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Perfect is best: Low leaf fluctuating asymmetry reduces herbivory by leaf

miners

Article  in  Oecologia · February 2005

DOI: 10.1007/s00442-004-1724-y · Source: PubMed

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Tatiana Cornelissen Peter Stiling

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Oecologia (2005) 142: 46–56
DOI 10.1007/s00442-004-1724-y

PL ANT ANI MA L IN TERACTION S

Tatiana Cornelissen . Peter Stiling

Perfect is best: low leaf fluctuating asymmetry reduces herbivory

by leaf miners

Received: 24 November 2003 / Accepted: 2 September 2004 / Published online: 17 September 2004
# Springer-Verlag 2004

Abstract Fluctuating asymmetry (FA) represents small, Introduction

random variation from symmetry and can be used as an
indicator of plant susceptibility to herbivory. We investi- Studies on the effects of plant quality on the attack rates of
gated the effects of FA of two oak species, Quercus laevis herbivorous insects have been performed extensively and
and Q. geminata, and the responses of three herbivore many hypotheses have been proposed to explain within
guilds: leaf miners, gallers, and chewers. To examine and between variations in herbivory rates among different
differences in FA and herbivory between individuals, 40 plant species. A frequently invoked factor influencing
leaves from each tree were collected, and FA indices were herbivory levels is stress, since stressors may affect plant
calculated. To examine differences in FA and herbivory nutritional quality for herbivores. The plant stress hypoth-
within-individuals, we sampled pairs of mined and esis (PSH) proposed by White (1984) argues that herbi-
unmined leaves for asymmetry measurements. Differences vore abundance is higher on stressed host plants due to an
in growth of leaf miners between leaf types were increased availability of nutrients, a decreased concentra-
determined by tracing 50 mines of each species on tion of defensive compounds and/or changes in the ratio of
symmetric leaves and asymmetric leaves. Asymmetric nutrients to chemical defenses. Evidence supporting the
leaves contained significantly lower concentrations of prediction that moderate stress benefits herbivores due to
tannins and higher concentrations of nitrogen than increased nutritional quality are abundant (e.g., McClure
symmetric leaves for both plant species. Both frequency 1980; Lewis 1984; Mattson and Haack 1987) and positive
of asymmetric leaves on plants and levels of asymmetry relationships between insect herbivory and plant stress
positively influenced the abundance of Brachys, Stilbosis have been found for some tree species, crops and
and other leaf miners, but no significant relationship herbaceous plants (e.g., Mattson and Haack 1987;
between asymmetry and herbivory was observed for Heinrichs 1988). Nevertheless, some authors claim that
Acrocercops. Brachys and Stilbosis mines were smaller experimental tests of the PSH have generated conflicting
on asymmetric leaves, but differences in mine survivorship results (e.g., Bultman and Faeth 1987; Louda and Collinge
between symmetric and asymmetric leaves were observed 1992; Koricheva et al. 1998; DeBruyn et al. 2002), and
only for Stilbosis mines. This study indicated that leaf many authors (e.g., Larson 1989; Koricheva et al. 1998)
miners might use leaf FA as a cue to plant quality, have championed the abandonment of the PSH and the
although differential survivorship among leaf types was search for alternative hypotheses. DeBruyn et al. (2002)
not observed for all species studied. Reasons for the argued that a major cause of the inconsistent support for
different results between guilds are discussed. the PSH is an inconsistency in the measurements of stress
used. Frequent measures of stress in plants include
Keywords Quercus . Fluctuating asymmetry . Plant different estimates of productivity, plant growth, biomass,
quality . Leaf miners shoot: root ratios and physiological parameters, such as
leaf water deficit and plant secondary chemistry. As
suggested by Moller (1995), an objective definition of
environmental stress would advance our understanding of
T. Cornelissen (*) . P. Stiling the relationship between plant stress and herbivory.
Department of Biology SCA 110, University of South Florida, During recent years, it has been claimed that develop-
4202 E Fowler Av, mental instability reflects the inability of organisms to
Tampa, FL, 33620-5150, USA
e-mail: [email protected] control developmental processes during ontogeny and to
Tel.: +1-813-9743754 achieve a predetermined phenotypic optimal expression
Fax: +1-813-9743263 (Moller and Swaddle 1997). One measure of develop-
 47

