Download Evolutionary Ecology of Arabidopsis thaliana: Interactions with Biotic

EVOLUTIONARY ECOLOGY OF ARABIDOPSIS THALIANA: INTERACTIONS
WITH BIOTIC AND ABIOTIC ENVIRONMENTAL FACTORS
by
Adebobola O. Imeh-Nathaniel
A Dissertation
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
DECEMBER 2012
Committee:
Dr. Maria G. Bidart- Bouzat
Dr. Juan L. Bouzat
Dr. Ron Woodruff
Dr. Dan Pavuk
Dr. Maria Rizzo (Graduate Faculty Representative)
ii
ABSTRACT
Dr. M. Gabriela Bidart-Bouzat, Advisor
Previous studies have demonstrated that plants may be affected by their interactions with
both biotic and abiotic environmental factors. An important biotic factor that usually affects the
performance of plants is insect herbivory. In addition, abiotic factors such as light, or more
specifically ultraviolet B (UVB) radiation, can also affect growth and physiological processes in
plants. Arabidopsis thaliana is an annual herbaceous plant species with documented defense
strategies including resistance and tolerance. Studies have also shown that these plant responses
to insect herbivory may be modified by other abiotic factors such as UVB radiation. In a series
of common garden experiments, the role of insect herbivory per se and in combination with
UVB radiation was evaluated. Using a population of recombinant inbred lines of A. thaliana, the
first experiment (described in Chapter 1) investigated the role of plant trichomes and size on
patterns of plant use by an insect community. In this experiment, it was also evaluated whether
insect herbivory imposes selection on trichome production and plant size. In a second
experiment (described in Chapters 2 and 3), the effect of UVB radiation on plant resistance and
tolerance to insect herbivory as well as on their potential fitness costs was assessed. This
experiment also evaluated the role of UVB on patterns of plant utilization by insect herbivores
and whether this important abiotic factor may influence plant phenotypic responses. Results
from these experiments revealed that plant trichomes influenced levels of herbivore damage and
plant size had an effect on colonization of plants by insect herbivores. In addition, results from
selection analyses revealed that insect herbivores exerted directional selection on trichome
density in A. thaliana. The second study showed that UVB radiation influenced the expression
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of resistance and tolerance as well as their associated fitness costs. Similarly, patterns of plant
utilization by insect herbivores and phenotypic responses of plants to insect herbivory were
modified by UVB radiation. Overall, these results highlight the importance of evaluating
complex environments, including both abiotic and biotic factors, as it relates to the evolution and
maintenance of traits related to plant defense against insect herbivory
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ACKNOWLEDGMENTS
First, my gratitude goes to my advisor, Dr. Gabriela Bidart-Bouzat for her unrelenting
help and guidance throughout my graduate studies at Bowling State University, Ohio, and more
importantly, in helping me to succeed in the completion of my Ph.D. research work. I am also
very grateful to all my committee members for finding the time to attend all meetings and their
useful and constructive comments throughout the course of this work. In addition, I will like to
express my gratitude to past and present members of Bidart-Bouzat’s lab including Lexie
Majorczyk, Sean Ryan and Julie Kemarly-Hopkins. I do appreciate the help I received from the
two undergraduate assistants Curtis McKinnon and Matthew Wilkinson to perform the field
experiments. I would also like to acknowledge Dr. Jeff Miner for providing a conductive
working environment throughout the period of the fieldwork at the BGSU Ecological Research
Station and to every member of the Department of Biological Sciences, who has in one way or
another contributed to the success of this study. The research conducted was supported by
BGSU and a Larry and Linda Oman Fellowship.
I appreciate the support I received from my siblings. I am sincerely grateful to my
mother, who was always available to help with my children during the past four years. I cannot
be thankful enough to my mother. To my three boys, life could not have been better without
you. Thank you for your understanding and love, especially when “mummy” did not have the
time to take you for soccer practice. To my husband, you are an inspiration to me. Thanks for
your support, encouragement, love, and active involvement in this study. I am so blessed to have
you. This dissertation will not be complete without acknowledging all friends and well-wishers.
I appreciate all your concerns, encouragement, and love. Above all, I am grateful to the Alpha
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and the Omega, the beginning and the ending of all things, who has made all grace sufficient for
me throughout the period of my graduate work at Bowling Green State University.
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TABLE OF CONTENTS
Page
CHAPTER 1. INTRASPECIFIC GENETIC VARIATION IN PLANT SIZE
AND TRICHOME DENSITY INFLUENCES PATTERNS OF PLANT USE BY
AN INSECT COMMUNITY
Abstract……………………………………………………………………………..
1
Introduction .……………………………………………………………………......
3
Methods .……………………………………………………………………………
5
Results ………………………………………………………………………………
10
Discussion…………………………………………………………………………..
12
Tables and Figures…...……………………………………………………………...
17
References……….…………………………………………………………………..
25
CHAPTER 2. CHANGES IN ULTRAVIOLET B RADIATION MODIFY
RESISTANCE AND TOLERANCE IN ARABIDOPSIS THALIANA TO INSECT
HERBIVORY
Abstract……………………………………………………………………………...
33
Introduction…………………………………………………………………………
34
Methods……………………………………………………………………………... 38
Results……………………………………………………………………………….
43
Discussion…………………………………………………………………………...
45
Tables and Figures …………………………………………………………….........
50
vii
References …………………………………………………………………………..
56
CHAPTER 3. EFFECTS OF ULTRAVIOLET-B RADIATION ON
PLASTICITY OF FITNESS-RELATED RESPONSES OF ARABIDOPSIS
THALIANA TO INSECT HERBIVORY
Abstract………………………………………………………………………….......
67
Introduction………………………………………………………………………….
69
Methods……………………………………………………………………………..
72
Results………………………………………………………………………………
75
Discussion…………………………………………………………………………..
77
Tables and Figures …...……………………………………………………………..
82
Reference ……….…………………………………………………………………..
85
viii
LIST OF TABLES
Table
Page
CHAPTER 1
1
ANOVA F values showing the presence of genetic variation in
measured phenotypic characters………………………………………….
2A
17
Analysis of variance for relative fitness showing the effects of insect
herbivores on directional selection for trichomes in A. thaliana………….. 18
2B
Analysis of variance for relative fitness showing the effects of insect
herbivores on stabilizing/disruptive selection for trichomes in A.
thaliana…………………………………………………………………………...
18
CHAPTER 2
1
ANOVA F-values showing the effects of herbivory, UVB radiation,
RILs and their interactions on trichome density (direct resistance) and an
indirect resistance estimate (1-herbivore damage)……………………….
2
50
ANCOVA F-values showing the relationship between relative fitness
and rosette damage across UVB environments and in each of the UVB
environments……………………………………………………………... 51
3
Pearson’s correlation coefficients showing the relationship between
relative fitness of undamaged plants and tolerance under ambient
(UVB+) and excluded (UVB-) ultraviolet B radiation level covariance… 52
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CHAPTER 3
1
Mixed model analysis of variance showing the effects of UVB radiation,
insect herbivory and RIL on plant phenotypic variables of A. thaliana…. 82
x
LIST OF FIGURES
Figure
Page
CHAPTER 1
1
Picture of Arabidopsis thaliana plant showing the presence of
19
trichomes
2
A split- plot experimental design with two levels of herbivory
20
(herbivory and sprayed) distributed in a checkerboard pattern among
ten plots
3
Analysis of covariance showing the relationship between trichome
density or plant size and plant utilization patterns by insect herbivores
(i.e. days to colonization and herbivore damage).................................... 21
4
Directional (β) and stabilizing/disruptive (γ) selection gradients
estimated by regression selection analyses on trichome density in the
presence and absence of insect herbivores ………………………….....
5
22
Directional (β) and stabilizing/disruptive (γ) selection gradients
estimated by regression selection analyses on plant size in the
presence of insect herbivores..................................................................
6
23
Directional (β) and stabilizing/disruptive (γ) selection gradients
estimated by regression selection analyses on number of days to first
herbivore colonization and insect herbivore damage…………………
24
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CHAPTER 2
1A
Picture of the experimental plots showing plant arrangements within
each plot ……………………………………………………………….
1B
53
Schematic representation of experimental plot layout showing two
levels of herbivory (herbivory and sprayed) and UVB (ambient and
excluded) treatments distributed among twenty plots…………………. 53
2
Absorption spectrum for Mylar and Clear acetate filters………………
3
Effects of the presence (UVB+) and exclusion (UVB-) of ultraviolet B
54
radiation on trichome density, indirect resistance, and tolerance of an
A. thaliana recombinant inbred line population ……………………
4
55
Genotypic correlations showing fitness costs of trichomes, resistance ,
and tolerance in A. thaliana plants grown under ambient and excluded
UVB radiation…………………............................................................
5
56
Genotypic correlations between resistance and tolerance in A. thaliana
plants grown under ambient and excluded UVB radiation…………….
57
CHAPTER 3
1
Reaction norms showing A. thaliana phenotypic responses to UVB
radiation and insect herbivory, in terms of total fruit production ,
rosette biomass, and number of days to first flowering ……………….
2
83
Reaction norms showing A. thaliana phenotypic responses, in terms of
days to herbivore colonization and rosette damage ………………….
86
1
CHAPTER 1
INTRASPECIFIC GENETIC VARIATION IN PLANT SIZE AND TRICHOME
DENSITY INFLUENCES PATTERNS OF PLANT USE BY AN INSECT COMMUNITY
ABSTRACT
Intraspecific genetic variation in plant traits can have important consequences for the
insect community utilizing plant resources for feeding and reproduction. For example, variation
in a plant structural defensive trait such as trichome density can influence levels of herbivore
damage. In addition, variability in plant size may affect host choice by insect herbivores since
this trait can serve as a visual cue for insects to locate a plant host species as well as provide
variable surface areas for insect oviposition and feeding. Despite the importance of these plant
traits, little is known regarding how genetic variation may influence plant damage levels and
colonization of plants by a natural insect community. Similarly, few studies have evaluated
whether insect herbivores impose selection on trichome production and plant size. Results from
a common garden experiment revealed the presence of genetic variation in trichome density and
plant size in a population of recombinant inbred lines of Arabidopsis thaliana. Genetic variation
in these traits significantly influenced both colonization of plants by insect herbivores and levels
of herbivore damage among plants. Selection analyses revealed directional selection favoring a
reduction in trichome density in the absence of insect herbivory (sprayed plots), which may be
indicative of a fitness cost related to the production of this defensive trait. In terms of plant size,
selection favored larger plants in field plots exposed to insect herbivory. These results
corroborate the role of plant trichomes as a resistant trait favored by selection in the presence of
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herbivory and emphasize the understudied role of plant size as an important cue in herbivore
colonization of plants in the field.
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INTRODUCTION
Plants have evolved different morphological structures such as trichomes and thorns, as
well as a wide variety of secondary chemical compounds including glucosinolates and phenolics,
which act as defense mechanisms against insect herbivore attacks (Levin 1973; Zangerl and
Bazzaz, 1992; Strauss and Agrawal, 1999; Rautio et al., 2002). Although chemical responses of
plants to insect herbivores have been widely studied (Bennett and Wallsgrove 1994; McConn et
al. 1997; Clauss et al. 2006; McConn et al. 1997; Kliebenstein 2004; Bidart-Bouzat et al. 2005),
little is known regarding how genetic variation in structural defensive traits may influence plant
damage levels and colonization of plants by natural insects. Trichomes are hair-like outgrowths
derived from the epidermal cells of plants that may have defensive function against herbivore
attacks (Figure1). Despite potentially important roles of trichomes in plant defense, most of the
evidence suggesting that trichomes may be involved in defense were obtained from studies
conducted under laboratory conditions using insect herbivores or through simulated herbivory
(Traw and Bergelson 2003; Clauss et al. 2006). Thus, these studies may not directly reflect the
role of trichomes on natural levels of insect herbivory. In addition, more information is required
on how genetic variation in trichome density influence patterns of plant utilization by insect
herbivores in natural populations. This is important because the ability of plants to respond to
herbivory in an evolutionary manner will mostly depend on the availability of genetic variation
in traits related to fitness.
