ANOVA model: percent leaf area removal vs. population ............. 45

Figure 2-10B: Palatability test (T. ni) ANOVA model: leaf weight change vs. population ....................... 45

Figure 2-11: Palatability test (T. ni) ANOVA model: herbivore weight change vs. population ................. 46

Figure 2-12A: Palatability test (C. nemoralis) ANOVA model:percent leaf area removal vs. population 47

Figure 2-12B: Palatability test (C. nemoralis) ANOVA model:leaf weight change vs. population ............ 47

Figure 2-13: Palatability test (C. nemoralis) ANOVA model: herbivore weight change vs. population .... 48

Figure 2-14: Palatability test (C. nemoralis) ANOVA model: trichome density vs. population ............... 49

Figure 2-15: Palatability test (C. nemoralis) ANOVA model: weight change vs. population (control) ..... 50

Figure 2-16: M. lapella freezing test ANOVA model: survival rate vs. temperature ................................ 51

Chapter 3

Figure 3-1: Burdock sample location map .................................................................................................. 76

viii

Figure 3-2: ANOVA model: percent leaf area damage vs. habitat .............................................................. 77

Figure 3-3: ANOVA model: number of miners per leaf vs. habitat ............................................................ 78

Figure 3-4: ANOVA model: number of larvae per capitulum vs. habitat ................................................... 79

Figure 3-5: ANOVA model: height vs. habitat ........................................................................................... 80

Figure 3-6: ANOVA model: circumference vs. habitat............................................................................... 81

Figure 3-7: ANOVA model: number of capitulae per plant vs. habitat ...................................................... 82

Figure 3-8: ANOVA model: trichome density vs. habitat ........................................................................... 83

ix

List of Appendices

Chapter 2

Appendix 2-I: Distribution of Data ............................................................................................................. 97

Appendix 2-II: Unequal Varariance of Data Test ..................................................................................... 103

1

Chapter 1: General Introduction

Every species has the potential to adapt to changes in its physical environment. For example,

Jensen et al. (2008) have shown that populations of brown trout have adapted to increasing water

temperature by having faster spawning time, faster growth rate, and greater offspring size. As a

result, populations at different water temperatures differed from each other in terms of these

traits. Similar results have been found in Daphnia magna which had greater thermal plasticity in

response to increased temperature (Doorslaer et al. 2009). Adaptations to the environment can

also be found in plants as well. For examples, leaves with sinuses have different shapes

depending on their locations on the tree (Dale 1992; de Casa et al. 2011): leaves that grow at the

shaded interior of the tree have greater surface area with smaller sinus areas than those at the

outer layer. This allows more sunlight to penetrate through outer layer of leaves and greater

photosynthesis by inner leaves with greater surface area. Therefore, such phenotypic plasticity is

beneficial to many trees.

Species also can adapt to other interacting species. Plants, for instance, can adapt to reduce

damage from herbivores. Many plants, such as milkweeds, have evolved the ability to produce

toxin to deter herbivores, while others produce physical defenses including trichomes or thorns

(Agrawal et al. 2012; Ronel and Lev-Yadun 2012). Conversely, some species have evolved high

tolerance to herbivory that allows them to withstand damage and survive (McNutt et al. 2012).

2

Such adaptation can also be important to a species moving into new habitat with unfamiliar

physical or biotic environments resulting in population differentiation; as a result, multiple

populations of a species with different characteristics can co-occur at the same time. This can be

seen in intercontinental introductions of a species, intracontinental populations of a species

within its range, and local populations in different habitats (Keane and Crawley 2002; Bruelheide

and Scheidel 1999; Bryant et al. 1983).

Intercontinental Variation - Invasive Species

As exotic species invade new habitats, they face new abiotic and biotic environments.

Populations in invaded areas therefore may diverge from their source populations as different

traits are selected in invaded areas. There may be common traits among invasive species that

give them a competitive edge in their new community (Daehler 1998), such as fast growth,

phenotypic plasticity, and tolerance to a wide range of environments. These traits may be

selectively favoured in invaded habitats, resulting in a differentiation from source populations.

For instance, Joshi and Tielborger (2012) have looked at the difference between the purple

loosestrife in North America (invaded range) and Europe (native range) and found that this plant

had greater growth rates. This indicates that this plant may have experienced different selective

regimes in its new habitat than in native habitats. This example is consistent with the Enemy

Release Hypothesis (ERH), which states that invasive species have higher fitness than native

species because they have lost specialized herbivores during migration (Keane and Crawley

2002), potentially resulting in faster growth than native species. After being released from

natural enemies, they can also become more competitive by investing less on defense and more

3

on reproduction and growth, as suggested by the Evolution of Increased Competitive Ability

(Blossey and Nötzold 1995; Maron et al. 2004). For example, Zou et al. (2008) have shown that

Sapium sebiferum has a greater growth rate in invaded habitats compare to their native habitats.

Increases in growth and reduced herbivory may be frequent in in introduced populations: 20 of

30 studies showed greater growth rate and 12 of out 22 showed lower resistance against

herbivores (Bossdorf et al. 2005).

Intracontinental Variation - Differences in Marginal Populations

Adaptation to novel conditions may result in differentiation of populations near a species’ range

limits. A stable range limit is a boundary beyond which a species cannot survive, often due to

unfavourable conditions (e.g. climate, resource availability, soil conditions, herbivory etc.)

(Kawecki 2008) Many studies have found differences in species traits or fitness between

populations at their range limit vs. more central habitats (Kawecki 2008). For instance, if range

limits are set by less favourable environments, marginal populations may not perform very well

compared to the central populations. Sexton et al. (2009) showed that 72% of studies have

observed fitness and abundance reductions when plants were transplanted near their range

margins.

However, there are also cases where populations at range margins perform better than central

populations. For instance, Garcia et al. (2010) showed that populations of lady slipper orchid

(Cypripedium calceolus) had greater numbers of individuals at the range limits than it in more

central populations. Such an improvement in performance may reflect geographic trends in

4

interactions with natural enemies. For instance, a study showed that, as brown argus butterflies

(Aricia agestis) expanded northward, they were less susceptible to parasitism and higher chance

of survival even when associated parasitoid species already existed in the new habitat (Menéndez

et al. 2008). These examples show that species can survive better in a seemingly unfavourable

environment as a result of differences in predation in range limits compared to central habitats.

One of the biological factors that may influence the range limit of plant species is herbivore

damage. For instance, if herbivores are less abundant at range margins, marginal populations

may gain a fitness benefit relative to more central populations. As an example, a study looked at

eastern woodland sedge (Carex blanda): sedge populations at the range margin were free from

specialized diseases and seed predators, allowing greater fitness (Alexander et al. 2007), and had

greater plant size and seed production (Alexander et al. 2007). Conversely, herbivory may help

to set range limits in the first place. A study of the altitudinal range limits of Arnica montana in

the Harz mountains and Lower Saxony indicated that this plant is less abundant at higher

elevations (Bruelheide and Scheidel 1999) because of greater slug herbivory.

Local Variation - Population Differences among Microhabitats

Studies also have suggested that populations may differ at a finer scale in response to different

microenvironments. For example, nutrient availability may play a significant role in shaping

defence traits. Coley et al. (1985) suggested that species would invest more in herbivore defense

in a nutrient-poor environment: when nutrients are scarce, it is very costly and difficult for

species to replace its lost tissues; therefore, plants should protect themselves in order to reduce

5

herbivore damage. Pearse and Hipp (2012) found such a pattern in oak (Quercus spp.): these

have more defenses in more stressful environments, including lower nutrient environments. In

contrast, populations may not have enough resources to produce adequate defences in areas with

low nutrients. Salgado-Luarte and Gianoli (2010) found that Chilean firetree (Embothrium

coccineum) seedlings from sunny areas had greater leaf thickness, which made the leaf less

palatable to herbivores. Denslow et al. (1990) study showed that the relationship between

phenolic content and the light availability differed depending on the shade tolerance of the

species considered: while shade tolerant species such as Miconia gracilis showed the greatest

phenolic concentration in low light exposure, the contrary was true for shade intolerant species,

such as Miconia barbinervis and Miconia nervosa (Denslow et al. 1990). For instance, Denslow

et al. (1990) showed greater herbivore damage to M. barbinervis in understory areas, which he

attributed to a lower phenolic concentrations in shaded populations. Plants-pathogen interactions

also may be influenced by resource supply (Schoeneweiss 1981); for example, Australian

Eucalyptus spp. are more susceptible to the root pathogen Phytopthora cinnamomi in habitats

with low nutrients and poor soil structure (Burdon and Shattock 1980; Weste 1986).

Local variation in resource availability also may influence the types of defenses that are

expressed. The Carbon-Nutrient Balance Hypothesis suggests that plants produce carbon-based

defenses under nutrient-limited conditions, such as low-nitrogen soils, and nitrogen-based

defences in carbon-limited environments, such as shaded areas (Bryant et al. 1983). Therefore,

herbivore damage to plants may depend on the type of defenses produced, which depends on the

environment. A meta-analysis indicated that woody and herbaceous plants developed different

types of chemical defenses in response to their limiting resources (Masaad et al. 2011). Due to

6

such effects of the environment on plant defences, a difference in herbivore damage may locally

exist between adjacent shady and open habitats.

Conversely, herbivores may favour plants in shade in order to hide from predators, access

moisture, and/or avoid heat from the sun (Van Valen 1973; Maiorana 1973; DeWalt et al. 2004;

Mantyla et al. 2008).Therefore, open and understory habitats may differ in herbivore damage due

to a different abundance of natural enemies instead of difference in plants. One study compared

the herbivore damage to Clidemia hirta between open and understory habitats in both native

(Costa Rican) and invasive populations (Hawaiian) (DeWalt et al. 2004). Greater herbivore

damage occurred in the understory than open habitats in Clidemia hirta’s native regions, but not

in invaded areas (DeWalt et al. 2004). This suggests that, while a greater abundance of natural

enemies is causing the absence of this species in native understory, loss of natural enemies has

facilitated the invasion of C. hirta into Hawaiian forests (DeWalt et al. et al. 2004). Similarly,

infection by pathogens often is favoured by moisture and shade, so that plants can be more

susceptible to pathogen attacks in the shady understory than open areas (Augspurger 1983;

Augspurger and Kelly 1984). These examples suggest that damage by natural enemies can

locally vary between habitats due to an environmental difference, such as herbivore abundance,

or a plant difference, such as a phenolic concentration.

My Research

Most studies of spatial variation in herbivory have focused on natives, while studies of variation

in herbivory on invasive species usually have focussed on differences between their native and

7

invaded continents. Comparatively, few studies have looked latitudinal or regional habitat

variation in herbivory among invasive populations; lack of these comparisons may overlook the

finer consequences of invasions. For example, do invasive plant species exhibit uniform fitness

and adaptations to herbivores throughout their latitudinal range in invaded regions? Does attack

on invasives vary depending on characteristics of invaded sites? It could be the natural enemies

are lacking in some, but not all areas, or that defences of invaders vary in response to the local

environment. Therefore, whether invasive species benefit by escaping enemies, and the selective

pressures they face, may not have a simple answer, but may vary according to location.

The objective of my M.Sc. research is to study herbivory and associated morphological trait and

fitness differences among populations of an invasive species, common burdock (Arctium minus),

within its new range. Dasvinder Kambo (2012) previously, found considerable latitudinal

variation in herbivore attack on this plant; here, I investigate causes and consequences of this

latitudinal difference, and ask whether similar differences may occur among local habitats. My

main questions are (1) does latitudinal variation in attack reflect genotypic variation in resistance

to herbivores? and (2) do local differences in environmental conditions affect herbivory?