mental instability is fluctuating asymmetry (FA) that otherwise symmetrical bilateral traits is a surrogate of
represents small, random variations from symmetry in plant stress and asymmetrical leaves should differ in
otherwise bilaterally symmetrical characters. Leaf fluctu- nutritional quality and herbivore susceptibility compared
ating symmetry has been used as an objective measure- to symmetrical leaves; (2) fluctuating asymmetry between
ment of the effects of environmental stress on plants (e.g., individuals and frequency of herbivory: if FA in leaves
Martel et al. 1999; Roy and Stanton 1999; Alados et al. predicts plant susceptibility to herbivores, plants with
2001). Individual- and population-levels of bilateral FA more asymmetric leaves or higher degrees of asymmetry
have been related to several biotic and abiotic stresses, should be subject to higher levels of herbivory than
including environmental factors, such as nutrition, tem- individual plants with a lower incidence of foliar asym-
perature, radiation, and pollution, as well as genetic metry; (3) fluctuating asymmetry within individuals and
factors, such as mutation, inbreeding, and hybridisation. frequency of herbivory: frequency of herbivory in asym-
FA is then suggested as a reliable stress estimator and metrical leaves should be higher than frequency of
measures of FA could thus represent sensitive indicators of herbivory in symmetrical leaves or leaves with lesser
the developmental performance of organisms in their degrees of asymmetry; (4) fluctuating asymmetry and the
environment and biomonitors of how organisms are able slow-growth, high-mortality hypothesis (Clancy and Price
to deal with deviant environmental and genetic conditions. 1987): herbivores feeding on leaves with lower nutritional
In addition to being an indicator of plant stress, some quality or digestibility should take longer to develop and
studies have shown correlations between FA and insect would be more susceptible to natural enemies. Therefore,
herbivore attack, suggesting leaf FA can be used not only insects feeding on asymmetrical leaves should exhibit
as an indicator of plant stress, but also plant susceptibility higher survivorship than insects feeding on symmetrical
to herbivory (e.g., Wiggins 1997; Zvereva et al. 1997). leaves.
Plants with more asymmetric leaves or higher levels of
leaf asymmetry should exhibit increased levels of herbi-
vory due to higher nutritional quality of asymmetric leaves Materials and methods
compared to symmetric leaves (Sakai and Shimamoto
1965; Lempa et al. 2000). Little is known as to how Study systems
developmental disorders are connected to plant metabo-
lism and the associated biochemical changes exhibited by Sand live oak, Quercus geminata (Fagaceae), is a semi-
asymmetric leaves (Lempa et al. 2000), but, since the left evergreen oak and, typically, old leaves abscise and new
and the right sides of a bilaterally symmetrical trait leaves appear in late April or early May, reaching full size
develop under the control of the same genes, minor in approximately 2 weeks. The leaves are rounded and
deviations from perfect symmetry actually represent persistent with deeply revolute, conspicuous impressed
developmental instability and may be responsible for veins on the underside and the bases and sides of the
differences in nutritional quality or secondary chemistry leaves are observed to be asymmetric in many instances.
between asymmetric and symmetric leaves. Positive Stilbosis quadripustulatus (Lepidoptera: Cosmopterygi-
correlations between FA and herbivory indicate either dae) is a moth whose larvae induce mines on the adaxial
that plants with asymmetric leaves are, on average, more surfaces of Q. geminata. S. quadripustulatus is a
susceptible to attack by herbivores, and/or that herbivory univoltine species, whose adults emerge in early summer
itself acts as a stressor and directly increases the level of (from May to June) from pupae that overwinter in soil and
leaf asymmetry. Although some authors favour the idea litter. Oviposition occurs approximately in early June,
that herbivores themselves can act as stressors increasing when females oviposit at the junction of the midvein and a
leaf asymmetry (e.g., Zvereva et al. 1997), correlations major lateral vein. Larvae take from 60 to 90 days to
between leaf FA and herbivory are not always likely to be complete their five instars and mines may reach 3.0 cm in
causal (Lempa et al. 2000). Instead, chemical and length (Simberloff and Stiling 1987). Many other
nutritional differences between symmetric and asymmetric herbivores comprise the insect community associated
leaves may influence leaf selection by herbivores, which with Q. geminata. Leaves are frequently found damaged
leads to positive correlations between herbivory and FA. by chewing insects such as the eastern buck moth
This study aimed to examine the relationship between Hemileuca maia (Lepidoptera: Saturniidae), and at least
leaf fluctuating asymmetry and herbivores on Quercus four cynipid species (Hymenoptera: Cynipidae) of galling
geminata and Q. laevis . We addressed the relationship insects are commonly observed on sand live oak leaves
between herbivory and FA by examining the community and stems: Andricus quercusfoliatus, Disholcaspis quer-
of herbivores attacking these two oak species and how cussuccinipes, Callirrhytis quercusbatatoides, and Belo-
they respond to random variations in leaf morphology. nocnema quercusvirens. A. quercusfoliatus induces white
Leaf miners (Stilbosis quadripustulatus, Brachys tessela- flower-like galls on sand live oak stems, whereas D.
tus, and Acrocercops albinatella), leaf gallers (eyespot quercussuccinipes wasps induce clusters of 5–20 yellow-
galls and Belonocnema quercusvirens) and leaf chewers ish brown galls usually crowded around a terminal oak
(Hemileuca maia, Orgyia leucostigma) were studied. The twig. C. quercusbatatoides wasps induce abrupt swellings
following hypotheses were tested: (1) fluctuating asym- of twigs, varying in form and size and B. quercusvirens
metry, plant stress and herbivory: fluctuating asymmetry in induces tan, globular pea-like galls on the underside of Q.
48