Trichomes occur in different parts of the plants such as stems, leaves, and reproductive
organs, and vary within and/or between populations (Strauss and Agrawal 1999; Handley et al.
2005). They may also vary in their structure, ranging from unicellular to multicellular, and
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anatomy, which may be branched, unbranched, spiral or hooked (Ouwerkerk et al. 1999;
Hulskamp 2000). In addition, trichomes can be characterized as glandular or non-glandular
based on their ability to secrete chemical materials such as terpenes, alkaloids, or fatty acid
derivatives (Schilmiller et al. 2008; Wang et al. 2008). The presence of trichomes in many plant
species has been associated with many physiological and defensive functions. Leaf trichomes
may reduce insect herbivory by making leaves less attractive to herbivores, trapping small
insects, and reducing mobility and nutritional quality of leaves, which may in turn, influence
oviposition rates, egg hatching, larval growth, and survival. Glandular trichomes also produce
exudates that may be toxic or serve as repellant to insect herbivores (Perez-Estrada et al. 2000;
Simmons et al., 2004; Wang et al. 2004; Handley et al. 2005). In addition to the role of
trichomes in defense against herbivores, previous studies have also shown that trichomes may be
beneficial to herbivores and pathogens trapping natural enemies of insect herbivores (Turlings
and Benrey 1998; Lovinger et al. 2000; Kennedy 2003).
Apart from the role of trichomes in plant-herbivore interactions, there is evidence that
they play multiple functions in plants other than that of defense against herbivores. For example,
leaf trichomes may serve the purpose of resistance to other abiotic stress such as drought by
reflecting light and reducing evapo-transpiration rates or by facilitating condensation of
moisture onto the plant surface (Ehleringer 1984; Larcher 2001), as well as removal of excess
salt (Gravano et al. 1998) and heavy metals (Sarret et al. 2006). In addition, trichomes may
protect living cells from the harmful effects of ultraviolet radiation and extreme temperature
changes (Skaltsa et al. 1994; Werker 2000; Agrawal et al. 2004). Since all of these functions
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may act in concert or separately in different plant species, experimental approaches are required
to determine whether trichomes play an important role in plant resistance to insect herbivory.
Although the presence of trichomes can reduce levels of herbivore damage (Clauss et al.
2006; Handley et al. 2005), less information is known about the role of other phenotypic traits
such as plant size on plant utilization patterns by insect herbivores in the field. Plant size is an
important phenotypic trait that can affect not only the levels of damage to plants, but also host
choice by herbivores. This is because herbivores use both olfactory and visual cues to choose
host plants for oviposition (Singer 2004; Costa et al. 2009; Reeves et al. 2009). Several studies
have also suggested that larger plants are more likely to be first (and more often) colonized than
smaller plants, since plant size may be a reflection of vigor and nutrient availability, which in
turn may enhance herbivore performance (Heisswolf et al. 2005; Lastra et al. 2006). It is also
possible that bigger plants would be a better choice for oviposition purposes due to their ability
to provide a larger surface area for oviposition, especially in herbivores that produce large
amounts of eggs. For example, herbivores may deposit a cluster of eggs on a host plant that has
a large surface area for oviposition (Wahlberg et al. 2004; Saastamoinen 2007), whereas less
eggs per plant may be deposited on plants with smaller surface areas (Courtney 1982). Thus,
time (and costs) expended by insects on searching for suitable plants for oviposition may be
reduced when insects locate larger sized plants with larger surface areas (Nieminen et al. 2003;
Kuussaari et al. 2004; Singer 2004).
In a common garden experiment, the presence of natural variation in trichome density
and plant size was assessed in a population of recombinant inbred lines of Arabidopsis thaliana.
In addition, it was determined whether this variation influenced the patterns of plant use by
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insect herbivores in the field (i.e., order of herbivore colonization and levels of herbivore
damage). It was also tested whether variation in trichome density, plant size, and herbivore
damage levels was significantly associated to plant fitness. Evaluation of such link between the
trichome density, levels of herbivore damage, and plant fitness is important because differences
in herbivore damage may impose selection for different types of defensive traits. Finally, this
experiment evaluated whether selection was acting on plant size and trichome density, and
whether insect herbivores were indeed an important selective pressure on these traits. As
summarized in Mauricio and Rausher (1997), three conditions should be met for showing that a
plant character that influences levels of insect herbivory (e.g., trichome density or plant size), is
under selection by the herbivores. First, the character has to be genetically variable (potential to
evolve). Second, variation in the studied character must influence levels of herbivore damage,
and has to be associated with plant fitness. Lastly, it has to be demonstrated that the presence of
insect herbivores indeed influences selection estimates; that is, that upon removal of the selective
pressure imposed by insect herbivory, estimates of selection are modified. In this experiment,
selection on plant size was measured only in the presence of herbivores but selection on
trichomes was evaluated in both the presence and absence of herbivores. Specifically, insect
herbivores were removed from half of the field plots, and potential changes in the patterns of
selection on trichome density were evaluated by comparing selection gradients for this character
in the presence and absence of insect herbivores.
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MATERIALS AND METHODS
Study Plant
Arabidopsis thaliana (L.) Heynh (Brassicaceae) commonly known as the mouse ear
cress is an annual herbaceous weed, which is native to Europe and Asia but has spread its
distribution to North America and Africa. This plant species is naturally found in open or
disturbed habitats, sandy soils, riverbanks, roadsides, rocky slopes, and meadows (Baskins and
Baskins 1983). Arabidopsis thaliana has a short life cycle lasting between 6-8 weeks from
germination to senescence and it is a mostly self-fertilizing species with less than 1% outcrossing
rates (Hoffmann et al. 2003). Trichomes in A. thaliana are usually unicellular, branched, and
non-glandular (Hulskamp and Kirik 2000). In this study, we used a population of recombinant
inbred lines (100 RILs), which was derived from a cross between the ecotypes Columbia (Col-0)
and Landsberg (Ler), and advanced to the eighth generation through a single seed descent (Lister
and Dean 1993). Arabidopsis thaliana RILs from this cross were used because a previous study
showed variation in trichome density between the two parental lines, Col-0 and Ler ecotypes
(Mauricio 2005). Therefore, the use of this recombinant inbred line population allows evaluating
whether genetic variation in trichome density may influence natural patterns of insect herbivory.
Experimental Design and Plant Growth Conditions
The experimental design consisted of a split-plot with two levels of herbivory treatment
(i.e., natural insect herbivores present and excluded) assigned to ten plots distributed in a
checkerboard pattern (Figure 2). Each of these treatments was replicated in five plots accounting
for a total of ten plots. Naturally occurring insect herbivores were excluded in control plots
using an insecticide (Proclaim by Syngenta Inc. USA) at intervals of ten days throughout the
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experimental period. Three replicates of the 100 RILs were randomly assigned to each of the 10
plots (total of 3000 plants). For both treatments, mammalian herbivores were excluded by using
wire mesh fencing.
Several species of insect herbivores are known to attack A. thaliana plants in the field.
Bidart-Bouzat and Kliebestein (2008) reported the presence of diamondback moths (Plutella
xylostella; Plutellidae), striped and western cabbage flea beetles (Psylloides striolata and
Psylloides pusilla, respectively; Chrysomelidae), cabbage aphids (Brevicoryne brassicae;
Aphididae) and onion thrips (Thrips tabaci; Thripidae) as natural herbivores observed feeding on
different A. thaliana genotypes. Other studies (Mauricio 1998; Mosleh Arany et al. 2005; Arany
2008) have also reported other insect herbivores feeding on filed-grown A. thaliana plants
including the cabbage looper Tricoplusia ni (Noctuidae), Spodoptera exigua (Noctuidae),
European cabbage butterfly Pieris rapae (Pieridae), and specialist weevils Ceutorhynchus
atomus and C. contractus (Curculinoidae). During this field study, the most common herbivore
observed attacking A. thaliana plants was the specialist diamondback moth (Plutella xylostella).
The generalist cabbage looper (Tricoplusia ni) and the western cabbage flea beetles Psylloides
pusilla were also observed feeding on the plants, but causing minor effects.
In terms of growth conditions, seeds were cold-treated for 5 days at 4°C to break
dormancy, and then, transferred to growth chambers for germination. Within the chambers,
plants were exposed to 10 hours of light and 14 hours of dark cycles, with day and night
temperatures of 20°C and 16°C, respectively. Plants were rotated within growth chambers every
other day and among chambers every week to avoid chamber and position effects. After five
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weeks of growth in the chambers, plants were transplanted to the field (Ecological Research
Station, Bowling Green State University, Bowling Green, Ohio).
Measurement of Plant Traits
In this experiment, the following plant traits were measured: trichome density, rosette
diameter, and total fruit number. Trichome density was measured using digital images of the
youngest fully extended rosette leaf per plant at bolting time. Leaf area was estimated using
image J software (rsbweb.nih.gov/ij/). Trichome density was calculated as the total number of
trichomes (on the upper side of the leaf) divided by leaf area. In order to evaluate if trichomes
are inducible in the presence of herbivores, we compared trichome density in sprayed plants to
plants exposed to insect herbivores. To estimate plant size, rosette diameter was measured as the
widest length of the plant rosette. A previous study has shown rosette diameter to be strongly
correlated with rosette area, a measure of plant size (Mauricio 1998).
Fitness was estimated as the lifetime fruit production and relative fitness as absolute
fitness divided by the mean absolute fitness per herbivory level. Total fruit production was
previously shown to be strongly correlated with total seed production in Arabidopsis thaliana
(Mauricio and Rausher 1997; Bidart-Bouzat 2004). Since selection operates at a population
level, and this selection is based on the differences in fitness between individuals; therefore, the
use of relative fitness is more appropriate than using absolute fitness (Cornner and Hartl 2004).
To assess whether plant genotype affected the order of colonization of insect herbivores among
plants, all plants were inspected daily for insect damage and the numbers of days to first
observed herbivore damage on each plant was recorded herein, referred to as colonization. To
estimate insect herbivore damage to the plant rosette leaves (i.e., percent area removed by
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herbivores), a qualitative leaf damage index adapted from Bidart-Bouzat et al. (2005) was used.
This index was calculated by assigning leaves to three size categories based on leaf length (i.e.,
small: 0- 3cm, medium: 3 - 6cm, and large: 6-9cm), and three damage categories based on the
amount of tissue removed (i.e., < 25%, 25-75 %, and > 75).
Statistical Analysis
To evaluate the effects of RILs (i.e., genotype) and block on trichome density, rosette
diameter, days to first observed herbivore damage, rosette damage, and total number of fruits,
analyses of variance using a linear random model (SAS statistical software, version 9.1) were
performed. Data was either log- or ranked-transformed to correct for normality and
heteroscedasticity, when necessary. In addition, we performed a series of analyses of covariance
to infer whether the associations between the response variables: herbivore colonization and
damage, and the covariates: trichome density and rosette diameter (estimate of plant size) were
affected by the plant genotype (RIL) using the model: Y = covariate + RIL + covariate × RIL +
error.
To evaluate patterns of selection on trichome density, plant size, order of herbivore
colonization and rosette damage, a regression analysis was used to estimate directional (β) and
stabilizing/disruptive (γ) selection gradients. In addition, it was determined whether herbivores
were exerting selection on trichome density, by performing analyses of variance on the
directional and stabilizing selection gradients to compare patterns of selection on trichome
density in the presence and absence of natural herbivores (Lande and Arnold 1983; Mauricio and
Rausher 1997). For this purpose, a mixed linear (for directional selection) and quadratic (for
stabilizing selection) models were used, considering the herbivory treatment as a fixed effect and
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RIL as a random effect. The effect of block and its interactions were removed from this model
because they were not significant.