This thesis has been divided into two main sections: differences in herbivore damage and plant

fitness among plants from different populations (Chapter 2) and differences in herbivore damage

between open and forest habitats (Chapter 3). Finally, in Chapter 4, I summarize my main

conclusions and make suggestions for future work. Together, my results suggest that variation in

herbivore damage to burdock is driven by environmental factors rather than plant traits.

8

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Bruelheide, H. and U. Scheidel. 1999. Slug herbivory as a limiting factor for the geographical

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Bryant, J. P., F. S. Chapin III, D. R. Klein. 1983. Carbon/nutrient balance of boreal plants in

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Burdon, J. J. and R. C. Shattock. 1980. Disease in plant communities. Annals of Applied Biology

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10

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Kambo, D. (2012) Differences in performance and herbivory along a latitudinal gradient for

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13

Chapter 2: Differences in herbivore damage and performance for

plants from a latitudinal range of populations

Abstract

Invasive plants do not always escape their herbivores in new regions; for instance, the Eurasian

biennial Arctium minus is attacked by a variety of native and introduced insects in its new North

American range. Previously, research has shown that damage by these herbivores strongly

decreases towards the northern range limit of this species. These population differences might

reflect a genetic cline in herbivore resistance and other plant traits, or environmental differences

such as varying herbivore abundance. To distinguish between these possibilities, herbivore

damage to leaves and seeds of A. minus was measured in a common garden experiment with

genotypes sampled from 11 populations from southern Ontario to near the northern range limit.

As well, a freezing tolerance experiment was performed with the important lepidopteran seed

predator Metzneria lapella, and palatability experiments were performed with two generalists,

the snail Cepaea nemoralis and the moth Trichoplusia ni. Results indicated that latitudinal

differences in herbivore damage cannot be explained by genotypic differences among plant

populations, but instead are likely to result from the loss of herbivores from colder sites. Whether

invasive populations of A. minus may benefit from enemy release therefore varies depending

upon the location of the population.

14

Introduction

A stable range limit is a boundary beyond which a species cannot survive, often due to

unfavourable conditions (e.g. climate, resource availability, soil conditions, herbivory etc.)

(Kawecki 2008). As species approach their range limits, the environment may increasingly

become unfavourable for them; as a result, the population size and fitness of a species may

decrease as it gets close to the range margin (Geber 2008). For instance, a study looking at the

fitness differences in purple loosestrife (Lythrum salicaria) along its latitudinal distribution

showed that, fitness was reduced as it approached to its range limit (Colautti et al. 2010). Such

patterns are common but not universal. For instance, Sexton et al. (2009) showed that 72% of

studies have observed fitness and abundance reductions when plants were transplanted near their

range margins.

One suggested reason for reduced fitness near range margins is the low genetic variation, which

reduces the likelihood of beneficial genes being present (Bridle and Vines 2006). Every dispersal

event of species would move only a subset of genotypes from the source population. Therefore,

when the species reaches its range limit, most of the genetic variation would be lost. As a result,

range margin populations may fail to adapt to their new environment. This is similar to the

genetic bottleneck effect or the founder effect, first outlined by Ernst Mayr (1963). Another

reason could be that continued gene flow from central populations, which hinders fixation of

locally adaptive genes (Kirkpatrick and Barton 1997). If range margin populations are not well-

isolated from source populations, locally maladaptive genotypes would consistently be dispersed

to the range margin habitat, hindering adaptation to the new environment.

15

Despite this evidence, there are also cases where populations at range margins perform better

than central populations. For instance, Garcia et al. (2010) showed that populations of lady

slipper orchid (Cypripedium calceolus) had greater numbers of individuals at the range limits

than its source populations. Such an improvement in performance may reflect geographic trends

in interactions with natural enemies. For instance, it could be that herbivores are less abundant at

range margins, so marginal populations are less damaged. A study on the altitudinal range limit

of Arnica montana at Harz mountains and Lower Saxony indicated that the lower abundance of

A. montana at higher elevation is caused by greater abundance of herbivores (Bruelheide and

Scheidel 1999). Another study looked at eastern woodland sedge (Carex blanda) specimens and

found similar patterns as well (Alexander et al. 2007). They found that the sedge populations at

the range margin were free from specialized diseases and seed predators, and had

correspondingly greater plant size and seed production (Alexander et al. 2007). The decoupling

between plants and herbivores could occur simply because herbivores may have different

climatic limits than their host species. For example, plant species may be more cold tolerant than

herbivores so that they can colonize colder northern sites. Alternatively, herbivores may face

geographical barriers, such as a forest or a body of water, which allow plants to disperse through

but prevent herbivores from catching up to plant dispersal (Case and Taper 2000). Herbivores

may also have difficult time finding new habitats of host species. Case and Taper (2000) stated

that, some plant species can spread as a patchwork of relatively small and isolated populations

instead as a long continuous network; as a result, herbivores may have difficult time locating

new marginal plant populations; therefore, insect herbivores may have difficult time tracking

their hosts.

16

Such a scenario is similar to what invasion biologists call the Enemy Release Hypothesis (ERH).

ERH states that invasive species have an advantage over native species because they have lost

specialized herbivores during migration (Keane and Crawley 2002; Mitchell and Power 2003).

The hypothesis was initially meant for large-scale intercontinental invasion of exotic species, but

studies on intracontinental invasions show that ERH can also be applied to smaller scale as well.

(Morriën 2010). For example, the Janzen-Connell hypothesis states that, because the parent

plants are surrounded by herbivores and pathogens, seedlings have greater fitness when they are

farther away from their parents (Connell 1971). This idea is similar to ERH in that: 1) there is a

source population (parent plants) and an invading population (seedlings moving away from

parents) and 2) the invading population experiences lower herbivory due to lower abundance of

herbivores. Escape of marginal populations may occupy an intermediate position between these

extremes. One potential difference between escape at range limits and other types of escape may

be that both specialists and generalists may be absent at range limits if they are unable to tolerate

the environment, whereas ERH and the Janzen Connell hypothesis primarily focuses on escape

from specialist herbivores.

Escape of marginal populations from enemies may have consequences for their defensive traits.

In the absence of natural enemies, a plant may respond by changing its defense level against

herbivores. According to Evolution of Increased Competitive Ability theory, an invading plant

may lose its resistance against herbivores in the absence of the natural enemies (Blossey and

Nötzold 1995; Maron et al. 2004). Similarly, if marginal populations are less attacked, they may

lose defences against herbivores. As well, because they were not exposed to high herbivory

pressure, high tolerance to herbivore damage may not have been selected in the population

17

unlike central populations with high herbivory. As a result, marginal populations may have

reduced tolerance compare to central populations.

Like native species, an invasive species might locally escape its enemies within its invaded range.

Invasive plants rarely escape all their enemies; instead, most invasive are subject to damage by

generalist enemies, and in some cases by specialists adapted to close relatives or co-introduced

with the invader (Hill and Kotanen 2012). After its initial colonization into non-indigenous

habitat, an invasive species will expand its range until it reaches its new range limits; its enemies

may or may not expand with it. As a result, within its introduced range, invasive populations

may face a significantly different environment and herbivory in different areas. Dasvinder

Kambo (2012) has surveyed leaf damage, seed damage, and reproductive output of the common

burdock (Arctium minus), an invasive species in North America, in more than 100 different

Ontario locations along latitudes ranging from 44.02°N to 49.04°N. He found that burdock has

greater damage by several different types of folivores and seed predators in southern central

populations, and the damage declines as it approaches northern habitats. Moreover, northern

populations have greater reproductive output than southern populations, and thus have higher

fitness. This within-range variation may be as large as the enemy-release experienced by many

new invaders from different continents.

Although many studies have shown the large-scale intercontinental population differences due to

different herbivory pressure, as explained by ERH and EICA, intracontinental examples of

variation in damage to invasive species are uncommon. Moreover, there are very few studies that

explain the cause and the consequence of spatial differences in herbivory on invaders. For

18

example, do the latitudinal trends in damage to burdock result from differences in herbivore

populations, or in the plants themselves? Understanding such local population differences along

the range can allow better understanding of characteristics and dynamics of invasive species

populations and may assist in predictions invasive species movements.

Here, I address the following questions: 1) do invasive populations of common burdock have

varying herbivore damage and fitness because of environmental factors or because of genetic

differences? 2) do differences in performance among populations have a genetic basis? And 3)

do populations have genetically different level of defenses? To answer the question, I performed

a common garden experiment with Arctium minus derived from 11 different populations from a

650km latitudinal gradient. I also performed a palatability experiment with 3 different

populations of burdock from this latitudinal gradient, and a seed predator freezing tolerance

experiment to supplement the common garden experiment. My results suggest that differences in

herbivore abundance likely cause the documented latitudinal herbivore damage gradient in

burdock populations.

Methods

Study Species

Common Burdock (Arctium minus (Hill) Bernhardi) (Asteraceae) is a Eurasian biennial that is

now found from the southern states of the United States to every province of Canada (Gross et al.

1980). It is self-compatible and considered an invasive species in many part of North America

and competitively more dominant than many native species (Gross et al. 1980).

19

Burdock produces 10-75 seeds in a capitulum (Kambo 2012). The outer layer of this capitulum is

covered in hooks which allow it to stick to animals and disperse its seeds (Gross et al. 1980). In

its first year, burdock grows to approximately 35-50cm height as a rosette. It then, overwinters,

grows to adult form, with height ranging from 50-150cm (Kambo 2012) and produces seeds

during summer.

Common burdock in Ontario is attacked by variety of species, ranging from snails to insects and

generalists to specialists. It gets attacked by wide range of miners in both Europe (native) and

North America (invasive) such as Phytomyza lappae Goureau (serpentine miner) and Calcomyza

flavinotum Frick (blotch miner). It also get attacked by many chewing folivores such as keeler

grasshoppers (Melanoplus keeleri (Thomas)), Meadow froghoppers (Philaenus spumarius

(Linnaeus)), and grove snails (Cepaea nemoralis Linnaeus). Finally, it is attacked by seed

predators such as burdock seedhead moths (Metzneria lapella (Linnaeus)), and tephritid flies

(Tephritis bardanae (Schrank) and Cerajocera tussilaginis (Fabricius)); both lay eggs in the

capitulum and larvae eat the seeds within.

Common Garden Transplant Experiment

Source of Samples: In summer of 2010, capitulae of five randomly chosen burdock plants were

collected from each 11 different location that Kambo (2012) surveyed for his research, along a

650km transect in Ontario (from Newmarket to Cochrane) (Figure 2-1). During May 2011, seeds

from these capitulae were germinated and grown in a greenhouse until they became seedlings,

approximately 10cm tall.

20

Common Garden Design: A common garden experiment was established at the Koffler Scientific

Reserve near Newmarket, Ontario (44.03°N, 79.55°W) during June 2011. This plot was

approximately 20mx20m and was plowed to remove all plant biomass. 20 seedlings from 11

locations were then planted in a grid 1m apart (total N= 220). To reduce transplant shock,

seedlings were watered for 2-3 days after planting. The plot was revisited in August 2011 to

observe preliminary herbivore damage for each population, then in May and August 2012 to

observe the overwinter survival, herbivory, and reproduction, and other data collection. Due to

an extremely hot summer during 2012 (Environment Canada 2012), many individuals withered

and were not available for leaf sampling, such as measurement of herbivore damage and physical

resistance measurements; therefore, the total individuals available for those measurements varied

among populations, ranging from 16 to 3 individuals.