geminata leaves. Galls are unilocular and occur in large from the leaf litter in the following spring (Waddell et al.
numbers during the fall. Eyespot galls (Diptera: Cecido- 2001). Turkey oak leaves are also attacked by a vast array
myiidae) are recognized as circular spots, usually 8– of herbivores, such as the leaf roller weevil Homoeolabus
10 mm in diameter. The adults emerge from the soil in the analis (Coleoptera: Atellabidae), the eastern buck moth H.
spring and lay eggs in the upper leaf surface. As the larva maia, the white tussock moth Orgyia leucostigma
grows, the leaf tissue surrounding it swells slightly and red (Lepidotpera: Lymantriidae) and other leaf miners such
rings are seen around the galls. Larvae complete their as Stigmella (Lepidoptera: Nepticulidae) and Cameraria
development in 8–12 days and pupate in the soil. This is (Lepidoptera: Gracillariidae).
the most common gall found on sand live oak leaves, often
reaching densities of five galls per leaf.
The turkey oak Quercus laevis is one of the character- Data collection
istic trees associated with the sand hill community over
much of Florida. Q. laevis is a moderately fast to fast- Patterns of leaf asymmetry, leaf quality and herbivory
growing tree and presents deciduous simple leaves, were examined for 30 individuals of Q. geminata and 30
alternately arranged with usually five lobes, although individuals of Q. laevis from March to October 2002 at the
this number may vary from three to seven. Although a University of South Florida Botanical Garden, Tampa,
common tree in Florida native vegetation, there are Florida. To verify the relationship between FA and leaf
relatively few studies concerning herbivory in this plant quality and to examine the frequency of occurrence of
species. Acrocercops albinatella (Lepidoptera: Gracillar- asymmetric leaves and levels of leaf asymmetry on each
idae) is a microlepidopteran species whose larval stages plant, 40 leaves were sampled from each individual plant
feed on young leaves, creating distinct linear-blotch mines in April 2002. Because herbivores themselves may act as
on the lower surface of Q. laevis leaves. Larvae typically plant stressors, these leaves were sampled before the
feed on the palisade parenchyma cells and deposit frass beginning of mine initiation and before leaves were
throughout the mine, completing their development in damaged by free-feeding herbivores. To quantify Quercus
approximately 10 days. Larvae emerge from the blotch geminata fluctuating asymmetry, widths of all leaves were
mine and usually pupate on the same leaf from which they measured on both the right and the left side, from the leaf
emerge (T. Cornelissen, personal observation). Brachys edge to the midrib, at the middle point of the leaf, which
tesselatus (Coleoptera: Buprestidae) is a univoltine species usually coincides with the widest part of the leaf. Q. laevis
that also forms distinct blotch mines in Q. laevis leaves. exhibited some variation in the number of leaf lobes, but
The adults emerge in mid-March to mid-April, coinciding 83% of the leaves we sampled exhibited three pairs of
with budburst of turkey oak. Adults initially feed on the lobes and measurements were taken between the first and
early leaves and flowers until mating and oviposition. second pairs of lobes (Fig. 1). These distances were
Eggs are deposited singly on the upper surface of the measured after photographing each leaf with a digital
leaves and after hatching the larvae mine into the camera at a standard distance of 30 cm in the laboratory
mesophyll creating distinct, characteristic damage. Con- and analyzing leaf length, leaf area, and right and left
trary from what happens in South Carolina (Waddell et al. widths using the software UTHSCSA Image Tool
2001) Brachys in our study sites go through two (University of Texas, USA). All the digital pictures were
generations instead of just one. The first mines appear in calibrated to the nearest 0.01 mm before measurements
early April and remain active until late June, when larvae were taken and the resolution set to the software did not
complete their development and pupate inside the mines. allow measurement errors greater than 1.0%. Absolute
New adults emerge in early July and oviposit to form new asymmetry in leaf width was defined as the unsigned
Brachys mines that remain active until September– difference between right width (RW) and left width (LW)
October. Pupation and overwintering of this second of a particular leaf as FAwidth = |RW − LW|. The absolute
generation occurs within the leaves after they have value of right–left traits is a good estimator of variance in
senesced and abscised from the tree. New adults emerge FA among leaves assuming that there is no directional

Fig. 1 Schematic representation

(not to scale) of measurements
used to define fluctuating
asymmetry in a Quercus laevis
and b Q. geminata. RW right
width and LW left width
 49