RESULTS
Analyses of variance revealed that all traits measured in this study were highly variable
(Table 1). Effects of RIL and herbivory on trichome density were significant (P= <0.0001) and
plant responses to herbivory were found to be dependent on the RIL (i.e., Herbivory × RIL
interaction: P= 0.0233). In addition, the effect of RIL on plant size, days to herbivore
colonization, and rosette damage were all statistically significant.
Analyses of covariance showed that variation in trichome density significantly influenced
herbivore damage levels but it did not affect days to colonization of plants by insect herbivores
(Figures 3A and 3B). On the other hand, plant size (rosette diameter) significantly influenced
both the number days to herbivore colonization and herbivore damage levels (Figures 3C and
3D). In any case, the effect of RIL (plant genotype) was significant in all ANCOVA models. In
addition, the relationship between the covariates and both damage and colonization was not
significantly affected by RIL (Figures 3A-D). These results suggest that trichome density and
plant size may play an important role in plant susceptibility/resistance to insect herbivory as well
as in determining patterns of plant colonization by insect herbivores in the field.
Regarding the patterns of selection on variables related to plant size and trichome density,
directional regression analyses revealed that relative fitness was inversely associated with
trichome density in sprayed plots but not in the plots exposed to insect herbivory (Figure 4). In
other words, selection favored genotypes with lower trichome density in sprayed plots but not in
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plants exposed to insect herbivores. Conversely, plant fitness was higher as plant size increased
(Figure 5), which implies that genotypes that produce larger plants are favored by selection.
There was no evidence of stabilizing or disruptive selection acting on either plant size or
trichome density. In addition, regression analyses did not indicate any form of selection acting
on days to herbivore colonization (Figure 6A); there was a borderline significant selection
differential however, indicating directional selection favoring a decrease in rosette damage
(Figure 6B).
In order to detect whether insect herbivores are exerting selection on trichome density,
we compared general patterns of selection on trichome density in the presence and absence of
natural herbivores using both directional and stabilizing gradients of selection (Tables 2A and
2B). Results revealed that the effect of herbivory on directional selection for trichome density
(i.e., trichome × herbivory interaction) was significant but the quadratic term for trichome
density was not significant, suggesting that insect herbivores are exerting directional selection
but not stabilizing selection on trichome density.
DISCUSSION
Herbivores are known to be attracted to their host plants through visual and olfactory
cues (Costa et al. 2009; Reeves et al. 2009; Singer 2004). These cues include plant volatiles
(Costa et al. 2009; Szendrei and Isaacs 2005), plant size (Handley et al. 2005) , shape, orientation
and plant age, which may in turn influence the type and quantity of volatiles released (Moreau et
al. 2003; Vergés et al. 2007; Wentzell and Kliebenstein 2008). This study assessed the role of
trichome density and plant size in determining patterns of plant utilization by insect herbivores in
13
the field. Results from this study showed that larger plants were the first to be colonized by
insect herbivores. This result further supports previous studies that have shown that plant size
may be important in host preference (Nieminen et al. 2003; Kuussaari et al. 2004; Heisswolf et
al. 2005; Lastra et al. 2006). First, larger plants may be colonized in preference of smaller plants
because they are visually more conspicuous to ovipositing females when compared with smaller
plants. Second, larger plants may be advantageous to herbivores in that they may be more
suitable for larval development. This is because larvae movement may be restricted during early
instars; thus, herbivores may show more preference for a plant that provides larger surface area
and more nutrients (Nieminen et al. 2003; Kuussaari et al. 2004). In addition, the plant vigor
hypothesis also predicts that vigorous plants with larger size will be preferred by herbivores
because they are more likely to have higher biomass and nutritional quality than smaller plants
(Heisswolf et al. 2005; Lastra et al. 2006)
Furthermore, this study experimentally demonstrated the role of trichomes in resistance
against insect herbivores in the annual crucifer A. thaliana. Herbivore damage levels
significantly decreased with increased trichome production. In addition, the exclusion of
herbivores resulted in a change in selection on trichome production, demonstrating that insect
herbivory is indeed an agent of selection on this character. These results are in agreement with
other studies that have shown that the presence of trichomes may lead to a reduction in the levels
of insect damage. For example, Mauricio (1988), in a field experiment, demonstrated that leaf
trichomes led to a decrease in herbivore damage in 144 natural populations of A. thaliana.
Similarly, leaf trichomes have been found to be associated with reduced level of herbivory in the
closely related Arabidopsis lyrata (Handley et al. 2005; Loe et al. 2007), as well as other
14
members of the Brassicaceae plant family (Lamb 1980; Agren and Schemske 1993). With
respect to plant size, larger plants had more damage than smaller plants. This further supports
the earlier observation that insect herbivores may link larger size to higher nutritional quality for
larval development. Additionally, larger plant size may indirectly imply better larval
performance by making them less vulnerable to plant defense traits if, as previously suggested,
more resources are allocated to vegetative growth at the expense of allocation to defense (Obeso
2002).
In order to investigate patterns of selection on trichome density, plant size, and plant
utilization by insect herbivores, regression analyses of these variables on relative fitness were
performed. Directional selection on trichome density was observed, which favored a decrease in
the production of this defensive character in sprayed plots. Conversely, no evidence of selection
acting on this character was found in plots exposed to insect herbivores. This result suggests the
existence of a cost of trichome production in the absence of insect herbivory (Bergelson and
Purrington 1996; Mauricio 1998). It is interesting to note that in contrast to what was expected,
there was no evidence of directional selection for increase in trichome production in the presence
of herbivory. In other words, since results showed that trichomes reduced levels of insect
herbivore damage and that there was negative selection acting against herbivore damage, then, it
was expected that selection would favor an increase in defense related characters such as
trichomes in the presence of herbivores (Valverde et al. 2001). The lack of positive selection for
trichome density in the presence of insect herbivores may imply the presence of a cost of
resistance; that is, production of trichomes is expensive and the benefit derived in terms of
resistance may not compensate for its cost. This may also explain the observed directional
15
selection for decreased trichome production in the sprayed plots when the benefit of resistance to
herbivory was absent. This result was in agreement with a previous study by Mauricio (1998) in
which trichome density was negatively associated with relative fitness, indicating the presence of
a fitness cost for trichome production (but see Valverde 2001). Another possible explanation for
this result could be that the levels of herbivore damage observed were not large enough to
reverse the direction of selection on trichome production in the A. thaliana populations used in
this study. Alternatively, there may have not been enough time for the A. thaliana population to
respond to the presence of natural herbivores (Mauricio and Rausher 1997). This is because
genotypes used in this study are recombinant inbred lines, which are not originated from the
study area. Therefore, it may require a longer period of time in order to observe a shift in the
direction of selection in this population. Anyhow, the direction of selection did show a shift
towards positive selection, but the shift was not sufficiently large to be significant. Taken these
results together, we predict that the cost of resistance may be acting to slow down the shift in the
direction of selection for trichome density and that given a longer period of time under the same
environmental conditions this selection gradient may become positive.
In spite of the positive association between plant size and both herbivore damage and
colonization, selection analyses revealed that selection favored larger plant size. Selection for
larger plant size suggests that mechanisms leading to tolerance of insect herbivory may be
favored (Paige 1992; Juenger and Bergelson 2000; Rautio et al. 2002; Freeman et al. 2003;
Gonzalez-Teuber and Gianoli 2007); for example, by increased vegetative biomass growth
compensating for plant tissue lost to herbivory. This is an alternative strategy of plants to
minimize the detrimental effects of herbivory on fitness. Paige and Whitham (1987) have
16
documented this phenomenon of compensation in the biennial Ipomopsis aggregata, which
showed a release of apical meristem dominance and the proliferation of lateral branches when
browsed by mammalian herbivores like mule deer and elk. This study also demonstrated that
insect herbivores impose selection on trichome density, which was documented by a significant
effect of insect herbivores in modifying patterns of directional selection for trichome density. To
date, only two studies have demonstrated this criterion for trichome density in the field, despite
the claim that co-evolution is the main driving force for the evolution of resistance to insect
herbivory (Mauricio and Rausher 1997; Valverde et al. 2001). This is because any claim that an
environmental factor is having an evolutionary impact on a character must be able to
demonstrate the existence of genetic variation in the character of interest, a link between
variation in the resistance character and fitness, and most importantly, that the presence of the
selective agent is imposing direct selection on the character. Two previous studies (Mauricio
and Rausher 1997; Valverde et al. 2001) as well as the present study have experimentally
manipulated the presence of the selective agent (insect herbivores) to show how its absence
influenced patterns of selection on trichome density. Although this study does not rule out the
possibility of other environmental factors simultaneously imposing selection on trichomes, it
does support the idea that insect herbivores are indeed important selective agents influencing the
evolution of defense structures in plants.
17
Table 1. ANOVA-F values showing the presence of genetic variation (RIL factor) in measured
phenotypic characters.
Source of variation
Block
Herbivory
RIL
Herbivory x RIL
Trichomes Plant size Days to
colonization
20.90***
21.67*** 0.08
21.16***
2.47***
3.36***
1.34*
1.31*
-
Significance levels * P<0.05, ** P<0.01, *** P<0.001
Rosette
damage
16.01***
1.81***
-
18
Table 2A. Analysis of variance for relative fitness showing the effects of insect herbivores on
directional selection for trichomes in A. thaliana.
Source of variation
RIL
Herbivory
Trichome Density
Herbivory x Trichome Density
Error
F-value
1.61
0.48
0.03
3.94
P- value
0.0095
0.4900
0.8654
0.0499
Table 2B. Analysis of variance for relative fitness showing the effects of insect herbivores on
stabilizing/disruptive selection for trichomes in A. thaliana.
Source of variation
RIL
Herbivory
Trichome Density
Trichome Density sq
Herbivory x Trichome Density sq
Error
F-value
1.52
0.56
0.20
0.10
0.18
P-value
0.0211
0.4546
0.6538
0.7579
0.6713
19
Figure 1. Picture of Arabidopsis thaliana plant showing the presence of trichomes
20
A
B
C
D
E
HERBIVORY
SPRAYED
A
B
C
D
E
Figure 2. A split- plot experimental design with two levels of herbivory (herbivory and sprayed)
distributed in a checkerboard pattern among ten plots. Different letters indicate different plots
per treatment.
21
Source of variation
RIL
Trichomes
Trichomes*RIL
(A)
Days to Colonization
16
F-value
1.43**
0.84
0.93
Source of variation
RIL
Rosdiameter
Rosdiameter*RIL
(C)
F- value
1.39*
7.34**
0.91
14
12
10
8
Rosette Damage (%)
30
Source of variation
RIL
Trichomes
Trichomes*RIL
(B)
F- value
1.65***
6.89***
1.22*
Source of variation
RIL
Rosdiameter
Rosdiameter*RIL
(D)
F- value
1.66***
7.12**
1.17
20
10
150
200
250
300
Trichome Density
350
5
6
7
8
Plant Size (cm)
Figure 3. Analysis of covariance showing the relationship between trichome density and days to
colonization (A) or rosette damage (B), and the relationship between rosette diameter and either
days to colonization (C) or rosette damage (D). * P<0.05, ** P<0.01, *** P<0.001
22
(A)
Relative Fitness
1.6
β = -0.01024
γ = -0.05079
1.4
1.2
1.0
0.8
0.6
-2
0
2
Standadized Trichome Density
(B)
Relative Fitness
1.6
β = -0.10302*
γ = 0.01621
1.4
1.2
1.0
0.8
0.6
-2
-1
0
1
2
Standardized Trichome Density
Figure 4. Directional (β) and stabilizing/disruptive (γ) selection gradients estimated by
regression selection analyses on trichome density in the presence (A) and absence (B) of insect
herbivores. * P<0.05
23
Relative Fitness
1.6
β = 0.16651***
γ = 0.01562
1.4
1.2
1.0
0.8
0.6
-2
0
2
Standardized Plant Size
Figure 5. Directional (β) and stabilizing/disruptive (γ) selection gradients estimated by
regression selection analyses on plant size in the presence of insect herbivores. *** P<0.001
24
(A)
Relative Fitness
1.6
β = 0.00849
γ = 0.05738
1.4
1.2
1.0
0.8
0.6
-2
-1
0
1
2
Standardized Days to Colonization
(B)
Relative Fitness
1.6
β = -0.08635†
γ = 0.03505
1.4
1.2
1.0
0.8
0.6
-2
0
2
4
Standardized Rosette Damage
Figure 6. Directional (β) and stabilizing/disruptive (γ) selection gradients estimated by
regression selection analyses on number of days to first herbivore colonization (A) and insect
herbivore damage (B). † P<0.1
25
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34
CHAPTER 2
CHANGES IN ULTRAVIOLET B RADIATION MODIFY RESISTANCE AND
TOLERANCE IN ARABIDOPSIS THALIANA TO INSECT HERBIVORY.