Herbivore Damage: Percent hole area damage caused by folivores was measured on a basal leaf,

the fifth, and tenth leaves by comparing the area of the leaf removed with leaf models with 1%,

2%, and 5% area removed. Lastly, 10 capitulae were chosen to count the number of seedhead

moth larvae per capitulum for each population.

Performance: Survival, height, stem circumference, total seed mass, and the total number of

seeds per individual were measured. One hundred seedheads were subsampled to measure the

average mass of a seedhead for each individual. Lastly, 10 seedheads were randomly selected per

individual (10 seedheads; 220 individuals; Total N=2200) and the average number of seeds per

capitulum was measured.

21

Physical Herbivory Resistance: Trichome densities and toughness were measured on three leaves

of each individual. Trichome densities were measured by counting trichomes along 0.7mm of the

leaf vein. Toughness of leaves was measured using a penetrometer (3mm in diameter; Chatillon);

this tool that punctures a leaf and measures the amount of force, in grams, required to do so.

Palatability Experiment

Herbivore Samples: Cabbage looper, Trichoplusia ni (Hübner) (Noctuidae), was bought from the

Canadian Forest Service (http://insect.glfc.forestry.ca/cart-panier/insects-insectes.cfm?lang=eng)

and the grove snail, Cepaea nemoralis, was caught in the wild for use in a herbivory experiment.

T. ni is a generalist herbivore that is also considered major pest in North America, feeding on

agricultural crops such as celery, lettuce, and spinach. C. nemoralis is also a common generalist

herbivore that was introduced to North America from Europe (Ozgo and Schilthuizen 2012).

Prior to the experiment, caterpillars and snails were exposed to burdock leaves for at least three

days in order to familiarize them with this species. They were then starved for one day and

weighed on the day before the experiment started.

Arctium minus leaf disks: Seeds from Newmarket (44.03°N, 79.55°W), Huntsville (45.33°N,

79.22°W), and Cochrane (49.04°N, 80.59°W) were germinated in the greenhouse in March 2012.

For the T. ni palatability experiment, two leaves from 10 individuals from each of 3 populations

were sub-sampled to make leaf disks 5cm in diameter (Newmarket, Huntsville, and Cochrane: a

total of 30 individuals and 60 leaf disks). The weight of each leaf disk was measured before it

22

was presented to herbivores. For the experiment with C. nemoralis, 60 leaf disks were made

from 20 individuals from each of the same population (20 individual leaf disks per population).

Experimental Design: One herbivore was randomly assigned to each of the 60 petri dishes with

leaf disks. Petri dishes with T. ni or C. nemoralis were then placed in the germination chamber

with 90% humidity a under 14:10 hour cycle of 25°C -light and 10°C-no light. Twenty extra leaf

disks from three populations were placed in the germination chamber without any herbivores as a

control to observe any differences in rate of water loss among populations. T. ni and C.

nemoralis were removed from the petri dish after three days and one week, respectively. At that

point, weight of leaf disks and herbivores were measured, and the percent leaf area damage on

leaf disks were measured by scanning and analyzing with ImageJ software

(http://rsb.info.nih.gov/ij/). For the T. ni experiment, since an individual provided two leaf disks,

data from leaf disks that were from the same burdock individual were averaged prior to statistical

analyses. For the grove snail experiment, data were not averaged because each leaf disk was

from separate individual as mentioned previously.

Freezing Tolerance Experiment

20 capitulae from each 20 individuals were sampled in Newmarket (total N=400 seeds) during

January 2012. They were divided evenly into four different treatment groups: -44°C, -34°C, -

29°C, and -24°C. These temperatures were chosen based on the coldest temperature recorded at

Cochrane, Barrie, and Newmarket during January 2011 (Environment Canada 2012). These

groups were then placed in freezers with their respective temperature for two days. Then, these

23

capitulae were opened, and mortality of Metzneria lapella from each treatment group was

measured. For those that were alive, the weight of each larva was also measured.

Statistical Analysis

For the common garden experiment, data were analyzed with ANCOVA using an REML

approach with the latitude as a variable and the population as a random effect (Crawley 2007).

Proportions, such as percent leaf area damage, were arcsine-transformed before analysis, using

following equation: y= 2*arcsine (√(x/100)), where x is the original value; other data were not

transformed. The transformed leaf area damage, performance, and physical resistance traits also

were compared among populations using one-way ANOVA to check any population effects.

Also, a chi-square and a linear regression were done to investigate differences in survival rate

during summer 2012 among populations.

For the palatability experiment, one-way ANOVAs with Tukey HSD were used to compare

transformed leaf area removal, leaf weight change, and herbivore weight change among the three

plant populations.

Finally, a chi-square test was done to investigate differences in mortality of larvae among

different treatments from freezing tolerance experiment. Herbivore weights were compared using

one-way ANOVA and Tukey-Kramer HSD analysis.

24

All analyses were performed with JMP 10 (SAS Institute Inc.).

Results

Common Garden Experiments

At the initial sampling in August 2011, there was no significant difference in herbivore hole

damage with latitude (ANCOVA: R2

= 0.0546, F1, 213 = 0.215, p-value = 0.654) (Figure 2-3A). In

May 2012, 163 individuals had survived after winter. A chi-square test indicates that there were

almost significant differences in terms of over-winter survival among populations with mid

populations, such as Huntsville (10), having high mortality rate and southern and northern

populations, such as Newmarket (4) and Timmins (3) respectively, having low mortality rate (X2

= 16.575, df = 10, p-value = 0.0840) (Figure 2-2). At minimum, 10 living individuals still were

available for sampling per population.

While some data from August 2012, such as leaf trichome or average seed mass data showed

normal residual distributions, height, leaf toughness, herbivore damage, total seed mass, and total

number of seedheads did not (Appendix 2-I). Also, total seed mass, total number of seedheads,

average capitulum mass, and number of seeds per capitulum data had unequal variance; therefore,

these data should be interpreted with caution (Appendix 2-II).

The ANCOVA of herbivore damage data of 2012 did not show any significant difference with

latitude (ANCOVA: R2 = -0.0649, F1, 124 = 1.876, p-value = 0.210) (Figure 2-3B). However, the

25

one-way ANOVA model of leaf area damage showed significant difference among the 11

populations sampled, with the most damage on the Manitoulin Island populations and the least

damage on the Cochrane population (ANOVA: F10, 367 = 2.738, p-value = 0.00290). There was no

significant difference in terms of number of larvae in capitulae for both ANCOVA and one-way

ANOVA as well (ANCOVA: R2

= 0.00460, F1, 124 = 1.876, p-value = 0.672; ANOVA: F10, 1539 =

0.895, p-value = 0.537) (Figure 2-3C).

Out of 163 individuals, 8 were still in rosette form at the end of the experiment in 2012, of which

half were from the Newmarket population. According to ANCOVA analyses, there were not any

significant differences in the height (ANCOVA: R2

= 0.0757, F1, 161 = 0.103, p-value = 0.756)

(Figure 2-4), and the stem circumference along the latitude (ANCOVA: R2

= -0.00535, F1, 161 =

0.301, p-value = 0.597) (Figure 2-5). Finally, mortality due to hot-dry weather significantly

differed among populations, with least mortality from Newmarket populations with (0%) and the

most from Cochrane (77%) (chi-square: X2

= 35.639, df = 10, p-value < 0.001) (Figure 2-2).

When percent mortality was regressed along latitude, there was a significant negative result

(linear regression: F1, 9 = 17.145, p-value = 0.00252, R2

= 0.656) (Figure 2-6).

There was no significant difference in total seed mass with latitude (ANCOVA: R2

= 0.0977, F1,

1548 = 0.0100, p-value = 0.922) (Figure 2-7A). Both the total number of seedheads (ANCOVA:

R2

= 0.142, F1, 1548 = 1.0227, p-value = 0.338) and the average mass of a capitulum (ANCOVA:

R2

= 0.317, F1, 1548 = 4.238, p-value = 0.0697) were not significantly differed with latitude as well

(Figure 2-7B, 2-7C). Finally, there was no significant difference in number of seeds per

26

capitulum (ANCOVA: R2

= 0.0888, F1, 1548 = 0.879, p-value = 0.373) (Figure 2-7D). There were

also no significant difference in total seed mass among population according one-way ANOVA

as well (ANOVA: F10, 152 = 1.807, p-value = 0.0639). As previously mentioned, seed data

showed unequal variance among populations; therefore, data should be interpreted with caution

(Appendix 2-II).

During 2012, Leaf toughness (ANCOVA: R2

= 0.0574, F1, 124 = 0.445, p-value = 0.521) and

trichome densities (ANCOVA: R2

= 0.108, F1, 124 = 0.00890, p-value = 0.927) were not

significantly different with latitude (Figure 2-8, 2-9). However, there were significant differences

in trichome densities among populations (ANOVA: F10, 367 = 6.808, p-value < 0.000100).

Palatability Experiment

For both the T. ni and C. nemoralis palatability experiments, there were no significant

differences in percent area removal (T. ni: F2, 25 = 2.153, p-value = 0.137; C. nemoralis: F2, 44 =

1.408, p-value = 0.255) or leaf weight changes among populations (T. ni: : F2, 25 = 0.957, p-value

= 0.398; C. nemoralis: F2, 44 = 2.399, p-value = 0.103) (Figure 2-10A, B, 2-12A, B). However,

there were significant differences in herbivore weight change for both experiments (T. ni: F2, 25 =

6.274, p-value = 0.00620; C. nemoralis: F2, 44 = 3.839, 0.0290) (Figure 2-11, 2-14). T. ni and C.

nemoralis gained weight the most when they fed on leaf disks from Cochrane and the least when

they were exposed to Newmarket leaf disks (Figure 2-11, 2-14). While Tukey-Kramer HSD

analysis on T. ni palatability experiment showed significant difference between Newmarket and

other populations, The C. nemoralis palatability experiment data showed a significant difference

27

between Cochrane and other populations. For the C. nemoralis palatability experiment,

Newmarket and Huntsville leaves had significant higher number of trichomes per 0.7cm of leaf

vein (F2, 56 = 6.626, p-value=0.00260) when Tukey-Kramer HSD was performed (Figure 2-13).

The control leaf disks showed that there was not any significant difference in leaf weight loss

due to water loss among populations (F2, 27 = 0.301, p-value = 0.743) (Figure 2-15).

Freezing Tolerance Experiment

There were totals of 82, 104, 109, and 114 M. lapella larvae found in the -24, -29, -34, and -44

degree Celsius treatment groups. A significantly greater number of larvae was alive when they

experienced -24 (93.90%) and -29 degree Celsius (88.46%) than -34 degree Celsius (28.44%)

and -44 degree Celsius (34.21%), which are equivalent to the winter temperature of Newmarket,

Barrie, and Cochrane (last two temperature), respectively (X2

= 149.793, df = 3, p-value < 0.01)

(Figure 2-16). The average mass of a larva was not significantly different among treatments (F3,

405 = 0.995, p-value = 0.395).

Discussion

After an exotic species invades a new habitat, it expands and colonizes further until it reaches its

new range limits (Geber 2008). Ecology of populations may differ across this new range; for

example, natural enemies may be scarcer in marginal sites (Bruelheide and Scheidel 1999;

Alexander et al. 2007; Menéndez et al. 2008). Previous work on Arctium minus showed an

increase in herbivory and reduced performance in southern populations compared to northern

28

range limit populations (Kambo 2012). Do these populations experience different herbivory and

fitness due to their genetic differences from each other, or due to environmental differences

among habitats, such as reduced herbivore populations? My results indicate that, although there

are evidences of population differentiation, environmental differences are more likely to be

causing the herbivory differences.