asymmetry (consistently larger left or right side) or widths was equal to zero leaves were categorized as
antisymmetry (consistent lack of symmetry, but in no symmetric. However, since we used only two decimal
particular direction). To examine differences in nutritional places when categorizing FA values and calculating FA
quality between symmetric and asymmetric leaves, all the indices, leaves with FA measurements ranging between
leaves sampled from each plant were analyzed for water, 0.001 and 0.09 mm were rounded to zero and categorized
nitrogen content, and tannin concentration. Water content as symmetric, in a more conservative approach. Leaves
was quantified by the difference between leaf wet and dry yielding absolute FA values equal or greater than 0.1 mm
weights after leaves were oven-dried. Leaves were then (either positive or negative) were all classified as
milled to a fine powder. Tannins were extracted from asymmetric since we believe the resolution of the
50 mg of dry tissue, and tannin concentration was equipment we used is quite accurate, as revealed by the
quantified using the radial diffusion assay (for details see small error range (1.00% or 0.001 mm) and high values
Hagerman 1987). The average of three replicates per leaf obtained for the Index of Repeatability for both plant
was used for statistical analysis. Nitrogen content was species (see Results). To examine herbivore distribution
determined using a CHN analyzer. between symmetric and asymmetric leaves, for each
To verify the relationship between plant fluctuating species of leaf miner each pair of leaves collected on
asymmetry and herbivory between individuals, we used each plant was categorized as (1) mined leaf, symmetric:
data on asymmetry from the 40 leaves collected from each unmined, symmetric, (2) mined leaf, symmetric: unmined,
plant to calculate two indices of FA (Palmer and Strobeck asymmetric, (3) mined leaf, asymmetric: unmined, asym-
1986): metric and (4) mined leaf, asymmetric: unmined, sym-
P metric. To test the relationship between FA within plants
jR i
Li j and leaf gallers, we sampled 20 leaves galled by
Index 1 ¼ Belonocnema and 20 leaves with eyespot galls and the
N
nearest 20 non-galled leaves and each pair of leaves was
Ph jRi
Li j
i
placed in one of the four categories as described above.
ðRi þLi Þ=2 To test for differences in mine survivorship between
Index 2 ¼
N symmetric and asymmetric leaves, 50 mines of each leaf
miner species were marked in asymmetric leaves and
where RI is the value of the right side, LI is the value of another 50 in symmetric leaves. Mined leaves were
the left side and N is the number of measurements taken. classified as symmetric or asymmetric after photographing
Index 1 is the absolute fluctuating asymmetry and it is the 300 mined leaves in the field and taking measurements as
most intuitive asymmetry measurement (Roy and Stanton described before. To account for individual variation in
1999). Index 2 is size-scaled, calculated as the absolute leaf miner development related to individual oak plants, no
value of right (RI) minus left (LI) sides divided by the more than six mines (three on symmetric leaves, three on
average (RI + LI)/2, to correct for the fact that asymmetry asymmetric leaves) were marked on each plant. All mines
may be size-dependent. These indices were then correlated (n=300) were permanently marked using a Sharpie pen as
with the density of leaf miners, galls, and chewed leaves soon as the eggs hatched and larvae initiated mine
recorded on each individual plant. Quercus plants were formation. Acrocercops mines were measured at 2-day
monitored for the occurrence of herbivores and the number intervals by tracing the numbered mines using acetate
of Acrocercops, Stilbosis, and Brachys mines were sheets, whereas Brachys mines were traced at 3-day
quantified in October 2002 by recording the number of intervals and Stilbosis mines were traced at 5-day
mines in 200 leaves on each plant. Leaf galls were counted intervals. Mine drawings were also digitalized and mine
in 200 leaves on each plant and stem galls were quantified size was measured using the software UTHSCSA Image
by counting 100 twigs on each individual in September Tool, with digital pictures calibrated to the nearest
2002. We also recorded the number of other leaf miners 0.01 mm. We compared mine size, developmental time
and chewed leaves on both plant species by counting 100 (days to pupation) and mine growth rate on symmetric and
leaves of each plant in October 2002. asymmetric leaves for each one of the leaf miner species.
Mine growth rate on each leaf type was calculated as
To examine the relationship between FA and herbivory growth rate = (final mine size − initial mine size)/number
within individuals, 40 mined leaves from each leaf miner of days mine was growing. After mine termination, all
species were collected from each plant and the 40 nearest leaves were inspected under a stereomicroscope to assess
neighbouring leaves without mines were collected from leaf miner survivorship and identification of mortality
the same individual plant. Leaves were oven-dried factors. Leaf miners offer a great opportunity to assess
between sheets of filter paper, mounted as herbarium population survivorship and mortality factors since a
specimens and asymmetry measurements of mined and record of the miner success is clearly observed on the
unmined leaves were calculated for each leaf miner leaves: parasitized mines have tiny circular exit holes left
species as described before. If the difference between by the parasitoid on mine’s surface and predated mines are
right and left widths (RW–LW) was different from zero usually found ripped open. Successfully emerged larvae
(either positive or negative) leaves were categorized as cut semi-circular exit holes on the mine underside towards
asymmetric, and if the difference between right and left the apex (Simberloff and Stiling 1987).
50

Data analysis a particular leaf type than would have been expected as a
result of a simple chance encounter with leaves of both
Leaf characters demonstrate FA if signed right-minus-left types (symmetric, asymmetric). To examine the relation-
values are normally distributed with a mean value of zero, ship between leaf types and herbivore survivorship,
reflecting randomly directed deviations from the optimal differences in mine growth, days to pupation and growth
symmetrical phenotype. One sample t-tests and Lilliefor’s rates of leaf miners in symmetric and asymmetric leaves
tests (Wilkinson 1999) were used to test whether mean were tested using One-Way ANOVAs.
values of signed right-minus-left values differed signifi- Some authors have stated that herbivores themselves
cantly from zero. Asymmetry was calculated as the may cause leaf asymmetry due to their feeding activities.
absolute difference between right and left widths of a We hypothesized that if leaf miners themselves cause
particular leaf. However, asymmetry of undamaged leaves asymmetry, we would expect that mined sides of leaves
of both plant species increased with leaf size (Q. laevis: would be larger/wider than unmined sides when the entire
r=0.412, P<0.01; Q. geminata: r=0.361, P<0.05) and so mine is encountered on a particular side (right or left) of
measurements of asymmetry on leaf width were further the leaf. To test this hypothesis, we performed paired t-
corrected for leaf size according to the formula FAwidth = tests over two scales: plants and leaves. For the leaf scale,
2×|RW − LW|/(RW + LW). According to Palmer (1996), we conducted a one-tailed paired t-test with all the mines
an important consideration in asymmetry studies is sampled that were located on a particular side of the leaf of
measurement error, since errors may look like asymmetry, Q. laevis (n=314 Brachys) and Q. geminata (n=1,017
requiring that either the symmetry differences measured Stilbosis mines). For the plant scale, we also performed a
are larger than the measurement error, or that subsequent paired t-test comparing measurements of asymmetry of 20
measurements taken on the same leaf are highly correlated. undamaged leaves collected before herbivory (April 2002)
We estimated measurement error by remeasuring 10% of with 20 undamaged leaves collected from each plant after
the leaves collected from each plant species 10 days after herbivory and after the second and partial flush of new
the first measurements were taken and the two measure- leaves in July 2002.
ments were correlated using an index of repeatability To verify whether increased herbivory in one year
(Falconer 1981). influences asymmetry in the following season, we
All the variables analysed were first submitted to compared levels of FA of 30 sand live oaks measured in
Lilliefor’s test for data normality and transformations 2002 with levels of FA of these same plants in 2003.
(angular, log-transformation and centering) were em- Thirty individuals between 0.7 and 1.3 m in height were
ployed to stabilize variances and normalize the data. monitored from April 2002 to June 2003. Twenty
However, for the sake of clarity, figure axes and means (+1 undamaged leaves were sampled from each plant in
SEM) show untransformed data. To test for differences in August 2002 for FA measurements and plants were
nutritional quality between symmetric and asymmetric monitored for Stilbosis occurrence from May to October
leaves, we averaged water, tannin, and nitrogen content of 2002, when we recorded the number of Stilbosis mines on
all symmetric and asymmetric leaves within each 200 leaves on each plant. In June of 2003 we again
individual plant (n=30 for each species) and differences sampled twenty new but fully developed undamaged
between the two leaf types were examined using one-way leaves from each plant for fluctuating asymmetry
ANOVAs. To examine the relationship between plant measurements as described before. These measurements
fluctuating asymmetry and herbivory between individuals, were further compared with leaf FA and herbivory rates
we regressed the percentage of asymmetric leaves and the recorded for the previous year.
two FA indices calculated for each plant species with the
density of leaf miners (Brachys, Acrocercops, Stilbosis,
other mines), galls, and percentage of leaves attacked by Results
folivores. Other mines included Cameraria, Buccalatrix,
and Stigmella mines that were present on the leaves, but in Tests for asymmetry on Q. laevis and Q. geminata
low abundance compared to the other leaf miners. A
stepwise interactive multiple regression (Wilkinson 1999) Q. laevis and Q. geminata demonstrated similar patterns of
was used to examine which factors predicted leaf miner leaf asymmetry before herbivory, as signed right-minus-
abundance and predictors with low tolerance values left character values were normally distributed (Lilliefor’s
(<0.10) and high collinearity were excluded from the tests, P=0.14–0.61) and did not deviate significantly from
model. To verify the relationship between FA and zero in all data sets tested (One-sample t-tests, P=0.29–
herbivory within-individuals we used One-Way ANOVAs 0.74), showing no evidence of antisymmetry or directional
to test for differences in FA between mined and unmined asymmetry. The data set used to test the relationship
leaves of Q. laevis and Q. geminata. Pairs of mined- between FA and herbivory within individuals also exhibit
unmined leaves and galled-non-galled leaves were classi- the normal distribution with a mean that does not
fied into four categories as described before (data collec- significantly deviate from zero, except for leaves mined
tion) and differences in the frequency of these categories by Acrocercops (Brachys: mean RW–LW = 0.017,
were tested using a χ2-test. Preference for each leaf type t=0.598, P=0.55; Stilbosis: mean RW–LW = 0.029,
was assumed if mines/galls were found more frequently in t=1.016, P=0.31; Acrocercops: mean RW–LW = −0.065,
 51