ABSTRACT
Plants have evolved two major mechanisms in response to frequent attacks from insect
herbivores. While resistance reduces the levels of damage suffered by plants, tolerance reduces
the negative effects of herbivory on plant fitness. Despite their roles in defense and consequent
fitness advantage, the evolution of these two characters may be constrained by the fitness costs
associated with their production. In addition, these costs may be influenced by variation in
environmental conditions. In a common garden experiment, the effects of ultraviolet B (UVB)
radiation on plant resistance and tolerance to insect herbivory was evaluated by exposing
recombinant inbred lines of Arabidopsis thaliana to natural levels of insect herbivory under two
levels of UVB radiation (ambient and excluded UVB). Specifically, this study evaluated the
existence of genetic variation in plant resistance and tolerance to insect herbivory under variable
UVB radiation and whether changes in UVB radiation modify fitness costs associated with
tolerance and resistance. Results from this study revealed the presence of genetic variation in
plant resistance and tolerance and that this variation is dependent on UVB radiation levels. In
addition, there were differences in the fitness cost of plant resistance and tolerance across UVB
environmental gradients. No trade-off between resistance and tolerance however, was observed.
These results highlight implications of changes in UVB radiation on future evolution of
important plant characters involved in plant defense to insect herbivory.
35
INTRODUCTION
Resistance and tolerance represent two major mechanisms by which plants may reduce
the detrimental effects of insect herbivores on their fitness. While resistance directly influences
the amount of damage inflicted on plants by their herbivores, tolerance reduces the fitness
consequences of damage (Rausher 1992; Strauss and Agrawal 1999; Stowe et al. 2000).
Resistance may be expressed in the form of production of structural barriers such as trichomes,
and thorns, or chemical toxins like phenolics and glucosinolates (Levin 1973; McConn et al.
1997; Strauss and Agrawal, 1999; Rautio et al., 2002 Handley et al. 2005; Clauss et al. 2006;
Kliebenstein 2004; Bidart-Bouzat et al. 2005). On the other hand, tolerance through
compensatory growth enables plants to maintain fitness despite biomass loss resulting from
herbivore damage. Compensatory responses may include the removal of apical dominance and
proliferation of lateral branches (Paige 1992; Juenger and Bergelson 2000; Rautio et al. 2002;
Freeman et al. 2003), extension of flowering period (Pilson and Decker 2002; Freeman et al.
2003), and shifts in resource allocation to different plant parts or plant architecture (GonzalezTeuber and Gianoli 2007).
Although herbivores may impose directional selection on resistance and tolerance
(Marquis 1992; Valverde et al. 2001), several studies have failed to find this trend in both field
and laboratory studies (Mauricio and Rausher 1997; Weinig et al. 2003). One major reason for
this observation is the potential presence of fitness costs, which may constrain the evolution of
resistance and tolerance (Fineblum and Rausher 1995; Stowe 1998; Pilson 2000). Fitness costs
may arise due to potential tradeoffs between defense and other functions, such as growth and
reproduction (Herms and Mattson 1992; Belsky et al. 1993; Vandermeijden et al. 1988; Obeso
36
2002; Agrawal et al. 2004). Consequently, in the absence of herbivory, more resistant or tolerant
plants would have lower fitness compared to the most susceptible ones. Yet, in the presence of
herbivory, the benefit of resistance or tolerance may be able to offset their associated costs.
Thus, the evolution of these characters will depend on the balance between their associated costs
and benefits (Obeso 2002).
Multiple environmental factors such as nutrient availability, competition, elevated CO2,
and ozone, have been shown to modify the expression of tolerance and resistance to insect
herbivore damage (Bolsinger et al. 1992; Bidart-Bouzat et al. 2005; Zvereva and Kozlov 2006;
Caldwell et al. 2007; Veteli et al. 2007; Lau and Tiffin 2009). Even though considerable work
has demonstrated how environmental factors may influence costs of resistance (Bergelson and
Purrington 1996; Prittinen et al. 2003), only few studies have evaluated environmental effects on
the costs of tolerance (Agrawal et al. 2004; Stevens et al. 2007). In addition, both empirical and
theoretical studies have not shown a consistent pattern on how the environment may modify
these costs (Bergelson and Purrington 1996). For example, while some authors have predicted a
higher cost of resistance or tolerance under more stressful conditions (Mutikainen et al. 2002),
others have predicted a reverse trend (Herms and Mattson 1992). An understanding of how
environmental factors may influence genetic variation in tolerance and resistance, as well as their
associated costs, may help in predicting evolutionary trajectories of these characters in future
changing environment (Tiffin 2000a).
Ultraviolet B (UVB) radiation represents a small fraction of the solar spectrum (290-315
nm). The level of UVB radiation reaching the surface of the Earth has been recently changing
due to the depletion of the trophospheric ozone layer, pollution, and cloud cover
37
(Intergovernmental Panel on Climate Change, IPCC 2007). Several studies have evaluated the
effect of UVB radiation on plant growth and physiological processes (Hader et al. 2000; Searles
et al. 2001), ranging from inhibition of photosynthetic activities to modification of flowering
patterns, biomass accumulation, and reproduction (Searles et al. 2001; Tegelberg et al. 2004).
With respect to plant chemistry, UVB radiation can increase accumulation of UVB-absorbing
compounds such as flavonoids and phenolics, which may in turn influence plant’s interactions
with higher trophic levels (Agrell et al. 2000; Searles et al. 2001; Hofmann et al. 2003; Izaguirre
et al. 2003; Stratmann 2003; Bassman 2004; Izaguirre et al. 2007). For example, Izaguirre et al.
(2007) reported that the same phenolics compounds commonly induced in response to insect
herbivory were found to be induced in Nicotiana attenuata and Nicotiana longifolia when
exposed to enhanced UVB radiation. Similarly, Stratmann (2003) detected an increase in
phenylpropanoids in plant plants exposed to ambient UVB radiation when compared with plants
grown under excluded UVB (using light filters) suggesting that UVB radiation may influence
plant-insect interactions.
Since variation in UVB levels has been shown to affect different plant characters related
to defense, growth and reproduction (Veteli et al. 2003; Bassman 2004; Rousseaux et al. 2004),
it is expected that these changes could have an effect on plant resistance and/or tolerance to
insect herbivory. Previous studies have focused on the effect of changes in UVB radiation levels
on the production of plant secondary chemicals, such as phenolics, which have a role in
resistance against insect herbivores (Agrell et al. 2000; Searles et al. 2001; Hofmann et al. 2003;
Izaguirre et al. 2003; Stratmann 2003; Bassman 2004; Izaguirre et al. 2007). No information
appears to exist however, on the role of UVB on structural defenses, such as trichomes or
38
characters related to plant tolerance against insect herbivory. With respect to the effects of
variable environments on resistance and tolerance, previous studies have shown variable
responses of characters related to resistance and tolerance (Ziska and Teramura 1992; Dube and
Bornman 1992; Tevini 1993; McDonald et al. 1999; Agrell et al. 2000; Lavola et al. 2000;
Hofmann et al. 2003). However, there is a lack of information regarding effects of the UVB
environment on potential fitness costs related to plant resistance and tolerance.
This study presents data from a common garden experiment in which a population of
recombinant inbred lines (RIL) of Arabidopsis thaliana was exposed to two different levels of
UVB radiation (i.e., ambient- and excluded-UVB) and insect herbivory (i.e., sprayed plants and
plants exposed to natural levels of insect herbivory). The main goal of this study was to
determine whether there was genetic variation in resistance and tolerance to insect herbivory in
this A. thaliana RIL population under variable UVB radiation levels, and to assess potential
fitness costs associated with plant resistance and tolerance. An understanding of the effects of
UVB on plants as well the fitness costs associated with plant response to this environmental
factor in terms of resistance and tolerance is important because they may constrain the future
evolution of these characters, which in turn may alter the dynamics of plant-insect interactions.
For example, if costs increase with enhanced UVB levels, plant resistance and tolerance may be
more commonly expressed as inducible strategies in order to minimize their costs (Simms and
Rausher 1987, Herms and Mattson 1992; Bergelson and Purrington 1996; Cipollini 1998;
Purrington 2000). In addition, the expression of fitness costs across different UVB environments
may provide information regarding whether either plant tolerance or resistance are favored over
the other, or whether these strategies are likely to be maintained at intermediate levels.
39
MATERIALS AND METHODS
Study Plant
Arabidopsis thaliana (L.) Heynh (Brassicaceae), also known as the mouse ear cress, is
an annual herbaceous plant native to Europe and Asia with a wide geographic distribution across
North America and Africa. This plant species is naturally found in open or disturbed habitats,
sandy soils, riverbanks, roadsides, rocky slopes, and meadows (Baskins and Baskins 1983).
Arabidopsis thaliana has a short life cycle lasting between 6-8 weeks from germination to
senescence and it is mostly a self-fertilizing species with less than 1% outcrossing rates
(Hoffmann et al. 2003). Because of the high selfing rates, natural populations seem to consist
majorly of a single genotype. However, a significant level of genetic variation exists among
different ecotypes in both reproductive and life history characters (Bergelson et al. 1998;
Mauricio 1998; Nordborg and Bergelson 1999). In A. thaliana, two types of resistance to insect
herbivory have been reported. These include structural defenses, such as trichomes, and
chemical defenses such as glucosinolates, which are known to vary across populations (Mauricio
and Rausher 1997; Valverde et al. 2001; Bidart-Bouzat et al. 2005; Clauss et al. 2006; Loe et al.
2007). In this study, 100 recombinant inbred lines (RILs) derived from a cross between the A.
thaliana ecotypes Columbia (Col-0) and Landsberg (Ler), and advanced to the eighth generation
through a single seed descent were used (Lister and Dean 1993). A RIL population was used in
order to assess intraspecific genetic variation in plant resistance and tolerance to UVB radiation.
Experimental Design
A common garden experiment was performed to assess the effects of UVB radiation and
plant genotype on tolerance and resistance in A. thaliana. This experiment included two UVB
40
treatment levels (total of 20 plots), which consisted of ambient (UVB+) and excluded (UVB-)
ultraviolet B radiation (Figure 1). Ultraviolet B radiation was excluded from the randomly
assigned UVB- plots using Mylar polyester film. This film transmits radiations only in the
wavelength greater than 315 nm, thereby blocking UVB radiation but transmitting radiations in
UVA regions. For the UVB+ treatment, plots were covered with clear acetate film that allowed
most UVB radiation to pass through (see Figure 2). Half of the plots assigned to each UVB level
were exposed to natural levels of insect herbivory and the other half were excluded from insect
herbivores by spraying the plots with a pesticide (Proclaim by Syngenta Inc. USA) at ten days
interval. Plants excluded from insect herbivory were inspected on a daily basis to ensure there
was no herbivore damage. Each plot was surrounded with a wire-mesh fence to exclude
mammalian herbivory.