Herbivory Differences among Populations

The overall herbivore damage in the common garden was about 50% lower than the amount that

was observed by Kambo in the wild populations (2012). This may be due to seasonal fluctuations

in herbivore abundance. As well, herbivores may not yet have located and fully colonized the

common garden plants even by the end of this experiment.

Preliminary sampling of the common garden during August 2011 showed only non-significant

differences in leaf damage among populations. This is expected because the burdock was

transplanted late in the season. As a result, it had had short period of time to accumulate enough

herbivore damage to show significant differences among populations. As well, greenhouse-

generated plants may not reflect field conditions; for instance, they may be less defended or more

palatable than wild plants.

By August 2012, the common garden experiment did not show any significant difference in leaf

damage among populations, even though previous work by Kambo (2012) suggested that there is

29

decline in herbivore damage northward. Moreover, the common garden showed no difference in

the number of burdock seedhead moth larvae among populations, while Kambo (2012) found

otherwise in wild populations. This suggests that the latitudinal gradient of herbivore damage is

likely not due to genetic differences among populations, but rather external environmental

differences. Such a difference may be simply the abundance of herbivores: burdock-feeding

herbivores may be less abundant in northern areas, reducing the total herbivory on the population.

This result is consistent with within-region ERH (Keane and Crawley 2002): as A. minus

expanded northward, it may have lost its major herbivores and experienced less herbivory. There

are two possible mechanisms that may have caused this decoupling. First, herbivores might have

not been able to locate northern populations of host species as they expanded their ranges (Case

and Taper 2000). This may be true for leaf miners such as Liriomyza arctii and Calycomyza

favinotum (Gross et al. 1980). Specimens at the Royal Ontario Museum herbarium (2012)

showed that there are approximately 60 year gap between the earliest record of burdock (1877)

and that of L. arctii (1940) and C. flavinotum (1935). This hints that there may have been a

significant period of time when burdock was able to expand northward in the absence of leaf

miners; this may still be the case at northern sites. Second, herbivores might be scarcer in

northern areas because the climate is not favourable for them (Case and Taper 2000). This

mechanism may be true for seed herbivory. The freezing tolerance experiment for M. lapella

showed that moth larvae from Newmarket were not able to tolerate the cold temperatures of

northern habitats. Therefore, the major seed-feeding herbivores of A. minus may not be able to

expand northward as much as the burdock populations did. This may have caused the reduction

in seed predation in range margins, which was documented in previous work (Kambo 2012).

30

However, the result may also have been caused by the acclimatization of the insect to the local

climate.

The history of the northward expansion of common burdock may influence herbivory as well.

For example, if burdock has recently colonized northern Ontario, local herbivores may not

recognize it as a potential food source; as a result, the plant may be less attacked. However, A.

minus was introduced to North America by 1638 (Gross et al. 1980), while the earliest record of

the burdock in Royal Ontario Museum (2012) is 1877. Finally, burdock disperses seeds by

sticking onto animals (epizoochory) (Sorensen 1986). This would allow seeds to travel for long

distances; in fact, evidence shows that long-distance dispersal is most frequent in species with

epizoochory (Sorensen 1986). Therefore, it is likely that A. minus have been present in northern

Ontario for a long time, and that herbivores are familiarized with the plant.

Even though there was no correlation between latitude of origin and herbivore damage in the

common garden, there were still significant differences in herbivore damage among populations.

This indicates that, although they do not follow a latitudinal gradient, genetic differences in

herbivore susceptibility are present among populations; however, it is not certain what traits are

involved. There was a significant difference in the trichome density among populations

according to one-way ANOVA, but it did not correspond to the herbivore damage (i.e.

populations with greater trichome densities did not have lower damage). Other differences, such

as chemical differences, may be responsible for the observed patterns.

31

The genetic divergence of populations may reflect the colonizing ability of burdock. As

previously mentioned, burdock seeds can travel for long distances by epizoochory (Sorensen

1986); therefore, burdock often may disperse sufficient distances to be isolated from their source

populations. This also may result in a founder effect in newly established populations,

contributing to divergence between populations (Mayr 1963). These factors may contribute to

the genetic distinctiveness of many burdock populations.

Performance Differences among Populations

The common garden showed non-significant differences in every seed measurement among

plants from different populations, while Kambo (2012) showed greater seed production in wild

northern populations. This suggests that the latitudinal gradient in seed production is also likely

due to environmental differences. Such difference may be outcome of the herbivore abundance

difference along the latitude as explained previously. Experiencing lower folivore damage may

have allowed northern populations greater photosynthesis and, in turn, reproduction. Reduction

in seed predator abundance may have further increased the fitness of the northern population as

well.

Increased in seed production may be due to climate differences over the latitude. Reinartz (1984)

showed that biennial plants do not necessarily finish their two-stage life cycle within two years.

Plants may spend two years or more in rosette form if environmental conditions are not

favourable. He further stated that when common mullein (Verbascum thapsus) remained as a

rosette for an extra year, it produced five times more seeds (Reinartz 1984). Similarly, it might

32

be possible that northern populations also spend longer time in the rosette form than southern

populations due to unfavourable climates in the north, such as colder temperature; as a result,

they may produce greater seeds when they finally flower. However, preliminary observations

suggest this may not be the case.

Northern populations in the common garden had significantly more individuals that prematurely

withered. This suggests that northern genotypes are not as tolerant as southern genotypes to

extreme heat. Indeed, northern habitats are significantly cooler and less dry compared to

southern habitats (Environment Canada 2012); therefore, individuals in the north may not be

selected for heat tolerance. However, individuals in the south would be selected for heat

tolerance, especially recently due to rapid climate change and extremely hot summers

(Environment Canada 2012). As a result, northern populations may lack traits that help plants to

survive under hot weather.

Resistance Differences among Populations

The common garden experiment showed no latitudinal trend or population differences in leaf

toughness; whereas, reduced leaf toughness in northern populations was found in wild

populations in the previous work done by Kambo (2012). The conflicting results between the

common garden experiment and the work by Kambo suggests that physically resistance, such as

toughness and trichome density, may be plastic responses to herbivory. Many plants have

phenotypic plasticity in chemical and physical defenses: the level of herbivore resistance can

differ depending on the level of herbivory (Agrawal 2001). Similarly, greater herbivore damage

33

in southern habitats may have triggered plastic responses and greater physical defenses in

burdock.

Trichomes can be one of the defensive structures that plants may produce to protect themselves

from herbivores (Bossdorf 2005). The common garden experiment showed no latitudinal trend in

trichome density; however, there was a significant difference in the trichome density among

populations. This again may indicate genetic differences among populations but not a latitudinal

gradient of trichome density.

Nonetheless, leaf damage among common garden populations was similar despite the difference

in trichome densities, contrary to the expectations that individuals with greater number of

trichomes should have lower leaf damage. One explanation may be that trichomes simply do not

play major role in preventing herbivores from damaging leaves; instead, secondary chemicals or

other types of physical resistances may be the major source of resistance against herbivore. The

palatability experiment suggested that southern populations may be better defended chemically.

Herbivores grew less when they were exposed to plants from the Newmarket population

compared to northern populations even though they consumed a similar amount, these three

populations also showed similar herbivore damage also in the common garden experiment

(Figure 2-3B, 2-10A, and 2-12A). Therefore, even if the herbivores feed on burdock regardless

of the latitudinal origin, southern populations are less suitable as food. This suggests that there

may be differences in levels of secondary chemical resistance between genotypes. Northern

34

populations may have adapted to locally reduced herbivory pressure by investing less resource

on resistance, as predicted by EICA (Blossey and Nötzold 1995).

There are small significant differences, about 10-15 trichomes difference, among populations,

while no differences in leaf damage. Why is there such difference in trichome density among

populations? Trichomes of some species, such as Atriplex halimus, have functions other than

resistance against herbivores, such as water absorption and water loss reduction (Mozafar and

Goodin 1970). A study on lyre-leaved rock-cress (Arabidopsis lyrata) suggested that individuals

that had greater trichome productions also had greater tolerance to drought (Sletvold and Agren

2012). Similarly, a greenhouse study done by Gonzales et al. (2007) showed that Chilean

tarweed (Madia sativa) trichomes are equally induced by damage and drought. Trichomes of

Arctium minus may have a similar function as well. This idea may be supported by patterns of

mortality among populations. Many northern populations in the common garden had

significantly more individuals that prematurely withered. Because northern populations have

fewer trichomes, they might not be as tolerant to extreme heat. Indeed, northern habitats are

significantly cooler and less dry compare to southern habitats (Environment Canada 2012);

therefore, individuals in the north may not need as many trichomes to prevent water loss.

Overall, it seems that the latitudinal gradients in leaf and seed damage, found in previous work

on Arctium minus (Kambo 2012) are not due to genetic differences, but instead are likely to be

due to differences in herbivore abundance with latitude (Kambo 2012). Instead, clines in

physical herbivore resistance traits, such as toughness, may be plastic response to herbivores

35

Also, trichomes may not be the major defensive mechanism of common burdock; they instead

may have a function in reducing water loss in this species. The results also may suggest that

burdock populations have adapted to their local climates, but adaptation to local herbivore

populations is much less clear.

36

Figures

Figure 2-1: Locations from which seed populations were collected for the common garden

experiment (https://maps.google.ca). Locations include: Newmarket (44.02°N, 79.31°W),

Peterborough (44.18°N, 78.19°W), Barrie (44.24°N, 79.40°W), Bracebridge (45.02°N, 79.32°W),

Huntsville (45.19°N, 79.13°W), Parry Sound (45.20°N, 80.02°W), Manitoulin Island (45.42°N,

82.02°W), North Bay (46.19°N, 79.26°W), Kirkland (48.09°N, 80.02°W), Timmins (48.28°N,

81.09°W), and Cochrane: (49.04°N, 81.01°W)

Cochrane

Timmins Kirkland

Lake

North Bay

Huntsville

Peterborough

Newmarket

Parry Sound

Bracebridge

Barrie

Manitoulin

Island

37

Figure 2-2: Survival of Arctium minus plants after 20 seedlings were planted in the common

garden at the Koffler Scientific Reserve for each 11 populations in 2011. Populations organized

from most southern to most northern source location. There was an almost significant difference

among populations in percent of individuals that survived over the winter of 2011(p-value =

0.084). However, there was a significant difference in mortality among populations during

summer 2012 without including winter mortality (p-value = 0.00252).

16

12 10 11

9 12 12 13

9 11

3

0

2 5 1

1

3 5 2 10 6

10

4 6 5

8 10

5 3

5

1 3

7

0

5

10

15

20

25N

um

ber

of

Indiv

iduals

Population

Surviving Dead after Summer 2012 Absent after Winter 2011

38

Figure 2-3: Herbivore damage on common burdock sampled from different populations in the

common garden experiment. A: Average percent hole area damage on leaves (mean ± standard

error) during August 2011. An ANCOVA analysis indicates that there is no significant difference

with latitude (p-value = 0.654). B: Average percent area hole damage on leaves (mean ± standard

error) during August 2012. An ANCOVA analysis indicates that there is no significant difference

with latitude (p-value < 0.210). C: The average number of larvae occurring in a capitulum (mean

± standard error) during August 2012. An ANCOVA analysis indicates that there is no

significant difference with latitude (p-value = 0.672).