t=−2.777, P=0.006). The mean repeatability of FA

measurements was high for both Q. laevis (index of
repeatability = 0.905; F119,120=19.46, P<0.0001) and Q.
geminata (index of repeatability = 0.966; F119,120=21.57,
P<0.0001), indicating small measurement errors and the
reliability of FA measurements. Q. laevis leaves were, on
average, more asymmetric than Q. geminata leaves.
Values of FA index 1, for example, ranged between
0.065 and 0.882 (average: 0.469±0.045) for turkey oaks
and between 0.100 and 0.576 (average: 0.266±0.020) for
sand live oaks.

Fluctuating asymmetry and leaf quality

Asymmetric leaves of both plant species exhibited better

nutritional quality for herbivores than symmetric leaves
within the same individual plant (Fig. 2). Asymmetric
leaves contained significantly lower concentrations of
tannins (Q. laevis: F1,58=18.19; P<0.0001; Q. geminata:
F1,58=12.14; P<0.001) and higher nitrogen content (Q.
laevis: F1,58=4.50; P<0.05; Q. geminata: F1,58=4.79;
P<0.05) than symmetric leaves. No differences in water
content were observed between symmetric and asym-
metric leaves of either plant species (P>0.05). When we
regressed tannin concentration and FA indices calculated
for each plant, we observed that variation in FA index 2
explained 13.0% of the variation in tannin concentration of
Q. geminata (F1,28=8.12, P<0.05), and 16% of the
variation in tannins of Q. laevis leaves (F1,28=5.49, Fig. 2 Differences in a tannin concentration and b nitrogen content
P<0.05), but no significant relationship was observed between symmetric and asymmetric leaves of Q. laevis and Q.
geminata. Bars indicate mean ± 1SE
between the variation in FA indices and nitrogen for both
plant species (P>0.05).
between plants (F1,28=17.72, P<0.001) and 37.8% of the
variation in Stilbosis mines among individual sand live
Fluctuating asymmetry between individuals and oak plants F1,28=17.04, P<0.001). Variations in levels of
herbivory asymmetry also influenced the abundance of other leaf
miners mines—mainly Cameraria and Stigmella—in both
Herbivore abundance on the two oak species exhibited a plant species (Q. geminata: r2=0.303, F1,28=12.16,
tendency to vary with both the frequency and the levels of P<0.005; Q. laevis: r2=0.263, F1,28=9.96, P<0.005). No
asymmetry on individual plants. Q. laevis plants with a significant relationship was observed between variation in
higher percentage of asymmetric leaves were significantly FA indices and variation in densities of Acrocercops mines
more attacked by Brachys (r2=0.279, F1,28=10.84, in Q. laevis, eyespot galls on Q. geminata and chewed
P<0.005; Fig. 3) and other leaf miners (r2=0.259, leaves on both plant species (all P>0.05).
F1,28=9.81, P<0.005). Similar patterns were observed for Although variation in both the percentage of asym-
herbivores attacking Q. geminata, and the percentage of metric leaves and levels of asymmetry of Q. geminata
asymmetric leaves explained 31.8% of the variation of tended to positively influence Belonocnema galls
Stilbosis (F1,28=13.06, P<0.001; Fig. 3) and other mines (r2=0.10, P=0.071), no significant relationship was
(r2=0.125, F1,28=11.46, P<0.005). No significant relation- observed between variation in levels of FA and variations
ship was observed between the percentage of asymmetric in densities of other leaf and stem galling species studied
leaves on turkey oaks and the abundance of Acrocercops (Andricus:r2=0.09, P=0.11; Disholcaspis: r2=0.06,
mines and between the percentage of asymmetric leaves P=0.32; Callirhytis: r2=0.10, P=0.09; eyespot galls:
and chewers and stem gallers for both oak species (all r2=0.04, P=0.42).
P>0.05). A Pearson correlation matrix revealed high collinearity
Variation in levels of asymmetry between plants also among predictors of leaf miner abundance between plants,
influenced leaf miners’ abundance. Plants with higher FA such as the percentage of symmetric and asymmetric
indices exhibited higher densities of Brachys and Stilbosis leaves in a plant (Q. laevis: r=0.98, Q. geminata: r=0.97)
mines (Fig. 4). Variation In FA index 2 (size-scaled) and the two FA indices used (Q. laevis: r=0.974, Q.
explained 38.8% of the variation in Brachys abundance geminata: r=0.913). These correlations generated multiple
52