One hundred recombinant inbred lines (RILs) of A. thaliana were used in this study.
Seeds were initially cold-treated for 5 days at 4°C to break dormancy, and then, transferred to
growth chambers for germination. Within the chambers, plants were exposed to 10 hours of
daylight and 14 hours of night, with day and night temperatures of 20°C and 16°C, respectively.
Plants were rotated every other day within growth chambers and every week among chambers to
avoid chamber and position effects. After five weeks of growth in the chambers, plants were
transplanted to the field (Ecological Research Station, Bowling Green State University, Bowling
Green, Ohio). Two sets of 100 RILs were randomly assigned to each of the 20 plots (total of
4000 plants).
41
Estimation of Resistance and Tolerance
To evaluate the effects of UVB radiation on plant resistance and tolerance, two resistance
measures (defined as direct and indirect resistance) and one measure of tolerance were estimated
for each RIL. The direct measure of resistance was estimated as trichome density. Trichome
density was measured using digital images of the youngest fully extended rosette leaf per plant at
bolting time. Leaf area of the selected rosette leaves was estimated using image J software
(www.rsbweb.nih.gov/ij). Trichome density was calculated as the total number of trichomes (on
the upper side of the leaf) divided by the leaf area. The indirect measure of resistance was
estimated by subtracting a proportion of the rosette area damaged from one (i.e., 1- damage).
This measurement assumes that the level of herbivore damage sustained is a reflection of the
level of resistance (i.e., a plant with a high resistance level will have low levels of herbivore
damage and vice versa). This indirect estimate of resistance is important because it accounts for
all types of resistance mechanisms present in this plant species (both structural and chemical).
Rosette leaf damage (i.e., percent area removed by herbivores on the rosette leaves) was used as
a measure of insect herbivore damage, which was estimated as a qualitative leaf damage index
(Bidart-Bouzat et al. 2005). This index was calculated by assigning leaves to three size
categories based on leaf length (i.e., small: 0- 3cm; medium: 3 - 6cm; and large: 6-9cm), and
three damage categories based on the amount of rosette leaf tissue removed (i.e., < 25%, 25-75
%, and > 75%).
To estimate plant tolerance, plant fitness was measured as total fruit number, which is an
estimate of lifetime fitness in A. thaliana, and further compared among damaged and undamaged
plants. Relative fitness was estimated as absolute fitness divided by the mean absolute fitness of
42
the population of RILs per UVB and herbivory treatment level. Tolerance was estimated by
comparing the relative fitness of the damaged plants (WD) to that of the undamaged plants (WU).
This may be estimated as the ratio of the relative fitness of the undamaged to the damaged plants
(WD/WU) or as the difference (WD-WU) between the relative fitness of damaged and the
undamaged plants (Strauss and Agrawal 1999). The choice between these two approaches
depends on how the damage data were collected. For example, when damage data are
categorical (i.e., presence or absence of damage), an approach using the difference in mean
relative fitness would be more appropriate; however, when damage data are continuous, the use
of the ratio would be more indicated. This is because tolerance estimated as a ratio is
conceptually similar to using a regression coefficient of fitness on damage. This is equivalent to
defining tolerance as a reaction norm along a gradient of variable levels of damage, such that the
slope describes to what extent plant fitness has been affected by damage (Mauricio and Rausher
1997; Simms and Triplett 1994; Tiffin and Rausher 1999; Strauss and Agrawal 1999). In this
study, rosette damage was estimated using a damage index, which vary in a continuous fashion.
Therefore, tolerance was estimated as the ratio of the relative fitness of the undamaged to the
damaged plants (WD/WU). Two approaches were used to evaluate costs associated to resistance
and tolerance, as well as the effect of UVB radiation on these costs. First, fitness costs were
detected through Pearson’s correlation coefficients as the presence of a negative association
between relative fitness of the undamaged plants and either resistance or tolerance under each
level of UVB radiation. Second, the existence of a potential trade-off between plant resistance
and tolerance was also assessed using correlation analysis.
43
Statistical Analysis
To evaluate the effects of UVB radiation and RIL on indirect resistance, and those of
UVB, herbivory and RIL on trichome density, analyses of variance were performed using the
PROC GLM procedure (SAS statistical software, version 9.1). Data was either log- or rankedtransformed to correct for normality and heteroscedasticity when necessary. For this purpose, a
mixed linear model was used for trichome density, considering UVB radiation and herbivory as
fixed effects, and block, RILs and its interactions as random effects. For indirect resistance,
herbivory was not included in the model since this measure was estimated only from plants
exposed to herbivory. In addition, higher-order block interactions were first included in the
model but then excluded because they were not significant. Regarding tolerance, since it was not
possible for a plant to be damaged and undamaged at the same time, tolerance was estimated as
means for each RIL. In order to show the presence of genetic variation for tolerance, an analysis
of covariance was performed including relative fitness as the dependent variable and rosette
damage as the covariate using the following model: Relative fitness = Block + RIL + Rosette
damage + RIL × Rosette damage + Error. Significant rosette damage × RIL interaction indicates
the presence of genetic variation in tolerance.
Cost of resistance was evaluated using Pearson’s correlation coefficients as estimates of
potential genetic associations between the RIL means of relative fitness of undamaged plants and
the two measures of resistance; that is, trichome density and indirect resistance (1-damage).
Relative fitness of the undamaged plants was used based on the premise that in the absence of
herbivory, the benefit of being resistant is removed and only the cost will be manifested. Thus, a
significant negative genetic correlation coefficient between relative fitness and resistance in the
44
absence of damage implies the presence of a fitness cost. In the case of tolerance, however, a
direct genotypic correlation between the fitness of the undamaged plant and tolerance would be
biased (Tiffin and Rausher 1999). This is because using the same data to estimate relative fitness
of the undamaged plants and tolerance would introduce an artifactual covariance (Tiffin and
Rausher 1999). This artifactual covariance was calculated and subtracted from the estimated
covariance between tolerance and relative fitness of undamaged plants. A bootstrap technique
was used to generate a 95% confidence limit in order to determine if the negative covariances
have confidence limits that do not include zero using a modified version of the SAS macro as
described in Stinchcombe (2005). This method compiles the bootstrap distribution of each
corrected covariance, and the 2.5 and 97.5 percentile of this distribution formed the 95%
bootstrap percentile confidence interval. Thus, if the confidence interval failed to include zero,
then the p-value is deemed to be less than or equal to 0.05, and the effect is said to be significant.
RESULTS
Analyses of variance revealed significant UVB effects on both direct (trichome density)
and indirect resistance measures as well as genetic variation in these characters’ responses to
changes in UVB variation (see Figure 3A, 3B, 3C and Table 1). Overall, exclusion of UVB and
herbivores resulted in a decrease in trichome density, although this effect was more pronounced
in the sprayed treatment (herbivory excluded) as indicated by a significant herbivory by UVB
interaction. In addition, plant responses to UVB radiation in terms of trichome density were
found to be dependent on both RIL and herbivory (i.e., significant UVB × RIL interaction)
(Table 1). Effects of herbivory on trichome density were found to be marginally dependent on
45
RIL and the three-way interaction (UVB × Herbivory × RIL) was statistically not significant
(Table 1). This non-significant three-way interaction indicates that UVB radiation affected
trichomes density in response to herbivory in the same way across the genotypes (Table1). With
respect to tolerance to insect herbivory, a significant genetic variation in tolerance was revealed
by a significant RIL × rosette damage interaction in an analysis of covariance using relative
fitness as the dependent variable (Figure 3D).
In order to evaluate fitness costs of resistance (both direct and indirect resistance
measures) and tolerance, as well as whether variation in UVB radiation influenced these costs,
genotypic correlations between relative fitness of the undamaged plants and resistance or
tolerance were performed. Overall, results suggest that changes in UVB radiation influence
fitness costs associated with both direct and indirect resistance as well as tolerance to insect
herbivory. In terms of direct resistance (trichome density), results from genotypic correlations
did not reveal any significant trade-offs between relative fitness and trichome density under
ambient UVB radiation levels (r=-0.06, P=0.596); (Figure 4A). However, a marginally
significant cost was observed in plants excluded from UVB radiation (r=-0.17, P=0.0875);
(Figure 4B). In terms of resistance, a negative genotypic correlation between relative fitness of
the undamaged plants and indirect resistance was detected under ambient UVB, which suggests
the presence of fitness cost in this environment (r=-0.31, P=0.0015); (Figure 4C). Conversely,
under the excluded UVB regime, although an inverse trend was observed, the correlation was not
significant (r=-0.13, P=0.1889); (Figure 4D).
In terms of tolerance, results revealed a significant negative genotypic correlation
between relative fitness of the undamaged plants and tolerance under both ambient and excluded
46
UVB radiation levels (r=-0.68, P<.0001; and r=-0.36, P=0.0003; respectively); (Figures 4E and
4F). However, after correcting for the artifactual covariance, the corrected covariance was
negative under ambient UVB but not under excluded UVB (Table 3). This result suggests that
cost of tolerance appears to be present only under ambient UVB. The significant negative
genetic correlation observed under excluded UVB may have been due to the presence of an
artifactual covariance as result of using the same data to estimate relative fitness of the
undamaged plants and tolerance. Despite the presence of fitness costs for both indirect
resistance and tolerance, evaluation of the potential presence of trade-offs between tolerance and
trichome density did not reveal any form of trade-offs under ambient UVB (r=0.13518,
P=0.1799) or excluded UVB (r=0.014, P= 0.888); (Figures 5A and B). Similarly, evaluation of
trade-offs between tolerance and indirect resistance did not reveal any trade-offs under ambient
(r=0.035, P=0.7281) or excluded UVB (r=-0.051, P=0.6176); (Figures 5C and D).
DISCUSSION
This study demonstrates that variation in UVB radiation influences resistance and
tolerance to insect herbivory in A. thaliana. Although previous studies have demonstrated
herbivore-induced variation in resistance characters such as trichome density and plant damage
levels (Mauricio and Rausher 1997; Weinig et al. 2003; Clauss et al. 2006), no studies to date
have shown whether variation in UVB radiation levels may influence intraspecific genetic
variation in resistance and tolerance to insect herbivory. This study revealed significant genetic
variation in the response of both direct resistance (trichome density) and indirect resistance
characters to variation in UVB levels. This significant RIL× UVB interaction is very important
47
because it indicates a potential for these characters to evolve under future changes in this
environmental factor. Another interesting finding from this study includes the significant
interaction between UVB and herbivory for trichome density, suggesting that future changes in
UVB radiation may influence plant structural defense responses to insect herbivory, which in
terms may affect coevolutionary plant-insect associations.
With respect to potential fitness costs associated with resistance, this study failed to find
costs associated with trichome production in both ambient and excluded UVB environments
(although a marginally significant result was found in excluded UVB plots). Given the
detrimental effects of UVB radiation on plant fitness and related characters, the inability to
detect a cost for the production of trichomes is not unexpected. This is because previous studies
have linked trichome production with plant protection from harmful effects of ultraviolet
radiation and extreme temperatures (Skaltsa et al. 1994; Werker 2000; Agrawal et al. 2004).