0

0.5

1

42 44 46 48 50

Tra

nsf

orm

ed L

eaf

Are

a

Dam

age (

%)

Latitude

Figure 2-3A

0

0.2

0.4

42 44 46 48 50

Tra

nsf

orm

ed L

eaf

Are

a

Dam

age (

%)

Latitude

Figure 2-3B

0

0.5

1

42 44 46 48 50

Num

ber

of

Larv

ae

Latitude

Figure 2-3C

39

Figure 2-4: The average height of a common burdock (mean ± standard error) sampled from

different populations in the common garden experiment. An ANCOVA analysis indicates that

there is no significant difference with latitude (p-value = 0.756).

0

20

40

60

80

100

120

43 44 45 46 47 48 49 50

Heig

ht

(cm

)

Latitude

40

Figure 2-5: The average stem circumference of a common burdock (mean ± standard error)

sampled form different populations in the common garden experiment. An ANCOVA indicates

that there is no significant difference with latitude (p-value = 0.597).

0

2

4

6

8

10

12

43 44 45 46 47 48 49 50

Circu

mfe

rence

(cm

)

Latitude

41

Figure 2-6: Linear regression between the proportion of individual surviving the summer in the

common garden and latitude. Results show a significant negative regression (p-value < 0.00252).

y = -10.205x + 541.71

R² = 0.6558

0

20

40

60

80

100

120

43 44 45 46 47 48 49 50

Pro

port

ion o

f Surv

ivin

g Indiv

iduals

Latitude

42

Figure 2-7: Performance of common burdock sampled from different populations in the common

garden experiment. A: Average total seedhead mass (mean ± standard error). An ANCOVA

analysis indicates that there is no significant difference with latitude (p-value= 0.922). B: The

average total number of seedheads per plant (mean ± standard error). An ANCOVA analysis

indicates that there is no significant difference with latitude (p-value= 0.338). C: The average

individual seedhead mass (mean ± standard error). An ANCOVA analysis indicates that there is

no significant difference with latitude (p-value< 0.0697). D: The average number of seeds per

capitulum (mean ± standard error). An ANCOVA indicates that there is no significant difference

with latitude (p-value < 0.373).

0

0.2

0.4

0.6

42 44 46 48 50

Tota

l Seedhead M

ass

(g)

Latitude

Figure 2-7A

0

10

20

30

40

42 44 46 48 50

Tota

l N

um

ber

of

Seedheads

Latitude

Figure 2-7B

0

0.2

0.4

0.6

42 44 46 48 50

Avera

ge S

eedhead M

ass

(g)

Latitude

Figure 2-7C

0

500

1000

42 44 46 48 50Tota

l N

um

ber

of

Seeds

per

Capitulu

m

Latitude

Figure 2-7D

43

Figure 2-8: The average toughness (penetration force) of a common burdock leaf (mean ±

standard error) sampled from different populations in the common garden. An ANOVA indicates

that there is no significant difference in average leaf toughness with latitude (p-value = 0.521).

0

5

10

15

20

25

30

35

40

45

43 44 45 46 47 48 49 50

Toughness

(g)

Latitude

44

Figure 2-9: The average number of trichomes along 0.7cm of main vein on the leaf (mean ±

standard error) sampled from different populations in the common garden experiment. An

ANCOVA analysis indicates that there is no significant difference in number of trichomes with

latitude (p-value < 0.927).

0

5

10

15

20

25

30

35

40

45

50

43 44 45 46 47 48 49 50

Num

ber

of

Trich

om

e p

er

0.7

cm

Latitude

45

Figure 2-10: Results of common burdock leaf palatability tests using Trichoplusia ni. Means are

shown ± standard error. A: Average percent area remaining after leaf disks were exposed to T. ni

for three days. There was no significant difference among populations (ANOVA: p-value =

0.137). B: Average leaf weight change after leaf disks were exposed to T. ni for three days.

There was no significant difference among populations (ANOVA: p-value = 0.398).

80

82

84

86

88

90

92

94

Newmarket Huntsville Cochrane

Perc

ent

leaf

are

a r

em

ain

ing

Population

Figure 2-10A

0

50

100

150

200

250

Newmarket Huntsville Cochrane

Leaf

weig

ht

change (

mg)

Population

Figure 2-10B

46

Figure 2-11: Trichoplusia ni weight change (mean ± standard error) after common burdock leaf

disks were exposed to T. ni for three days. There was a significant difference in weight gain

depending on the treatments (ANOVA: p-value = 0.00620). Different letters indicate significant

differences (p-value < 0.0500) according to Tukey-Kramer HSD analysis.

A

B B

0

5

10

15

20

Newmarket Huntsville Cochrane

Herb

ivore

weig

ht

change (

mg)

Population

47

Figure 2-12: Results of common burdock leaf palatability tests using Cepea nemoralis. Means

are shown ± standard error. A: Average percent area remaining after leaf disks were exposed to

C. nemoralis for three days. There was no significant difference among populations (ANOVA:

p-value = 0.255). B: Average leaf weight change after leaf disks were exposed to C. nemoralis

for three days. There were no significant differences among populations (ANOVA: p-value =

0.103).

0

20

40

60

80

100

Newmarket Huntsville Cochrane

Perc

ent

leaf

are

a r

em

ain

ing

Population

Figure 2-12A

0

0.1

0.2

0.3

0.4

Newmarket Huntsville Cochrane

Leaf

dis

k w

eig

ht

change

Population

Figure 2-12B

48

Figure 2-13: The number of trichomes along 0.7cm of a vein on the common burdock leaf from

palatability experiment using Cepea nemoralis (mean ± standard error). Populations are

organized from most southern to most northern location. There was a significant difference in the

number of trichomes among populations (ANOVA: p-value = 0.00260).

A A

B

0

5

10

15

20

25

30

35

40

Newmarket Huntsville Cochrane

Num

ber

of

tric

hom

es

per

0.7

cm

Populations

49

Figure 2-14: The weight change of Cepea nemoralis (mean ± standard error) after common

burdock leaf disks were exposed to C. nemoralis for three days. There was a significant

difference in weight gain depending on the population (ANOVA: p-value = 0.0290). Different

letters indicate significant differences according to Tukey-Kramer HSD analysis (p-value <

0.0500).

A

A B

B

-0.1

0

0.1

0.2

0.3

0.4

0.5

Newmarket Huntsville Cochrane

Herb

ivore

weig

ht

change

Leaf disk populations

50

Figure 2-15: The common burdock leaf weight change (mean ± standard error) due to water loss

during palatability experiments. An ANOVA showed that leaf disks from three populations have

similar water loss rates (p-value = 0.743).

0

50

100

150

200

250

300

350

Newmarket Control Huntsville Control Cochrane Control

Leaf

weig

ht

Diffe

rence

Leaf disk populations

51

Figure 2-16: The proportion of M. lapella individuals that died in four different temperature

treatments of the freezing tolerance experiment. A greater proportion of individuals died when

they were exposed to -34 degree Celsius and higher, which is equivalent to the winter

temperature at Cochrane (chi-square: p-value < 0.00100).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-24 -29 -34 -44

Mort

ality

(pro

port

ion)

Temperature (degree Celsius)

A A

B

B

A

52

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57

Chapter 3: Differences in herbivore damage to Arctium minus in

open and forest habitat

Abstract

Herbivore pressure and plant defenses can be influenced by biotic factors, abiotic factors, and the

interaction between them. Although many studies have investigated variation among habitats in

the effects of herbivores on native plants, fewer have studied habitat-dependent variation in

herbivory on exotics. In this study, herbivory, performance, and trichome densities of common

burdock (Arctium minus) were compared between open and understory habitats, with high and

low sunlight exposure respectively, on five sites. The result indicated that open habitat

populations showed reduced herbivore damage, faster growth rate, and lower trichome densities.

This may be due to: 1) herbivores occurring more abundantly in open habitats 2) burdock

expressing greater defences in understory sites, as expected under Resource Availability

Hypothesis, and 3) burdock may be able to escape from the herbivory by fast growth in open

habitats. These results emphasize that enemy release is not an absolute effect, but can depend on

an individual’s habitat.

58

Introduction

Invasions of exotic species have become a major problem in conservation biology. Exotic

species can disrupt the natural ecosystem by outcompeting and, ultimately, replacing some native

species. (Gurevitch and Padilla 2004; Hedja et al. 2009; Miller and Gorchov 2004). Despite this,

most potential invaders never spread or reach high abundance. One factor promoting

invasiveness may be predation differences between native and exotic habitats. The Enemy

Release Hypothesis (ERH) suggests that, as species colonize new habitats, they lose their

specialized natural enemies so that they experience less predation in new areas (Elton 1958;

Keane and Crawley 2002; Mitchell and Power 2003; Torchin and Mitchell 2004). As a result,

their mortality rates are reduced and fitness is increased. The Evolution of Increased Competitive

Ability (EICA), an extension of ERH, further postulates that, compared to native species, exotic

species invest less on resistance against predation as a response to reduced damage by enemies

(Blossey and Nötzold 1995; Maron et al. 2004). As a result, they can invest more resources into

growth and reproduction. Both of these mechanisms may increase invasiveness. For example, a

study showed that Norway maple (Acer platanoides) has significantly lower leaf herbivory and

fungal damage in North America compared to its native habitats, in Europe (Adams et al. 2009).

Similarly, Genton et al. (2005) showed that North American native ragweed (Ambrosia

artemisiifolia) experiences significantly less herbivore damage in France (invaded habitat)

compared to its native environment. These reductions in herbivory and predation may help

species to colonize new areas, achieve greater fitness, and ultimately become invasive.

59

It also may be that abiotic factors indirectly influence exotic species by affecting the herbivory

they experience in exotic habitats. For example, the physical environment can affect the level of

resistance that exotic species express. Coley et al. (1985) propose that species living in nutrient-

poor area would invest more in herbivore defense. They hypothesize that, because there are

limited amounts of nutrients, it is difficult to replace lost tissues to herbivores (Coley et al. 1985);

as a result, plants must protect their tissues from damage. For instance, Larbat et al. (2012)

showed that when nitrogen is limited, roots of patio tomato (Solanum lycopersicum) in

greenhouse conditions had greater phenolic defences. A study of multiple oak (Quercus) species

showed similar results: Pearse and Hipp (2012) found that oak trees expressed greater defense in

more stressful environments.

Conversely, individuals that are in a poor resource environment may have reduced defences

simply because they lack resources to produce them. For example, if the canopy cover is high, a

plant will receive very little sunlight; as a result, it may develop relatively reduced carbon-based

defence against herbivores. Salgado-Luarte and Gianoli (2010) reported that Chilean firetree

(Embothrium coccineum) seedlings from sunny areas had greater leaf thickness, which may have

made the leaf less palatable to herbivores. A study on Liriodendron tulipifera and Cornus florida

also showed greater phenolic contents in sunny areas, which led to reduced herbivory (Dudt and

Shure 1994). Another study suggested that the effect of light availability on chemical defence

differ depending on the shade tolerance of the species (Denslow et al. 1990). While shade-

tolerant species, such as Miconia gracilis, showed the greatest phenolic concentration under low

light exposure, the contrary was true for shade intolerant species, such as M. barbinervis and M.

nervos (Denslow et al. 1990). A similar pattern has also been seen in plant-pathogen interactions

60

where favourable conditions allowed better defense against pathogens (Schoeneweiss 1981). For

example, a study has shown that Australian Eucalyptus is more susceptible to the root pathogen,

Phytopthora cinnamomi, in habitats with low nutrients and poor soil structure (Burdon and

Shattock 1980; Weste 1986).