Fig. 3 Relationship between the abundance of a Brachys mines and

the percentage of asymmetric leaves on the host plant Q. laevis
(r2=0.279, P<0.005) and b Stilbosis mines and the percentage of
asymmetric leaves on Q. geminata (r2=0.318, P<0.001). Mine
abundance was determined by counting the number of mines of each
leaf miner species on 200 leaves per plant

regressions coefficients with low tolerance and all

correlated predictors were standardized by centering each
value to generate a mean of zero for each predictor (Quinn
and Keough 2002). An interactive stepwise multiple
regression revealed that 67.4% of the variation in Brachys
abundance among plants was explained by variation in FA
index 1, the amount of tannins in leaves and variation in
Q. laevis leaf area (r2=0.674, F5,24=9.902, P<0.0001).
Variation in Stilbosis abundance was also influenced by
variation in asymmetry among plants, since almost 62% of
the variation was explained by variation in FA index 1, the Fig. 4 Relationship between the abundance of mines caused by a
Brachys (r2=0.475, P<0.001), b Stilbosis (r2=0.394, P<0.001), and c
percentage of asymmetric leaves in a plant and the Acrocercops (r2=0.018, P>0.05) and the levels of relative asymme-
presence of other leaf miners (r2=0.619, F4,25=10.16, try (FA index 2) on host plants. FA index 2 refers to the asymmetry
P<0.0001). For Acrocercops mines asymmetry was not index that is size-scaled, calculated as the absolute value of right (RI)
important because 38% of the variation among plants was minus left (LI) sides divided by its average (RI + LI)/2
explained by tannin concentration and the amount of
nitrogen on leaves alone (r2=0.380, F2,27=8.288,
P<0.005).
 53

Fluctuating asymmetry within individuals and each one of the leaf miner species. For Brachys mines, for
herbivory example, in approximately 61% of the cases the mined leaf
was asymmetric and the unmined leaf in the pair was
Leaves attacked by both Brachys and Stilbosis were, on symmetric, whereas for Acrocercops mines, a more even
average, more asymmetric than unmined leaves within the distribution of the mines was observed among the four
same individual plant (Fig. 5a). Leaves mined by Brachys possible categories (Fig. 5b). For Stilbosis mines, in 61.4%
were, on average, 4.3 times more asymmetric than of the cases, the mined leaf was asymmetric and the
neighboring unmined leaves (F1,58= 39.67, P<0.0001), unmined leaf was symmetric, and in approximately 30%
while leaves mined by Stilbosis were approximately 2.6 of the cases, both leaves in a pair were asymmetric. In
times more asymmetric than unmined leaves (F1,58= cases where both leaves were asymmetric, for both
43.39, P<0.0001). However, no significant differences in Brachys and Stilbosis, mean asymmetry of the mined
asymmetry were observed between neighbouring mined leaf was significantly higher than asymmetry of the nearest
and unmined leaves attacked by Acrocercops (average unmined leaf (Brachys: F1,58=11.78; Stilbosis:
absolute FA of mined leaves: 0.265 mm±0.027, average F1,58=14.63; both P<0.005).
absolute FA of unmined leaves: 0.276 mm±0.035). For both Belonocnema and eye spot galls, we observed
Leaf miners were more frequently encountered in no significant differences in frequency of occurrence
asymmetric leaves compared to symmetric ones in both among the four categories of galled-nongalled leaves
plant species (Q. laevis: χ2=65.84, P<0.0001; Q. gemina- (Belonocnema: X2=2.78, P>0.05; eyespot galls: X2= 4.31,
ta: χ2=60.31, P<0.0001; Fig. 5b), although frequency of P>0.05) and no differences in asymmetry were found
occurrence of categories of leaf types was different for between the galled and the nearest non-galled leaf
(Belonocnema: average FA galled leaf = 0.213 mm
±0.019, average FA of non-galled leaf = 0.209 mm
±0.026; eyespot galls: average FA galled leaf = 0.236 mm
±0.023, average FA of non-galled leaf = 0.239 mm±0.025;
all P>0.05).