Thus, trichomes may be serving a dual role in plants by protecting them from both herbivores
and harmful UVB radiation, thereby reducing the negative fitness consequence of these factors
on plants. In terms of indirect resistance, however, a significant cost was detected in the
presence of ambient UVB but not under excluded UVB, indicating that UVB radiation modified
the cost of indirect resistance in this system. This finding is concordant with previous studies
that have demonstrated that environmental factors may modulate cost of resistance (Bergelson
and Purrington 1996; Koricheva 2002; Siemens 2003; Osier and Lindroth 2006). For example,
Osier and Lindroth (2006) demonstrated that nutrient availability modified cost of resistance in
Populus tremuloides. Similarly, Siemens (2003) found the cost of resistance to differ in Arabis
perennans that grew alone and Arabis perennans that grew under competition with Bouteloua
48
gracilis. It is also important to note that in this study, the measure of indirect resistance was
related to the level of herbivore damage, and thus, it includes potential costs related to both
structural and chemical defenses. Since costs related to structural defenses were absent in this
study, the presence of this indirect resistance cost may have arisen from the constitutive
chemical resistance mechanisms present in A. thaliana (Marak et al. 2003; Cipollini et al. 2003;
Sampedro et al. 2011; Stowe and Marquis 2011). For example, Stowe and Marquis (2011)
provided empirical evidence for the presence of constitutive costs of foliar glucosinolates in
Brassica rapa by artificially selecting for foliar glucosinolates. Their results revealed that lines
selected for high foliar glucosinolates showed decrease in number of flowers and mean seeds per
fruit. It is also possible that the presence of fitness costs may be associated with the process of
induction of chemical resistance (see Cipollini et al. 2003)
With respect to fitness costs associated with tolerance, this study revealed a significant
negative genetic correlation between relative fitness of undamaged plants and tolerance under
both levels of UVB radiation. After correcting for an artifactual covariance however, which
likely resulted from using the same data to estimate relative fitness of the undamaged plants and
tolerance, the corrected covariance was only negative in plots exposed to ambient UVB
radiation. This result suggests that there is evidence that costs of tolerance may exist under
ambient UVB radiation levels. Little information exists on the effects of abiotic environmental
changes on the cost of tolerance to insect herbivory and different results have been obtained in
terms of how cost of tolerance may vary across environmental gradients. For example, while
Stinchcombe (2002) detected a cost related to tolerance to be present under low soil nutrients but
not under high nutrient level in the ivy leaf morning glory exposed to deer herbivory, Steven et
49
al. (2007) failed to find costs of tolerance to differ between low and high nutrient levels in aspen
plants exposed to artificial defoliation. In addition, Siemens et al. (2003) found that costs of
tolerance to insect herbivory in Arabis perennans were present in a non-competitive environment
but absent in a competitive environment. The inconsistent patterns in the expression of fitness
costs associated with tolerance in these studies suggest that these costs may be dependent on
multiple factors including the plant species and the specific selective factor/s acting in each type
of environment.
Since theoretical models predict that tolerance and resistance serve the same function, it
can be expected that plants should be either resistant or tolerant but not both (Fineblum and
Rausher 1995; Tiffin 2000b; Fornoni et al. 2004). Yet, some models have predicted that both
resistance and tolerance may be maintained at intermediate levels under certain conditions.
These models, as summarized by Fornoni et al. (2004) and Nunez-Farfan et al. (2007), include
the cost of tolerance and resistance across environmental gradients, fluctuations in both time and
space with respect to insect herbivory, and characters that are under the selective pressures of
factors other than herbivory. In addition, the type of damage on plants, such as rosette damage
or apical damage, and type of herbivores present may also be exerting different pressures on
tolerance and resistance. In this study, trade-offs between resistance and tolerance to insect
herbivory were not detected. This result was in agreement with previous studies that have
assessed the presence of trade-offs between resistance and tolerance. For example, Tiffin and
Rausher (1999) did not detect any trade-offs between resistance and tolerance to foliovory and
apical meristem damage in the common morning glory. Likewise, Weinig et al. (2003) did not
find any significant trade-offs between tolerance and resistance to apical damage in A. thaliana,
50
suggesting the possibility of these two characters evolving independently of each other. Most of
these studies, however, did not include potential changes in these trade-offs across environments
(but see Stevens et al. 2007).
The inability to detect tradeoffs between resistance and tolerance in this study could be
explained by two factors. First, fitness costs for resistance and tolerance varied across
environments. For example, fitness costs of tolerance and indirect resistance were only observed
in plots exposed to ambient UVB but not in plots excluded from UVB. This type of variation in
fitness costs across variable environments may lead to the maintenance of genetic variation in
resistance and tolerance, and thus, to selection for intermediate levels of these characters across
populations (Fornoni et al. 2004). Second, it is possible that defense traits used in this study may
be under selection by selective agents other than insect herbivory (Stowe et al. 2000; Fornoni et
al. 2003; Loe et al. 2007; Nunez-Farfan et al. 2007). For example, enhanced UVB radiation in
the absence of herbivory led to an increase in trichome density. This result therefore suggests
that trichomes may be playing a protective role against UVB radiation (Skaltsa et al. 1994;
Werker 2000; Agrawal et al. 2004) and may be under selection in the presence of UVB radiation.
Nevertheless, a selection experiment will be required to confirm whether UVB radiation is
indeed imposing direct selection on trichome density.
In conclusion, results from a common garden experiment revealed that changes in UVB
radiation influence plant characters related to resistance and tolerance to insect herbivory, and
that this environmental effect is dependent on plant genotype (RIL). This study also showed the
existence of fitness costs associated with plant resistance and tolerance, which were dependent
on the UVB environment; that is, they were present only under ambient UVB radiation.
51
Although previous studies have not shown a consistent pattern on the expression of fitness cost
associated with resistance and tolerance (i.e. whether costs are more pronounced in the more
stressful environments or less stressful environments), results from this study support the view
that cost of tolerance and resistance may be more pronounced under a more stressful
environment (Mutikainen et al. 2002; Sampedro et al 2011). This is because growth and other
physiological processes may be compromised if resources decline or conditions become more
stressful. In addition, plant responses to UVB may also come at an additional fitness cost, which
becomes more pronounced in the presence of insect herbivory. The presence of these fitness
costs under enhanced UVB radiation may have important evolutionary implications because
selection for higher levels of resistance and tolerance to insect herbivory under enhanced UVB
may result in lower fitness, and thus, may constrain the evolution of these characters.
52
Table 1. ANOVA F-values showing the effects of herbivory, UVB radiation, RILs and their
interactions on trichome density (direct resistance) and an indirect resistance estimate (1herbivore damage).
Source of variation
Block
Herbivory
UVB
Herbivory x UVB
RIL
Herbivory x RIL
UVB x RIL
Herbivory x UVB x RIL
Trichome Indirect
Density
Resistance
38.59***
1.14
220.48***
73.79*** 92.94***
75.74***
0.85
1.27
1.35†
2.32***
1.39*
1.00
-
Significance levels: † P<0.1, * P<0.05, ** P<0.01, *** P<0.001
Tolerance
0.55
0.86
0.90
-
53
Table 2. ANCOVA F-values showing the relationship between relative fitness and rosette
damage across UVB environments and in each of the UVB environments. A significant
association in rosette damage × RIL indicates the presence of genetic variation in tolerance.
Source of variation
Block
RIL
Rosette Damage
Rosette Damage x RIL
Across UVB
environments
17.50***
1.27*
3.29†
1.28*
UVB+
UVB-
25.84***
1.26†
7.24**
1.07
7.09***
1.33*
1.50
1.26†
Significance levels: † P<0.1, * P<0.05, ** P<0.01, *** P<0.001
54
Table 3. Pearson’s correlation coefficients showing the relationship between relative fitness of
undamaged plants and tolerance under ambient (UVB+) and excluded (UVB-) ultraviolet B
radiation level. Corrected covariance represents the covariance adjusted for the artifactual
covariance.
Correlation coefficient
Covariance
Artifactual covariance
Corrected covariance
95% Confidence interval
UVB+
-0.68*
-0.0474
-0.0317
-0.0157
(-0.036,0.003)
UVB-0.36*
-0.0151
-0.0229
0.0079
(-0.004,0.019)
Significance levels for Pearson coefficient of correlation: * P<0.001
55
Figure 1A. Picture of the experimental plots showing plant arrangements within each plot.
UVB+
UVB-
UVB+
UVB-
UVB+
UVB-
UVB+
UVB-
UVB+
UVBHERBIVORY
SPRAYED
UVB+
UVB-
UVB+
UVB-
UVB+
UVB-
UVB+
UVB-
UVB+
UVB-
Figure 1B. Schematic representation of experimental plot layout showing two levels of
herbivory (herbivory and sprayed) and UVB (ambient and excluded) treatments distributed
among twenty plots.
56
Mylar
Clear acetate
Absorbance (AU)
3
2
1
0
200
250
300
350
400
Wavelength (nm)
Figure 2. Absorption spectra for Mylar and Clear acetate filters.
450
0.94
600
Resistance
0.92
400
*
0.90
0.88
0.86
200
0.84
(B)
UVB+
UVB-
(D)
1.8
600
UVB+
UVB-
UVB+
UVB-
Tolerance
1.6
400
**
Trichome Density (Sprayed)
(C)
(A)
**
Trichome Density (Herbivory)
57
200
1.4
1.2
1.0
0.8
0.6
UVB+
UVB-
Figure 3. Effects of the presence (UVB+) and exclusion (UVB-) of ultraviolet B radiation on
trichome density in the presence of herbivory (A), trichome density in the absence of herbivory
(B), indirect resistance (C), and tolerance (D) of an A. thaliana recombinant inbred line
population. Means of RILs are represented with closed circles and asterisks on lines represent
significant differences in phenotypic means between treatments. * P< 0.05, **P<0.001
2.0
(A)
UVB+
Relative Fitness (Undamaged)
Relative Fitness (Undamaged)
58
r = -0.06
1.8
1.6
1.4
1.2
1.0
0.8
0.6
200
300
400
500
(B)
UVBr = -0.17
1.4
1.2
1.0
0.8
600
200
(C)
UVB+
Relative Fitness (Undamaged)
Relative Fitness (Undamaged)
2.0
r = -0.31**
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.88
0.90
0.92
UVBr = -0.13
1.4
1.2
1.0
0.8
0.88
0.94
0.90
Relative Fitness (Undamaged)
Relative Fitness (Undamaged)
UVB+
r = -0.68***
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.8
1.0
1.2
Tolerance
0.92
Resistance
(E)
0.6
600
(D)
Resistance
2.0
400
Trichome Density
Trichome Density
1.4
1.6
(F)
UVBr = -0.36**
1.4
1.2
1.0
0.8
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Tolerance
Figure 4. Genotypic correlations showing fitness costs of trichomes (A and B), resistance
(C and D) and tolerance (E and F) in A. thaliana plants grown under ambient and
excluded UVB radiation. Each point represents genotypic means of RILs. ** P<0.01,
*** P<0.001
59
UVB+
(A)
1.6
UVB+
(C)
r = 0.04
P= 0.73
r = 0.13
P= 0.18
Tolerance
1.4
1.2
1.0
0.8
0.6
1.8
UVB-
(B)
r = 0.01
P= 0.88
1.6
Tolerance
UVB-
(D)
r =-0.05
P= 0.62
1.4
1.2
1.0
0.8
0.6
200
400
Trichome Density
600
0.84
0.86
0.88
0.90
0.92
0.94
Resistance
Figure 5. Genotypic correlations between resistance and tolerance in A. thaliana plants grown
under ambient and excluded UVB radiation. Each point represents genotypic means of RILs.