The resource availability of the environment may also influence the types of defenses that are

expressed. The Carbon-Nutrient Balance Hypothesis suggests that plants produce carbon-based

defences in a nutrient-limited environment (such as low-nitrogen soils) and nitrogen-based

defences in a carbon-limited environment (such as shaded areas) (Bryant et al. 1983). Therefore,

herbivore damage to plants may depend on the type and amount of defenses produced, which in

turn depends on the environment. A meta-analysis summarized 96 experiments that manipulated

the carbon availability and its effect on carbon-based defenses (Masaad et al. 2011) showing that

plants produced more carbon-based defenses in carbon-rich environments (Masaad et al. 2011).

Abiotic factors can directly influence predators or herbivores as well. Food choice by herbivores

often is affected by the nutrient contents of plants, which may depend on habitat (Behmer 2009).

As well, herbivores may avoid risky habitats, such as open sites. For instance, herbivores may

favour plants in shade in order to: 1) hide from predators, 2) access moisture, and 3) avoid heat

from the sun (Van Valen 1973; Maiorana 1973). A study comparing herbivory on Clidemia hirta

between open and understory habitats showed greater herbivore damage in understory than open

habitats in native areas due to greater abundance of herbivores (DeWalt et al. 2004). Mantyla et

al. (2008) studied whether passerine birds (Parus major and Cyanistes caeruleus) are attracted to

61

trees that have insect foliar damage. They showed that, while birds did not have any particular

preferences in shaded forest patches, they did show clear preference for trees with foliar damage

in sunnier forest patches. This suggests that birds can detect insect prey better in open areas than

shaded areas. Insect herbivores therefore may be selected to prefer plants in shaded habitats,

while invasive plants may respond to greater herbivory in shaded habitats by investing greater

resources in herbivore defence.

Such three-way interactions between resources, enemy populations, and defence may have

significant consequences for invasiveness. For example, if a plant species depends on habitats

that are relatively rich in resources and/or exposed to sunlight, it may be favoured by herbivores,

which may hinder their invasion as postulated by Blumenthal (2006) in his R-ERH theory. He

suggested that herbivores would prefer invasive plants in resource-rich environments over those

in resource-poor environments because, aside from being more nutritious, plants with high

resources would be selected for reduced defenses, as predicted by Coley et al. (1985). For these

reasons, enemy release may not apply to many species, and even in species where it does occur,

this effect may be habitat-dependent. Fully assessing enemy release requires consideration of

invaded habitats, as well as invasive species.

To study the effect of interactions between abiotic and biotic factors on herbivory of an invasive

species, I measured herbivore damage, performance, and trichome density of common burdock

(Arctium minus) in open habitats and forest understory at five different sites in Southern Ontario.

Straw (1990) studied pre-seed predation by tephritid flies on common burdock and its relation to

62

habitat type (i.e. open area or forest understory) in its native range in the United Kingdom. This

study showed that burdock seedheads were more attacked by tephritid fly larvae in shaded areas

than open habitats. He suggested that this may be due to differences in physical and chemical

defences of plants in the understory and open populations. He also speculated that shaded areas

may provide a better food supply and microclimate for adult insects. I investigated whether

herbivore damage to burdock (including both seed predation and folivory) shows similar patterns

in its introduced range, in the presence of both introduced specialists from Europe and new

enemies from North America. More specifically: 1) does herbivory differ between open and

understory habitats of Arctium minus? 2) Does herbivory resistance of Arctium minus differ

between open and shaded area? 3) Do A. minus in open habitat perform better than in understory

habitats? I hypothesized that individuals in understory habitats would have higher herbivore

damage, lower performance, and greater defenses.

Methods

Study Species

Common burdock (Arctium minus (Hill) Bernhardi: Asteraceae) is a Eurasian biennial that is

found from the southern states of the United States to every province of Canada (Gross et al.

1980). It is considered an invasive species, and is competitively more dominant than many native

species (Gross et al. 1980).

Common burdock in Ontario is attacked by variety of species, ranging from snails to insects and

generalists to specialists (Gross et al. 1980; Kambo 2012). Herbivores include native Agromyzid

63

miner herbivores such as Phytomyza lappae Goureau (serpentine miner) and Calcomyza

flavinotum Frick (blotch miner). It also is attacked by many chewing folivores such as native

keeler grasshoppers (Melanoplus keeleri (Thomas)) and exotic grove snails (Cepaea nemoralis

(Linnaeus), and sucking herbivores such as introduced meadow froghoppers (Philaenus

spumarius (Linnaeus). It also is attacked by introduced pre-dispersal seed predators such as

Gelechiid burdock seedhead moths (Metzneria lapella (Linnaeus)), and Tephritid flies (Tephritis

bardanae (Schrank) and Cerajocera tussilaginis (Fabricius)); all of these lay eggs in capitulae,

where their larvae then consume the seeds.

Study Sites

Common burdock was sampled from five mixed forest sites in Southern Ontario: Palgrave

Conservation Area (43.57°N, 79.51°W), the Koffler Scientific Reserve (KSR) (44.03°N,

79.55°W), Erindale Park (43.32°N, 79.39°W), Pomona Park (43.48°N, 79.24°W), and Northwalk

Conservation Area (44.02°N, 79.06°W) (Figure 3-1). These forest habitats were dominated by

maple, oak, and beech trees, interrupted by disturbances such as fields and roads. Five burdock

populations were sampled in open habitats (including fields, forest edges, and gaps) and five in

understory habitats for each site (5 sites; 2 habitats per site; 5 populations per habitat; 10 plants

per population; Total 500 plants). To distinguish open from understory sites, the transmission

coefficient for diffuse light penetration was measured for each population using a Plant Canopy

Digital Imager (CID Bio-Science). The population was considered an understory site if it had a

coefficient lower than 0.25.

64

Herbivore Damage: Percent area leaf damage for a basal leaf, the fifth, and the tenth leaf above

the basal leaf were measured for each individual plant (n = 30 leaves per population) by

comparison with leaf models with 1%, 2%, and 5% area removed. Presence and number of

serpentine and blotch mines also were observed on every leaf. The number of moth larvae per

seedhead was also measured from the 10 randomly-selected capitulae per population.

Performance: Stem circumference and height of all individuals (n= 500) were measured using

measuring tape. Reproductive success was estimated by counting the number of capitulae per

plant, and seeds were counted for 10 capitulae per population.

Trichome Densities: Trichome densities of all leaves were measured by counting the number of

trichomes along 7mm of the main vein using a dissecting microscope (n = 30 leaves per

population).

Statistical Analysis

Data were analyzed with Analysis of Variance using an REML approach. The model used was a

completely randomized partly hierarchical design (Kirk 1995) with habitat type (understory vs.

open habitat) crossed with site, and population nested in the site x habitat interaction; population

was treated as a random effect, while site and habitat were fixed. Proportions were arcsine-

transformed before analysis, using following equation: y = 2*arcsine (√(x/100)), where x is the

65

original % herbivore damage value; other data were not transformed. All analyses were

performed using JMP 10 (SAS Institute Inc.).

Results

The percentage of leaf area damaged by herbivores was greater in forest understory habitats than

open habitats (p-value= 0.0109) (Table 3-1A; Figure 3-2). There also were significant

differences among sites, and a significant site x habitat interaction indicating that the effect of

habitat type varies among sites (Table 3-1A; Figure 3-2); in particular, plants from one site

(Pomona Park) experienced less damage in understory than in open sites. In contrast, there were

no significant differences in the average number of serpentine miners (p-value =0.114) and

blotch miners (p-value= 0.157) per plant between habitats, though the overall trend showed

greater damage in understory habitats (Table 3-1B and 3-1C; Figure 3-2 and 3-3); the site and

habitat x site effects were non-significant. Finally, individuals in understory habitats had, on

average, a greater number of moth larvae in each capitulum (p-value= 0.00890) (Table 3-2A) and

significantly greater proportion of seeds damaged (p-value= 0.0359) (Table 3-2B) than open

habitats (Figure 3-4); there also were significant differences among sites, but no habitat x site

interactions (Table 3-2).

Individuals in open habitats were taller (p-value= 0.000300) (Table 3-3A) and had a greater stem

circumference (p-value < 0.000100) (Table 3-3B) than those in understories (Figure 3-5 and 3-6);

there were also significant differences among sites, and a significant site x habitat interaction.

Individuals in open habitats also had greater number of capitulae (p-value < 0.000100); the site

66

and habitat x site also were significant effects (Table 3-4A) (Figure 3-7). However, there were no

significant difference in number of seeds (p-value= 0.3865) (Table 3-4B) per capitulum between

habitats.

Finally, trichome density was significantly greater in understory populations than open

populations (p-value< 0.000100); though there was a significant site x habitat interaction (Table

3-5) (Figure 3-8).

Discussion

Understory populations may have lower photosynthetic rates due to lower leaf surface area and

lower light availability, leading to lower capitulum production. As well, results showed that there

were significant differences in leaf damage between open and understory habitats (Figure 3-2),

which may have affected the reproductive success of the burdock (Figure 3-7). Since levels of

folivore damage were very low, the effect of this damage on the seed production is questionable.

However, some studies have indicated even minor damage may have significant effects (Marquis

1984; Schoonhoven et al. 2005); in particular, even low levels of leaf damage observed may be

significant in understory habitats where low light intensity already restricts photosynthesis.

Plants in understory habitats also had a significantly greater number of moth larvae in their

capitulae (Figure 3-4); seed damage is especially important because it directly influences the

fitness of this biennial species, whereas the leaf damage may not. This result is similar to those

of Straw (1990), who found that understory plants in the UK had greater losses of seeds than

67

plants in openings, even though these were attributable to a different family of insects

(Tephritidae).

These results suggest that the herbivory experienced by this invasive species depends on which

habitats it has colonized even in areas where it is not native. This need not be the case: while

DeWalt et al. et al. (2004) showed that the Clidemia hirta experienced greater herbivore damage

in forests than open habitats in its native range, this pattern disappeared in invaded regions. They

explained this result by suggesting Clidemia’s natural enemies were lost during invasion; in

contrast, Arctium has been accompanied in North America by at least some of its herbivores,

perhaps explaining why the same pattern of damage (albeit due in part to different enemies)

occurs in both invaded and native areas.

Differences in herbivore damage among habitats also may affect an invader’s habitat

associations. For instance, many invasive species are disturbance-dependent or perform better in

disturbed, low biomass, open areas (Hobbs and Huenneke 1992; Blumenthal 2006) than in

undisturbed sites with higher plant biomass and diversity. This could be indirectly due to

differences in herbivory between two areas, if herbivore abundance and diversity also was

greatest in undisturbed sites. For instance, burdock’s association with disturbed or open sites

(Gross et al. 1980) may reflect, in part, lower levels of herbivory in these areas.

68

Metzneria lapella is an introduced species whereas both serpentine and blotch miners are natives,

yet they all showed at least a tendency towards reduced attack rate in open habitats, hinting that

shared habitat preferences of herbivores may be more important than co-evolved interactions.