Fluctuating asymmetry and mine survivorship

Mines of all three species were, on average, smaller on

asymmetric leaves than on symmetric ones (Brachys:
F1,98=5.90, P<0.05, Stilbosis: F1,98=32.77, P<0.0001,
Acrocercops: n.s.; Fig. 6a) and Brachys and Stilbosis
mines on asymmetric leaves exhibited significantly slower
growth rates, consuming less plant tissue than mines
growing on symmetric leaves (Brachys: F1,49=13.89,
P<0.001; Stilbosis: F1,22=15.601, P<0.001; Fig. 6b).
Although Brachys and Stilbosis mines were smaller and
consumed less tissue on asymmetric leaves, no significant
differences in the number of days necessary to pupation
were observed between the two leaf types (Fig. 7a, all
P>0.05). Also, no significant differences in mine size,
growth rates or days to pupation were observed for
Acrocercops mines growing on symmetric and asymmetric
leaves of Q. laevis.
Mortality imposed by top-down or bottom-up factors
differed among the leaf miners studied. Acrocercops mines
exhibited the highest survivorship among the species
studied, with 68% of the mines developing until pupation,
compared to 51% of survivorship of Brachys mines and
49% of survivorship for Stilbosis mines. Parasitism by
micro hymenopterans was responsible for mortality of
Fig. 5 Differences in a fluctuating asymmetry between mined and 23% of Brachys mines, while predation and plant
unmined leaves attacked by Acrocercops, Brachys and Stilbosis and resistance (larvae found dead inside the mine) accounted
b frequency of occurrence of combinations of asymmetric and for 38% of the mortality of Stilbosis mines. For the
symmetric leaves on pairs of mined and unmined leaves for each Stilbosis mines that survived to pupation, we observed a
species studied (ms-us mined leaf, symmetric: unmined, symmetric,
ms-ua mined leaf, symmetric: unmined, asymmetric, ma-ua mined significantly higher survivorship of mines on asymmetric
leaf, asymmetric: unmined, asymmetric, ma-us mined leaf, asym- compared to symmetric leaves (X2=24.51, P<0.05;
metric: unmined, symmetric). Bars indicate mean ± 1SE. Fig. 7b). No significant differences in survivorship
54

Fig. 6 Differences in a mine size and b mine growth rate between Fig. 7 Differences in a leaf miners developmental time and b
symmetric and asymmetric leaves attacked by Acrocercops, Brachys survivorship in symmetric and asymmetric leaves of oak species.
and Stilbosis leaf miners. Bars indicate mean ± 1SE. Bars indicate mean ± 1SE.

The number of Stilbosis mines per 200 leaves on the 30

between symmetric and asymmetric leaves were observed plants studied between 2002 and 2003 ranged between 7
for Brachys or Acrocercops mines (P>0.05). and 48 mines (mean: 26.23±2.27) but higher attack rates in
some plants did not influence FA measurements in the
following year, since we observed a high correlation
Do herbivores cause asymmetry? between FA measurements in 2002 and 2003 for each one
of the 30 plants studied (r2=0.86, n=30). These results
At the plant scale, paired t-tests revealed that the mean demonstrated that although herbivory rates varied among
difference in relative FA before and after herbivory did not individuals, plants with higher number of mines in one
depart significantly from zero for both Q. laevis (mean year did not exhibit higher FA in the following year,
difference before and after herbivory: 0.003 mm, 95% CI: demonstrating a consistency of variation in FA among
= −0.005 to 0.011, t=0.689, P=0.469) and Q. geminata individual plants during 2 years.
(mean difference before and after herbivory: 0.002 mm,
95% CI: = −0.001 to 0.006, t=1.271, P=0.214). Paired t-
tests conducted at the leaf scale also revealed no Discussion
significant differences in leaf width between mined and
unmined sides of Q. laevis leaves attacked by Brachys Although literature relating the effects of plant quality to
(mean width of mined side of leaf = 7.429; mean width of variation in herbivore abundance between and within
unmined side of leaf = 7.412; t=0.604, P=0.550) or plants is plentiful, few studies have related random
between mined and unmined leaves of Q. geminata leaves variations in leaf morphology and its effects on leaf
attacked by Stilbosis (mean width of mined side of leaf = quality to variation in herbivory rates in individual plants
2.612; mean width of unmined side of leaf = 2.587; (but see Wiggins 1997; Martel et al. 1999). We suggest
t=1.192, P=0.243). These results suggest that there was no that leaf fluctuating asymmetry may be used by herbivores
direct relationship between the presence of these leaf as a predictor of plant quality, positively influencing insect
miners and changes in width of the mined side of the abundance. Several findings of our study reinforce the
leaves. hypothesis that herbivores may use asymmetry as a cue to
 55