60
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CHAPTER 3
EFFECTS OF ULTRAVIOLET-B RADIATION ON PLASTICITY OF FITNESSRELATED RESPONSES OF ARABIDOPSIS THALIANA TO INSECT HERBIVORY
ABSTRACT
The amount of ultraviolet-B (UVB) radiation reaching the Earth’s surface has been
changing over the past few decades due to the destruction of the stratospheric ozone layer,
increased air pollution, and cloud coverage. Current changes in the environment may lead to the
evolution of adaptive plastic responses in plants as well as influence interactions of plants with
other organisms, such as insect herbivores. Potential evolutionary responses of organisms to
environmental change however, will likely depend on the underlying genetic variation that is
present in a population. Here, data from a common garden experiment is presented in which
recombinant inbred lines (RILs) of Arabidopsis thaliana were exposed to two different levels of
UVB radiation (i.e., ambient and excluded UVB) and natural levels of insect herbivory. This
study evaluated whether variation in UVB radiation influenced fitness-related traits of A.
thaliana genotypes as well as their phenotypic responses to insect herbivory. Results from this
study indicate that A. thaliana RILs grown under excluded-UVB were the first to be colonized
by insect herbivores and received higher levels of insect damage. Results from this study
revealed that variation in UVB and herbivory levels influenced all fitness-related traits measured
in a common garden experiment. These traits were affected not only by changes in the UVB
environment, but were also genetically dependent, which may have implications for the
evolution of these traits in future changing environments. More interestingly, UVB levels
influenced plant responses to insect herbivory, in terms of total fruit production and flowering
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time. This result is important because it implies that changes in UVB radiation may influence
the dynamics of plant-insect interactions.
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INTRODUCTION
Multiple environmental changes influencing organisms and their interactions have been
witnessed during the past few decades. Such changes in the environment may lead to the
evolution of plastic responses, which may be reflected in variation in phenotypic traits related to
growth, development, and/or reproduction. These plastic changes represent mechanisms by
which organisms can respond, at least in the short term, to challenges imposed by variable or
heterogeneous environments (Bradshaw 1965; Travis 1994; Schmitt et al. 1999; Sultan 2000).
Phenotypic plasticity is a widely accepted adaptive strategy, particularly useful to sessile
organisms like plants, which have limited capacity to escape in space from the potential
detrimental effects of currently changing environments (Bradshaw 1965). To predict to what
extent such plastic responses may influence the evolutionary trajectory of a species and its
interactions with other species, it is imperative to evaluate whether there is genetic variation in
plasticity as well as genetic variation in the plastic response to environmental factors. This is
essential because the extent, to which a population may evolve in the face of a changing
environment, will likely depend on the underlying genetic variation present at the population
level.
Ultraviolet-B radiation represents a small fraction of the solar spectrum ranging from 290
to 315 nm. The level of UVB radiation reaching the Earth’s surface has been consistently
changing during the last decades due to the depletion of the trophospheric ozone layer, pollution
levels, and cloud coverage (Intergovernmental Panel on Climate Change, IPCC 2007). Several
studies have evaluated the effect of UVB radiation on plant growth and physiological processes
as well as plant chemical responses to this factor, which appear to vary among plant species
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(Hader 2000; Searles et al. 2001a; Searles et al. 2001b). Whereas some studies failed to find
significant effects of UVB radiation on plant growth and photosynthetic processes (Searles et al.
2001a; Tegelberg et al. 2004), other studies have shown UVB-induced decreases in leaf area and
growth rate (likely as a result of decreases in photosynthetic rate and increases in leaf thickness
(Bornman and Teramura 1993; Stratmann 2003). Changes in UVB radiation may also modify
flowering patterns, biomass accumulation, and reproductive traits. With respect to plant
chemistry, UVB radiation is known to increase accumulation of UVB-absorbing compounds,
such as flavonoids and phenolics, which in turn may interfere with plant interactions with their
herbivores (Agrell et al. 2000; Searles et al. 2001; Hofmann et al. 2003; Izaguirre et al. 2003;
Bassman 2004; Izaguirre et al. 2007). On the other hand, levels of secondary chemicals, such as
terpenoids may be unaffected or even decreased in some plant species when exposed to enhanced
UVB radiation levels (Singh 1996; Ambasht and Agrawal 1997).
Several studies have evaluated the interactive effect of UVB radiation with other abiotic
agents on plant responses. For example, abiotic factors such as elevated CO2 (Ziska and
Teramura 1991; McDonald et al. 1999; Agrell et al. 2000; Lavola et al. 2000), nutrient and water
stress (Hofmann et al. 2003), temperature changes (Tevini 1993), and heavy metals (Dube and
Bornman 1992) have been shown to modify plant response to UVB radiation. On the other hand,
only few studies have addressed the interactive effect of UVB radiation with biotic agents such
as insect herbivores (Ballare et al. 1996; McCloud and Berenbaum 1999; Caputo et al. 2006;
Caldwell et al. 2007). These studies, although informative, have mostly been conducted under
laboratory conditions, but see Caputo et al. (2006). More importantly, they did not evaluate
intraspecific variation in plant fitness responses to insect herbivory under variable UVB radiation
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levels. In general, previous studies have mainly focused on assessing effects of UVB radiation
on insect performance and the amount of insect damage suffered by plants. For examples,
studies have shown that UVB may affect the nutrient levels of plants thereby leading to poor
larval performance, increase in the number of days to pupation and late eclosion (Leonard and
Juliano 1995). Additionally, UVB radiation may also induce responses similar to those induced
by herbivory, such as production of secondary chemicals, which may be directly toxic to
herbivores, and thus, reduce the amount of damage suffered by plants (Agrell et al. 2000; Searles
et al. 2001; Hofmann et al. 2003; Izaguirre et al. 2003; Bassman 2004; Izaguirre et al. 2007).
Furthermore, it is not known whether effects of UVB radiation on patterns of insect distribution
and damage among plants in the field are genetically dependent. This information, in addition to
potential genetic variation in the plant phenotypic response to both UVB radiation and insect
herbivory is important because the interactive effect of these factors may lead to natural selection
favoring some genotypes over others, which may in turn alter the co-evolution of plant-insect
interactions.
This study presents results from a common garden experiment in which recombinant
inbred lines (RILs) of Arabidopsis thaliana were exposed to two different levels of UVB
radiation (i.e., ambient- and excluded-UVB) as well as to the presence and absence of natural
levels of insect herbivory. The interactive role of plant genotype, UVB radiation, and insect
herbivory was assessed on plant fitness-related responses of the annual plant A. thaliana.
Patterns of insect distribution and herbivory in the field were also evaluated to assess whether
these could explain observed changes in plant phenotypic responses to insect herbivore attacks.
Information from this experiment would allow us to make predictions regarding whether changes
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in UVB radiation may influence phenotypic responses of plants to a natural insect community,
and thus, the evolution of plant-insect interactions in future changing environments.
MATERIALS AND METHODS
Study Plant Species
Arabidopsis thaliana (L.) Heynh (Capparales: Brassicaceae), also known as the mouseear cress, is an annual herbaceous plant native to Europe and Asia with a wide geographic
distribution across North America and North-western Africa (Baskins and Baskins 1983;
Hoffmann et al. 2003). This species is a self-fertilizing plant, characterized by a short life cycle,
which is naturally found in open or disturbed habitats (Baskins and Baskins 1983; Hoffmann et
al. 2003). Because of the high selfing rates (less than 1% outcrossing rates), natural populations
seem to consist majorly of single genotypes. However, significant levels of genetic variation in
phenotypic traits exist among different local populations of this species (Bergelson et al. 1998;
Mauricio 1998; Nordborg and Bergelson 1999). Recombinant inbred lines of A. thaliana (100
RILs) derived from a cross between the ecotypes Columbia (Col-0) and Landsberg (Ler), and
advanced to the eighth generation through a single seed descent were used in this study (Lister
and Dean 1993). These RILs were used to generate a genetically variable population since the
two parental lines (Col-0 and Ler) are known to differ in several characteristics related to growth,
reproduction, phenology, and both chemical and structural defenses (Mauricio 2005; Weinig et
al. 2003). Thus, genetic and phenotypic variation among these RILs allows for the assessment of
intraspecific variation in the response of this plant species to both insect herbivory and UVB
radiation.
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Experimental Design
To address the question of whether UVB radiation can influence plasticity of plant
phenotypic responses to insect herbivory, the model plant Arabidopsis thaliana was used in a
common garden experiment in which the presence/absence of natural insect herbivory and the
levels of UVB radiation were manipulated. Specifically, a split-plot design was used to assess
the effect of UVB radiation, insect herbivory, and plant genotype on plant phenotypic responses.
The two UVB treatment levels consisted of ambient (UVB+) and excluded (UVB-) ultraviolet-B
radiation randomly assigned to 20 different plots (i.e., 10 plots assigned to each UVB level).
While ultraviolet-B radiation was excluded from the randomly assigned UVB- plots using Mylar
polyester film, UVB+ plots were covered with clear acetate film allowing most of the UVB
radiation to pass through. Mylar film transmits radiations only in the wavelength greater than
315 nm thereby blocking all the UVB radiation but transmitting radiations in the ultraviolet-A
regions. On the other hand, clear acetate film allowed most UVB to pass through. Arabidopsis
thaliana genotypes consisted of 100 recombinant inbred lines (RILs), which were planted at
random within each of the plots. Two sets of 100 RILs were randomly assigned to each of the 20
plots (total of 4000 plants). Insect herbivores were excluded from sprayed plots by spraying with
a pesticide (Proclaim by Syngenta Inc. USA) at ten days interval. Plants excluded from insect
herbivores were inspected on daily basis to ensure no herbivore damage. Each plot (both
herbivory and sprayed plots) was surrounded with a wire-mesh fence to exclude mammalian
herbivory.
In terms of growth conditions, seeds were cold-treated for 5 days at 4°C to break
dormancy. Prior to transplanting to the field, plants were initially raised in a 50 well flats in
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laboratory growth chambers. Within the chambers, plants were exposed to 10 hours of day and
14 hours of night, with day and night temperatures of 20°C and 16°C, respectively. Plants were
rotated within growth chambers every other day and among chambers every week to avoid
chamber and position effects. After five weeks of growth in the chambers, plants were
transplanted to the field (Ecological Research Station, Bowling Green State University, Bowling
Green, Ohio).
Plant phenotypic traits
In order to assess responses of plant RILs to herbivory under different UVB levels,
variables related to phenology (flowering time), plant growth (rosette biomass), and reproduction
(total fruit production) were measured in plots with herbivores present and excluded. Similarly,
the effects of UVB radiation, and RIL were assessed on days to plant colonization and percent
area of rosette leaves removed by insect herbivory. Flowering time was estimated as the number
of days required for the first flower per plant to be fully opened. Plant rosette biomass was
measured as rosette dry weight by oven drying the rosette leaves for two days at 70°C. Plant
fitness was estimated as the total fruit number produced over the plant lifetime, since this
measure is highly correlated with total seed production (Bidart-Bouzat 2004). To document the
order of colonization of insect herbivores among plants, all plants were inspected daily for insect
damage and the numbers of days to first observe herbivore damage on each plant was recorded
herein, referred to as colonization.
Statistical Analysis
To evaluate responses of plant genotypes to herbivory under different UVB levels,
analyses of variance were performed for each of the measured variables, flowering time, rosette
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biomass and total fruit production using PROC GLM (SAS statistical software, version 9.1).
Data was either log- or ranked-transformed to correct for normality and heteroscedasticity when
necessary. For this purpose, a mixed linear model was used, considering the herbivory and UVB
treatments as fixed effects, and plant genotype (RIL) as a random effect. In addition, similar
analyses were performed on the order of insect herbivore colonization among RILs and plant
rosette damage in order to evaluate the effects of UVB radiation, and genotype on patterns of
plant utilization by insect herbivores.
RESULTS
Analyses of variance revealed that A. thaliana differed in their fitness-related responses
to UVB exclusion and insect herbivory (Table 1). With respect to the number of fruits produced,
both UVB exclusion and herbivory significantly affected this trait (see Table 1 and Figure 1A).