Van Valen (1973) stated that herbivores prefer shaded plants because they can hide from

predators; as a result, herbivores may have increased fitness by: 1) avoiding enemies, 2)

spending less energy on behaviour or traits to avoid predators, and 3) using less time escaping

from predators and more time on feeding. The same logic may apply to burdock-feeding

herbivores as well. Even if herbivores do not necessarily prefer shaded plants, fewer herbivores

may be present in the open habitats because they were more conspicuous and were eaten by

predators. Finally, herbivores may prefer shaded areas in order to avoid heat during summer

(Maiorana 1973). This might have been especially true during extreme hot summer of 2012

(Environment Canada 2012).

In addition to representing safer sites for herbivores, it also may be that open-habitat burdock

plants were more nutritious than understory ones. Burdock in open habitats is exposed to greater

sunlight, allowing greater photosynthesis, and potentially resulting in a higher C: N ratio and

more C-based chemical defences. As well, the extreme drought might have caused open area

burdock to be less healthy and wither; as a result, burdock in open areas might have had reduced

nitrogen and water content that it was less favourable for herbivores to feed upon (Behmer 2009).

Many studies of the effects of drought on plant-herbivore interaction show herbivores that feed

on plants in a dry environment have reduced performance, including growth and survival, due to

reduced leaf nitrogen, soluble protein content, and relative water content (Herms and Mattson

1992; EnglishLoeb et al. 1997; de Bruyn et al. 2002; Walter et al. 2012). More generally, the

69

plant vigour hypothesis predicts that herbivores would perform better on vigorously growing

plants (Price 1991).

Reduced herbivory in open habitats may also have been due to faster growth. Results indicate

that burdock in open habitats was taller and had greater circumference (Figure 3-4 and 3-5).

Growing faster may have also helped to reduce herbivore damage as well. Kursar and Coley

(2003) differentiated species into two syndromes: defense and escape. Plants with the defense

syndrome have greater defenses against herbivores, but also a slower leaf expansion rate. Escape

syndrome is characterized by faster leaf expansion and lower defenses, which reduce the time

that leaves are young and most vulnerable to herbivory. As a result, herbivores have a limited

amount of time to feed on young leaves; therefore, faster-growing burdock in sunnier sites may

have outpaced its herbivores.

Understory populations had greater trichome densities than populations in open areas, though

there was a habitat x site interaction (Figure 3-8). Since understory populations also have greater

herbivore damage, this may indicate that a greater trichome density is a response to herbivory.

Trichomes can be used as a physical resistance against herbivores by protecting leaf tissues

(Levin 1973; Bossdorf 2005). If there are few herbivores, it would be beneficial for open habitat

populations to reduce investment in herbivory resistance, as expected by the Evolution of

Increased Competitive Ability Hypothesis (EICA) (Blossey and Nötzold 1995). EICA states that

as the herbivory pressure is reduced in invasive populations, plants reduce their investment on

defenses and focus on growth and reproduction (Blossey and Nötzold 1995).

70

Greater physical resistance, such as trichomes, also may be important for understory plants

because they have less sunlight. Coley et al. (1985) states plants that are exposed to poor

resource environments will invest more on defenses, since damaged tissues are harder to replace.

Endara and Coley (2010) reassessed the hypothesis by meta-analysis and showed that, indeed,

plants in resource-poor habitats invest more resources on defenses. Because of low sunlight

availability, it may be more difficult for understory burdock populations to replace lost tissues

due to herbivory; therefore, they may produce more trichomes, compare to open habitat

populations, in order to protect their tissues. This is consistent with the poorer growth of plants in

shaded sites.

These results have implications for enemy release. Even after escaping from most of their natural

specialist enemies, invasive plants, such as burdock, also face new herbivores in invaded habitats.

If damage by these new herbivores is very high, ERH would not apply. Therefore, both the

applicability of ERH and the success of invasion may vary depending on habitats that an

invasive species colonizes (Blumenthal 2006). For instance, if an exotic species colonizes an

area with high forest cover, its population growth might be limited by herbivores specific to that

habitat. If the trends in herbivore damage that I observed are general, this also may help to

explain the resistance of native woodland to plant invasion (Drake et al. 1989).

71

Tables

Table 3-1: ANOVA results comparing leaf damage of common burdock at multiple sites and in

two habitats (understory and open). Models also included Populations Models also included

Population (Site, Habitat) as a blocking factor.

A: ANOVA results comparing percent area removal of a common burdock leaf.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 7.1375 0.0109

Site 4 40 4.8433 0.0028

Site X Habitat 4 40 2.8284 0.0371

B: ANOVA results comparing the average number of serpentine miners on a burdock leaf.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 2.6078 0.1142

Site 4 40 1.6326 0.1849

Site X Habitat 4 40 1.3734 0.2604

C: ANOVA results comparing the average number of blotch miners on a burdock leaf.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 2.0809 0.1569

Site 4 40 0.7827 0.5432

Site X Habitat 4 40 0.5145 0.7255

72

Table 3-2: ANOVA results comparing seed damage in common burdock capitulum at multiple

sites and in two habitats (understory and open). Models also included Population (Site, Habitat)

as a blocking factor.

A: ANOVA results comparing the number of M. lapella larvae in a common burdock capitulum.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 25 8.0517 0.0089

Site 4 25 5.4082 0.0028

Site X Habitat 4 25 0.3691 0.8283

B: ANOVA results comparing proportion of damaged seeds per common burdock capitulum.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 25 4.9169 0.0359

Site 4 25 2.7803 0.0487

Site X Habitat 4 25 0.223 0.923

73

Table 3-3: ANOVA results comparing morphology of common burdock at multiple sites and in

two habitats (understory and open). Models also included Population (Site, Habitat) as a

blocking factor.

A: ANOVA results comparing average height of a burdock.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 15.3182 0.0003

Site 4 40 18.0588 <.0001

Site X Habitat 4 40 9.1573 <.0001

B: ANOVA results comparing average stem circumference of a burdock.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 30.2449 <.0001

Site 4 40 13.9006 <.0001

Site X Habitat 4 40 8.6323 <.0001

74

Table 3-4: ANOVA results comparing seed production of common burdock at multiple sites and

in two habitats (understory and open). Models also included Population (Site, Habitat) as a

blocking factor.

A: ANOVA results for number of capitulae per plant.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 25 115.7149 <.0001

Site 4 25 37.2926 <.0001

Site X Habitat 4 25 18.7286 <.0001

B: ANOVA results for average number of seeds per capitulum.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 25 0.7768 0.3865

Site 4 25 1.6151 0.2016

Site X Habitat 4 25 1.0519 0.4007

75

Table 3-5: ANOVA results comparing trichome density of burdock leaf at multiple sites and in

two habitats (understory and open). Models also included Population (Site, Habitat) as a

blocking factor.

Source df numerator df denominator F Ratio Prob > F

Habitat 1 40 19.7951 <.0001

Site 4 40 3.0413 0.028

Site X Habitat 4 40 8.2706 <.0001

76

Figures

Figure 3-1: Sites at which both understory and open populations of Arctium minus were sampled

(http://goo.gl/maps/q4cM9). Locations include: Palgrave Conservation Area (43.57°N, 79.51°W),

the Koffler Scientific Reserve (KSR) (44.03°N, 79.55°W), Erindale Park (43.32°N, 79.39°W),

Pomona Park (43.48°N, 79.24°W), and Northwalk Conservation Area (44.02°N, 79.06°W).

Palgrave

Conservation Area

Northwalk

Conservation Area

Pomona Park

Erindale Park

Koffler Scientific

Reserve

77

Figure 3-2: The average percent area damage (± standard error) on common burdock leaves for

open and understory habitats at five different sites. Damage was significantly greater in

understory than open sites (ANOVA: p-value < 0.0500), but a significant interaction indicated

that this effect differed between Pomona Park and other populations (Table 3-1A).

0

1

2

3

4

5

6

7

8

9

Open Understory

Perc

ent

are

a d

am

age (

%)

Habitats

Erindale Park

Pomona Park

Palgrave Conservation

Area

Northwalk Conservation

Area

KSR

78

Figure 3-3: The average number of serpentine and blotch miners (± standard error) on common

burdock leaves for open and understory habitats at five different sites. There was no significant

difference in number of miners in a leaf between habitats.

0

0.5

1

1.5

2

Open Under

Avera

ge N

um

ber

of

Serp

entine

Min

e p

er

leaf

Habitats

Erindale Park

Pomona Park

Palgrave

Conservation

AreaNorthwalk

Conservation

Area

0

0.1

0.2

0.3

0.4

0.5

0.6

Open UnderAvera

ge N

um

ber

of

Blo

tch m

ine

per

leaf

Habitats

Erindale Park

Pomona Park

Palgrave

Conservation

AreaNorthwalk

Conservation

Area

79

Figure 3-4: The average number of M. lapella larvae in the common burdock seedhead (±

standard error) for open and understory habitats at five different sites. Capitulae in understory

habitats contained significantly greater number of M. lapella than capitulate in open habitats

(ANOVA: p-value= 0.00890).

0

0.5

1

1.5

2

2.5

Open Understory

Avera

ge N

um

ber

of

larv

ae in t

he

Capitulu

m

Habitats

Erindale Park

Pomona Park

Palgrave Conservation

Area

Northwalker Conservation

Area

KSR

80

Figure 3-5: The average height of a common burdock (± standard error) for open and understory

habitats at five different sites. Individuals in open habitats were significantly taller than those in

understory habitats (ANOVA: p-value = 0.000300), but a significant interaction indicated that

this effect differed Palgrave Conservation Area and other populations (Table 3-3A).

0

20

40

60

80

100

120

140

160

Open Under

Heig

ht

(cm

)

Habitats

Erindale Park

Pomona Park

Palgrave Conservation

Area

Northwalk Conservation

Area

KSR

81

Figure 3-6: Average stem circumference (± standard error) of a common burdock with standard

error for open and understory habitats at five different sites. Individuals in open habitats had

larger stem circumference than those in understory habitats (ANOVA: p-value < 0.000100), but

a significant interaction indicated that this effect differed Palgrave Conservation Area and other

populations (Table 3-3B).

0

2

4

6

8

10

12

Open Under

Circu

mfe

rence

(cm

)

habitats

Erindale Park

Pomona Park

Palgrave Conservation

Area

Northwalk Conservation

Area

KSR

82

Figure 3-7: Average number of capitulae per common burdock (± standard error) with standard

error for open and understory habitats at five different sites. Individuals in open habitats

produced greater number of capitulate than those in understory habitats (ANOVA: p-value <

0.000100), but a significant interaction indicated that this effect differed Northwalk Conservation

Area and other populations (Table 3-4A).

0

20

40

60

80

100

120

140

160

180

Open Understory

Num

ber

of

Capitula

e

Habitats

Koffler Scientific Reserve

Pomona Park

Northwalk Conservation

Area

Palgrave Conservation

Area

Erindale Park

83

Figure 3-8: The average number of trichomes along 0.7cm of main vein on the common burdock

leaf for open and understory habitats at five different sites. Individuals in understory habitats had

greater trichome density than those in open habitats (ANOVA: p-value < 0.0001), but a

significant interaction indicated that this effect differed Erindale park and Pomona Park from

other populations (Table 3-5).

0

10

20

30

40

50

60

Open Understory

Trich

om

es

per

0.7

cm

Habitats

Erindale park

Pomona Park

Palgrave

Conservation Area

Northwalk

Conservation Area

Koffler Scientific

Reserve

84

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Walter, J., R. Hein, H. Auge, C. Beierkuhnlein, S. Loffler, K. Reifenrath, M. Schadler,

M. Weber, and A. Jentsch. 2012. How do extreme drought and plant community composi

tion affect host plant metabolites and herbivore performance? Arthropod-Plant Interactions

6: 15-25.

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Chapter 4: General Conclusion

Many studies have compared enemies of exotic invasive species with those of natives, or

compared damage to plants in their native and invaded areas (Mitchell and Power 2003; Colautti

et al. 2004; Liu and Stiling 2006). In contrast, relatively few studies have considered

intracontinental variation in herbivory on exotic species in their invaded areas. Therefore, it is

unclear how often invasive species experience gradients in damage across their new range, as

can occur for natives (Sexton et al. 2009; Bruelheide and Scheidel 1999).

As well, there have been few studies of effects of habitat type on damage to invasive species (but

see DeWalt et al. 2004). This is particularly unfortunate since variation in habitat-related factors

such as resource supply may be linked to the importance of enemy release. For instance,

Blumenthal (2006) suggested that, herbivores would prefer invasive plants in resource-rich

environment over those in resource-poor environments, because, as well as being more nutritious,

plants in high-resource environments would be selected for reduced defenses according to Coley

et al. (1985). Therefore, whether natural enemies have the potential to limit invasions may differ

among sites and habitats.

Kambo (2012) documented latitudinal differences in performance and herbivore damage of

common burdock populations within its invaded Ontario range. He showed that this species

experiences less herbivore damage and produces more seeds at more northerly sites. Similarly,

Straw (1991) found insect damage to burdock varied among habitats in its native range: plants in

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shaded sites were more damaged by seed predators. The purpose of my thesis was to explore and

explain such variation in damage among invasive burdock populations.

Does latitudinal variation in attack reflect underlying variation in resistance to herbivores?

In Chapter 2, I used a common garden experiment, to show that although there is a significant

difference in susceptibility of plants from different populations to herbivore damage, there was

no evidence that genotypes from more northern sites were better defended. This suggests that

environmental factors, such as herbivore abundance, may have the greatest effects on the

latitudinal gradient in herbivore damage observed by Kambo (2012). My feeding experiment

also failed to find evidence that more northern genotypes were less palatable, though there was

some evidence that they reduced the performance of herbivores. Therefore, variation in

herbivore populations may best explain the patterns observed in the field. These results are

consistent with an experiment in which I showed that a specialist seed predator was not able to

survive temperature typical of northern sites.

I did find that plants from southern populations have a significantly greater trichome density,

though this did not result in reduced herbivory. Instead, this pattern may be related to latitudinal

variation in drought tolerance, which was greater in more southern plants: burdock may express

greater trichome density to reduce water loss.

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Finally, overall reproductive output was similar for plants from different populations, suggesting

that the gradient observed in the field may result from variation in herbivore pressure. As well,

evidence of different reproductive strategies was found for northern vs. southern populations:

while northern populations produced many smaller seedheads, southern populations produced

fewer large seedheads.

Do local differences in environmental conditions affect herbivory?

In Chapter 3, I report a field survey which showed that plants in understory habitats had greater

herbivory and trichome density than plants of open habitats. Understory habitats may provide

herbivores with a refuge from their own predators, or plants growing in the understory may be

less nutritious; greater trichome density in understory populations may be response to the poor

growth in light-limited sites, according to resource availability hypothesis (Coley et al. 1985).

These results are similar to those of Straw (1991), who observed greater herbivory in native

understory habitats than open habitats.

Implications

Overall, the study shows invasive species may experience several source of variation in attack in

their new range. Attack may be reduced in habitats where herbivores are scarce, such as range

margins or (likely) open sites. This variation in attack may resemble the enemy release

hypothesis (Keane and Crawley 2000) on much smaller scale. For example, if burdock colonizes

southern forest habitats, it may be heavily attacked by its new natural enemies, reducing its

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success and selecting for improved defences; conversely, plants in open northern sites may be

largely damage-free, instead favouring allocation to reproduction as predicted by EICA (Blossey

and Nötzold 1995). Many invasive plants, including common burdock, experience significant

herbivore attack in at least part of their new range (Sexton et al. 2000; Blumenthal 2006). Simply

classifying such a plant as native or exotic therefore may overlook significant implications for

both its invasiveness and evolution. To truly understand the greater performance and

competitiveness of exotic species in their new habitats, multiple populations in different

locations and habitat types must be considered.

If the performance of invasive plants improves approaching their range limits because herbivores

become scarce, this advantage may be lost in future with a warming climate. For instance, the

limited the freezing tolerance of the major seed predator of the burdock (Metzneria), suggests

that this herbivore is scarce at northern sites because it is limited by cold winter temperatures.

However, if the winter temperatures rise in future due to global warming, this may allow this

herbivore to reach northern burdock populations; as a result, damage may increase and

production of viable seeds may decline. Therefore, the observed performance increase

approaching Arctium’s range limit may only be temporary.

Finally, the greater herbivore damage in the understory habitats further emphasizes the

importance of healthy forest against invasion. Well-established forests with high biomass often

are resistant to invasion (Hobbs and Huenneke 1992). This study shows that, compared to open

sites, understory habitats attract more herbivores of invasive species. Maintaining healthy forest

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structure may therefore encourage natural enemies of invaders as a form of conservation

biological control (Debach and Rosen 1991).

Future Work

1) Are there any chemical defenses that have significant influence on herbivory?

This study did find a difference in trichome density of plants from different latitudes and habitats;

however, this did not result in differences in folivory, implying that trichomes may have little

influence on resistance to herbivores. However, I did find significant differences in herbivore

weight gain among populations. These results hint that burdock leaves may produce secondary

chemicals that confer resistance to herbivory. The nature of any such chemicals is unknown.

Future research should determine the role of secondary chemicals in Arctium defense, and

investigate whether these contribute to differences in damage among populations.

2) Are biocontrol herbivores effective against invasive plants regardless of the latitude and

habitat types?

This study suggests that northern populations of burdock are less damaged because its herbivores

cannot tolerate the physical conditions at these sites. If this is a general phenomenon, this could

have implications for bio-control: introduced herbivores might be much less effective in northern

locations. Similarly, introduced biocontrol agents may be only effective in particular habitats.

Louda et al. (1990) found that beetles as a bio-control are only effective for invasive Hypericum

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perforatum in dry, sunny habitats. Future work should look at whether the utility of bio-control

may be limited in northern or otherwise unsuitable sites.

3) Will herbivory gradients approaching the range limit of invasive species change in

response to climate warming?

The study indicated that a specialist seed-eating moth cannot tolerate northern winter

temperatures, which explains its scarcity in northern populations of burdock; similar constraints

may explain the latitudinal decline of other guilds of herbivores (Kambo 2012). This may change

as northern latitudes warm in future. Similarly, global warming may also change the temperature

difference between open and the understory habitats, altering differences between them in plant

performance and herbivory. Future research should focus on whether differences in herbivore

damage among latitudes and habitats will be maintained or lost in the face of climate warming.

95

References

Blossey, B. and R. Nötzold. 1995. Evolution of increased competitive ability in invasive

nonindigenous plants: a hypothesis. The Journal of Ecology 83: 887-889.

Blumenthal, D. M. 2006. Interactions between resource availability and enemy release in plant

invasion. Ecology Letter 9: 887-895.

Bruelheide, H. and U. Scheidel. 1999. Slug herbivory as a limiting factor for the geographical

range of Arnica montana. Journal of Ecology 87:839-848.

Colautti, R. I., C. G. Eckert, and S. C. H. Barrett. 2010. Evolutionary constraints on adaptive

evolution during range expansion in an invasive plant. Proceedings of the Royal Society B 277:

1799-1806.

Debach, P. and D. Rosen. 1991. Biological control by natural enemies. Cambridge University

Press, Cambrdige, UK.

DeWalt et al., S. J., J. S. Denslow, and K. Ickes. 2004. Natural-enemy release facilitates habitat

expansion of the invasive tropical shrub Clidemia hirta. Ecology 85 (2): 471-483.

Garcia, M. B., D. Goni, and D. Guzman. 2010. Living at the Edge: Local versus Positional

Factors in the Long-Term Population Dynamics of an Endangered Orchid. Conservation Biology

24(5): 1219-1229.

Liu, H. and P. Stiling. 2006. Testing the enemy release hypothesis: a review and meta-analysis:

biological Invasions 8: 1535-1545.

Keane, R. M. and M. J. Crawley. 2002. Exotic plant invasions and the enemy release hypothesis.

Trends in Ecology and Evolution 17(4): 164-170.

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Kambo, D. (2012) Differences in performance and herbivory along a latitudinal gradient for

common burdock (Arctium minus). M.Sc. Thesis, University of Toronto.

Louda, S. M., K. H. Keeler, and R. D. Holt. 1990. Pages 414-444 in Grace, J. B. and D. Tilman,

editors. Perspectives on Plant Competitions. Academic Press. New York, USA

Maiorana, V. 1981. Herbivory in sun and shade. Biological Journal of the Linnean Society 15:

151-156.

Mitchell, C. E. and A. G. Power. 2003. Release of invasive plants from fungal and viral

pathogens. Nature 421: 625-627.

Sexton, J. P., P. J. McIntyre, A. L. Angert, and K. J. Rice. 2009. Evolution and Ecology of

Species Range Limits. Annual Review of Ecology and Evolution Systematics 40: 415-436.

Straw, N. A. 1991. Resource limitation of tephritid flies on lesser burdock, Arctium minus (Hill)

Bernh. (Compositae). Oecologia 86: 492-502.

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Appendix 2-I: Residual Distribution of data from Common Garden

The x-axis represents the number of observations for each value. The box plot represents

quantiles of each data with a mid-line as a median and a diamond as a mean.

A. Residual distribution of stem circumference data (cm). The result suggests a normal

distribution.

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B. Residual distribution of plant height data (cm). The result suggests a non-normal distribution.

C. Residual distribution of leaf toughness data (g). The result suggests a non-normal distribution.

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D. Residual distribution of transformed leaf damage data (%). The result indicates a non-normal

distribution.

E. Residual distribution of number of trichomes per 0.7mm data. The result suggets a normal

distribution.

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F. Residual distribution of total number of seedheads. The result suggests a non-normal

distribution.

G. Residual distribution of total seed mass data (g). The result suggests a non-normal distribution.

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H. Residual distribution of average capitulum mass data (g). The result suggets a normal

distribution.

I. Residual distribution of number of seeds per capitulum data. The result suggets a normal

distribution.

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J. Residual distribution of number of larvae per capitulum data. The result suggests a non-normal

distribution.

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Appendix 2-II: Test of Unequal Variance of Common Garden Data

Results of O’Brien tests for equal variance using JMP 10 (SAS Institute Inc. 2012) (O’brien and

Fleming 1979). They indicate that, while most data meet the requirement of equal variance, total

seed mass, total number of capitulate, average capitulum mass, and number of seeds per

capitulum data have unequal variance. Log transformation did not fix this problem.

Data F Ratio df numerator df denominator Prob > F

Stem Circumference 1.146 10 152 0.332

Stem Height 0.608 10 152 0.805

Leaf Toughness 0.470 9 114 0.892

Transformed Leaf Area Damage 1.324 9 114 0.232

Leaf Trichome 0.881 9 114 0.545

Total Seed Mass 5.807 10 1539 <0.000100

Total number of Capitulae 6.656 10 1539 <0.000100

Average Capitulum Mass 53.905 10 1539 <0.000100

Number of Seeds per Capitulum 26.639 10 1539 <0.000100

Number of Larvae per Capitulum 1.500 10 1538 0.133