Exclusion of UVB resulted in a significant increase in total fruit production and herbivory led to
a general decrease in the total number of fruits (Figure 1A and B). Additionally, plant responses
to herbivory in terms of total number of fruits produced were found to be dependent on UVB
radiation levels (Table 1). This significant interaction between herbivory and UVB radiation
levels has particular evolutionary significance since future changes in UVB radiation levels may
modify plant phenotypic response to their herbivores. The effect of genotype (RIL) on total fruit
number was statistically not significant (Table 1). However, separate analyses of variance for
each UVB levels (when pooling herbivore treatments) revealed a significant effect of RIL on
total fruit number (P= 0.0107 for UVB+; and P= <0.0001 for UVB-), indicating the existence of
genetic variation in this trait. All phenotypic responses of plants to UVB radiation were found to
be genotype dependent (significant UVB × RIL interaction), but those to insect herbivory were
78
not (see Table 1). In addition, the three-way interaction UVB × herbivory × RIL was statistically
not significant indicating that UVB radiation levels affected total number of fruits produced in
response to herbivory across the genotypes in the same manner.
With respect to rosette biomass, UVB exclusion and herbivory significantly affected this
trait (Table 1). Exclusion of UVB resulted in a significant increase in rosette biomass, and
herbivory led to a general decrease in rosette biomass (Figure 1C and D). Overall, the effect of
genotype (RIL) on rosette biomass was statistically not significant (Table 1). However, separate
analyses of variance for each UVB levels revealed a significant effect of genotypes on rosette
biomass (P= <.0001 for UVB+; and P= <.0001 for UVB-), indicating the existence of genetic
variation in this trait. Rosette biomass responses to UVB were found to be dependent on
genotype (significant UVB × RIL interactions), but not on herbivory (Table 1). Furthermore,
responses of plant to herbivory were not genotype dependent. Analysis of the three-way
interaction (i.e., UVB × herbivory × RIL) was not significant indicating that UVB radiation
levels affected rosette biomass produced in response to herbivory across the genotypes in the
same manner.
Analyses of variance also revealed that UVB, herbivory, and genotype significantly
affected the number of days to first flowering (Table 1). Overall, exclusion of UVB radiation
resulted in a decrease in the number of days to first flowering, and herbivory led to an increase in
this trait value (Figure 1E and F). For this trait, a significant interaction between herbivory and
UVB radiation was observed, indicating that plant responses to UVB radiation in terms of
flowering time is dependent on herbivory (Table 1). Moreover, while phenological responses of
plants to UVB variation were genotype-dependent (significant UVB × RIL interaction),
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responses to herbivory did not significantly depend on the genotype (Table 1). Finally, the threeway interaction of UVB radiation, herbivory, and genotype was significant, which implies that
UVB radiation differentially affected plant response to herbivory across the genotypes.
Exclusion of UVB led to a significant decrease in the number of days to plant
colonization by insect herbivores (Table 1 and Figure 2A). The effects of genotype on days to
herbivore colonization were marginally significant, and the effect of UVB radiation on this
variable was not dependent on plant genotype (Figure 2A). In addition, changes in UVB
radiation significantly affected levels of herbivore damage (Table 1 and Figure 2B). Plants
excluded from UVB radiation had more damage than plants exposed to ambient UVB.
Although, the effect of RILs was not significant on the levels of insect herbivore damage,
separate analyses of variance under the two UVB radiation levels revealed a significant effect of
RILs on the levels of herbivore damage (P=0.0055 for UVB+; and P<.0001 for UVB-). In
addition, variation in damage levels as a result of differences in UVB radiation levels was also
dependent on plant genotype (significant UVB × RIL; Table 1 and Figure 2B).
DISCUSSION
Even though several studies have investigated the effects of UVB radiation and insect
herbivory on plants, information is lacking regarding the effect of these two important
environmental factors on plant fitness-related responses. This study is also of evolutionary
significance because the ability of plant populations to evolve and the extent to which a
population may evolve in a changing environment will depend on the available genetic variation
within such population. In addition, this study revealed a significant UVB × RIL interaction for
80
all measured variables suggesting the presence of evolutionary potential of these traits to respond
to changes in UVB radiation. This significant interaction is important as it may lead to the
maintenance of genetic variation under variable environments, and allow populations to evolve
in changing environments (Via and Lande 1985; Via and Lande 1987; Gillespie and Turelli
1989).
Overall, the effect of UVB radiation was detrimental to plant fitness-related variables
measured in this study. Exclusion of UVB radiation led to an increase in the total number of
fruits and rosette biomass produced, as well as a delay in the number of days required for plants
to first flower. In the same vein, insect herbivory also had a negative effect on measured
phenotypic variables. This result is consistent with other studies that have shown that plants may
suffer fitness consequences because of exposure to high levels of UVB (Teramura et al. 1990;
Teramura and Sullivan 1991; Grammatikopoulos et al. 1994). This may be through growth
inhibition, alteration of photosynthetic enzyme activities, modification of stomata closure, and
alteration of growth patterns (e.g., decreases in stem length and leaf area, and changes in
flowering phenology) (Teramura 1993; Hader 2000; Searles et al. 2001a; Searles et al. 2001b
Bornman and Stratmann 2003). The negative impact of UVB radiation on fitness may also be
due to energy costs of synthesizing protective secondary metabolite in response this type of
stress (Logemann et al. 2000). In the same manner, insect herbivory may also have negative
consequences on plant fitness such as reduced seed production , increased fruit abortion,
decreased growth rate, changes in plant architecture, and delayed senescence (Marquis 1992;
Bidart-Bouzat 2004; Rudgers and Whitney 2006). It is important to emphasize that this study
revealed a significant interaction between herbivory and UVB, indicating an effect of variation in
81
UVB levels on plasticity of plant-fitness responses to insect herbivory. Thus, future changes in
UVB radiation levels may significantly affect plant-insect interactions and the fitness outcome of
plants exposed to natural insect communities.
With respect to patterns of plant utilization by insect herbivores, increase in UVB led to a
decrease in insect herbivore damage and an increase in the number of days to herbivore
colonization. This result may be due to two major reasons. Insect herbivores may detect
differences in the levels of UVB radiation and present avoidance behavior to high levels of UVB
radiation (Mazza et al. 1999; Mazza et al. 2002). Alternatively, UVB may mediate changes in
plant morphology and chemistry, which in turn influences the amount of insect herbivory. For
example, previous studies have suggested that trichomes, which are important structural barriers
for insect herbivores, may also be involved in the protection of plants against harmful effects of
UVB radiation. This protective mechanism is due to the ability of trichomes to reflect radiations
in the UVB region (Ehleringer 1984; Karabourniotis and Bornman 1999). There is experimental
evidence that trichome production was induced by UVB radiation in this study (see Chapter 2).
In addition to induction of physical barriers to herbivores, UVB radiations may also increase the
concentration of UVB absorbing compounds such as phenolics, flavonoids, and other secondary
chemicals that may directly influence plant utilization by herbivores (Lavola et al. 2003). For
instance, Izzaguire et al. (2007) found that UVB radiation induced the same phenolics
compounds that were induced by herbivory, suggesting that UVB radiation may trigger the same
defense pathway as insect herbivory. Information also exists on effects of UVB radiation
influencing concentrations of glucosinolates, a well-studied chemical defense system in the
Brasssicaceae plant family (Barro et al. 2002; Schreiner et al. 2009). For instance, Schreiner et
82
al. (2009) found up to six-fold increase in glucotropaolein, the only type of glucosinolates
present in nasturtium (Tropaeolum majus) after exposure to UVB radiation. Likewise, Baro et
al. (2002) observed similar increases in glucosinolate contents in the Ethiopian mustard
(Brassica carinata) when exposed to UVB radiation. This UVB-mediated increase in
glucosinolates has been suggested to be a stress response similar to that observed in plant
defense responses to insect herbivory and pathogen attack (Schreiner et al. 2009). Taken
together, these findings suggest that UVB radiation may have induced both physical and
chemical defenses in our system, which in turn may have pronounced effects on patterns of plant
use by insect herbivores.
Intuitively, one would expect that lower herbivory impact (less plant damage) under
UVB+ should be associated with enhanced plant fitness in this environment; this was not the
case however, in this study. Plants grown under UVB- had more herbivore damage, yet had
higher fitness. There are two possible reasons for this observation. First, it is possible that the
detrimental effects of UVB radiation are more severe than the effects of herbivory on plant
fitness; that is, potential fitness gains due to a reduction in levels of herbivory in UVB+
environments could not compensate for the reduction in fitness due to UVB radiation. Second,
there might be a cost associated to plastic responses of plants to UVB radiation. This cost, which
may be related to the production of UVB absorbing compounds or due to genetic causes such as
potential deleterious effects of genes involved in plasticity (Pigliucci 2001) may account for the
lower plant fitness in UVB+ plots even though insect damage was decreased in this environment.
Given that the interaction between UVB and insect herbivory was significant and there was a
marked decrease in the plasticity of total fruit production (estimate of fitness in this study) in
83
response to UVB exclusion, it is reasonable to infer that the effects of UVB radiation were likely
more detrimental than those of insect herbivory on plant fitness. Nonetheless, potential
physiological and genetic costs on plant fitness cannot be discarded, and may also at least partly
explain the observed results.
In conclusion, this study addressed how variation in UVB radiation levels may influence
potential fitness consequences of insect herbivory in plants. Although overall, UVB radiation
had a negative effect on plant fitness, this study revealed the presence of genetic variation in both
UVB environments suggesting a potential for plants to evolutionary respond to future changes in
UVB radiation levels. It is also important to note that although UVB radiation tended to reduce
the amount of herbivore damage inflicted on plants (likely due to increased induction of
protective secondary metabolites and physical structures), this reduction was not enough to
compensate for the negative effects of UVB radiation on plants.
84
Table 1. Mixed model analysis of variance showing the effects of UVB radiation, insect
herbivory and RIL on plant phenotypic variables of A. thaliana.
Source of Variation
Block
Herbivory
UVB
Herbivory x UVB
RIL
Herbivory x RIL
UVB x RIL
Herbivory x UVB x RIL
Flowering
date
1.76
181.27***
649.93***
6.98**
4.00***
0.92
2.38***
1.29*
Fruits
22.60***
817.48***
246.48***
98.98***
1.01
0.92
1.45*
1.03
Significance levels †P<0.1, *P<0.05, **P<0.01, ***P<0.001
Rosette Colonization Rosette
Biomass
Damage
6.98***
11.49***
0.61
235.99***
16.48***
155.80*** 157.43***
0.03
0.71
1.30†
1.28
0.98
17.65***
1.18
2.02***
0.49
-
85
(A)
(B)
600
Total Fruit Number
Total Fruit Number
300
*
200
500
*
400
400
300
200
100
UVB+
UVB-
Rosette Biomass
0.06
*
0.04
0.02
UVB+
UVB+
0.12
0.10
0.08
0.06
0.04
UVB-
UVB-
Days to Flowering
68
68
*
Days to Flowering
(F)
UVB-
0.02
(E)
70
UVB+
66
64
62
60
66
*
Rosette Biomass
0.08
(D)
0.14
0.10
*
(C)
64
62
60
58
58
UVB+
Herbivory
UVB-
UVB+
Sprayed
UVB-
Figure 1. Reaction norms showing A. thaliana phenotypic responses, in terms of total fruit
production (A-B), rosette biomass (C-D), and number of days to first flowering (E-F) to variation
in UVB radiation levels in herbivory and sprayed plots. Means of RILs are represented with
closed circles and represent significant differences in phenotypic means between UVB treatment
levels. * P<0.001
86
(A)
22
*
Days to Colonization
24
20
18
16
14
12
10
12
10
*
Rosette Damage (%)
(B)
8
UVB+
UVB-
Figure 2. Reaction norms showing A. thaliana phenotypic responses, in terms of days to
herbivore colonization (A), and rosette damage (B). Means of RILs are represented with closed
circles represent significant differences in phenotypic means between UVB treatment levels.
* P<0.001
87
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