plant quality: (1) Asymmetric leaves offered better nutri- (Lempa et al. 2000). We are unaware of other studies that
tional quality for herbivores, such as lower tannin have tested differences in nutritional quality between
concentration and higher nitrogen content; (2) plants symmetric and asymmetric leaves and how these affect
with more asymmetric leaves or higher levels of asym- herbivore preference and performance. Differences in
metry were attacked more by both Brachys and Stilbosis nutritional quality between asymmetric and symmetric
leaf miners; (3) FA indices calculated before herbivory leaves reinforce the idea that the relationship between
were reasonable predictors of leaf miners abundance at the asymmetry and herbivory is not causal, i.e., herbivory
end of the season; (4) Within a plant, leaf miners were does not cause asymmetry in our study system. Instead,
more frequently found in asymmetric leaves and mined herbivores may use asymmetry as a cue to plant quality
leaves exhibited higher levels of FA than unmined leaves; and suitable oviposition sites. Other studies that found a
(5) Mines were smaller on asymmetric leaves compared to positive relationship between fluctuating asymmetry and
symmetric leaves, and Stilbosis mines exhibited higher herbivory rates have sampled only after herbivory has
survivorship on asymmetric leaves. Most of our findings occurred, eliminating the possibility to assess whether
reinforce the hypothesis that herbivores are not responsible asymmetric leaves were present before herbivory and
for asymmetry and the relationship between herbivory and whether herbivores preferentially attack these leaves. With
asymmetry is not causal: (1) individual oak plants were our sampling design, we demonstrated that asymmetric
consistent in their levels of asymmetry before and af ter leaves were present in both Q. laevis and Q. geminata
herbivory; (2) there was no direct relationship between the before leaf miners attacked the leaves. Our results also
presence of a mine and changes on the width of the side of showed that mines caused by Brachys and Stilbosis were
the leaf where the mine had developed; (3) naturally smaller in asymmetric leaves compared to symmetric
increased levels of herbivory on Q. geminata plants did leaves and this may be explained by the lower consump-
not increase asymmetry in the following season. Since we tion rates observed in asymmetric leaves compared to
have used natural variation in asymmetry between and symmetric ones. Our results partially support the slow-
within individuals, our study did not address the sources of growth, high-mortality hypothesis since Brachys and
physiological and developmental stress in the Quercus Stilbosis mines growing on symmetric leaves with reduced
species studied, but it has been suggested that FA in leaves nutritional quality exhibited higher consumption rates
may be influenced by several abiotic and biotic factors, resulting in bigger mines at the end of the season.
such as nutritional deficiencies, water shortage, pollution Nevertheless, we found only limited support for higher
and plant competition (Palmer and Strobeck 1986). mortality rates caused by natural enemies on mines
Higher attack rates on plants with more asymmetric growing on symmetric leaves, since survivorship of
leaves and higher levels of asymmetry may be attributed to Brachys and Acrocercops mines did not differ between
the observed differences in nutritional quality between symmetric and asymmetric leaves of Q. laevis and only
symmetric and asymmetric leaves, although it is not Stilbosis mines exhibited higher survivorship on asym-
known how these differences arise. Since the left and the metric leaves of Q. geminata. These results indicate that
right sides of a particular bilaterally symmetrical trait although bottom-up factors, such as asymmetry and plant
develop under the control of the same genes, deviations quality may influence the choice of oviposition sites by
from perfect symmetry actually represent developmental insects, the positive effects of better nutritional quality on
instability, and the ability to develop symmetrical traits mine survivorship are not always realized when top-down
may be related to the ability to produce defensive factors, such as predation and parasitism, are strong and
chemicals (Moller 1995), if resource allocation to devel- other studies with leaf miners have demonstrated the
opmental control competes with allocation to production strength of top-down pressures on insect survivorship
of defensive chemicals. Also, FA may be determined by (e.g., Auerbach and Simberloff 1988; Mopper et al. 1995;
the same genes as those affecting resistance to herbivores Hawkins et al. 1997; Forkner and Hunter 2000; reviewed
or genes giving rise to elevated levels of FA may have by Connor and Taverner 1997).
pleiotropic effects on plant resistance (Moller and Swaddle Although variation in herbivory rates in both Q. laevis
1997). Although it is not well known how FA is associated and Q. geminata may be related to fluctuating asymmetry
with biochemical changes and plant metabolism, differ- and its consequential changes in plant quality, our study
ences in nutritional quality between symmetric and has also demonstrated idiosyncratic responses to FA both
asymmetric leaves arise and may be responsible for between and within guilds. Leaf chewers and leaf galls
differential attack rates in these two leaf types. This was were not influenced by levels of FA in both plant species.
first demonstrated by Sakai and Shimamoto (1965) Galling insects may not be as influenced by aspects of
studying tobacco plants and further supported by Lempa plant quality as leaf miners are, since their special mode of
et al. (2000) studying several chemical compounds in feeding within the gall allows them to manipulate plant
birch plants. For birch plants, it was observed that plants characteristics, and possibly avoid defensive strategies of
with higher levels of FA contained significantly lower the host and circumvent differences in nutritional quality
amounts of hydrolysable tannins, gallic acid, and flavo- between symmetric and asymmetric leaves. Manipulation
noid-glycosides. Laboratory feeding trials also showed of host plants by gall-formers may extend to control over
that Epirrita autumnata consumed more from birch leaves the chemical composition of gall tissues, and galling
collected from high FA trees compared to low FA trees herbivores may alter the physiological state of host tissues,
56

especially the tissues nearest to the developing larvae Heinrichs EA (1988) Plant stress-insect interactions. Wiley, New
(Price et al. 1987; Shorthouse and Rohfritsch 1992; York
Koricheva J, Larsson S, Haukioja E (1998) Insect performance on
Hartley 1998). Among leaf miners, strong responses experimentally stressed woody plants: a meta-analysis. Annu
were found for both Stilbosis and Brachys, but Acrocer- Rev Entomol 43:192–216
cops mines on turkey oaks seem not to be influenced by Larson S (1989) Stressful times for the plant stress-insect
variation in asymmetry between and within plants. This performance hypothesis. Oikos 56:277–283
Lempa K, Martel J, Koricheva J, Haukioja E, Ossipov V, Ossipova
variation in leaf miner response to fluctuating asymmetry S, Pihlaja K (2000) Covariation of fluctuating asymmetry,
may be explained by differences in life-history traits of the herbivory and chemistry during birch leaf expansion. Oecologia
species studied. Acrocercops cause relatively small linear- 122:354–360
blotch superficial mines just under the leaf epidermis of Lewis AC (1984) Plant quality and grasshopper feeding: effects of
sunflower condition on preference and performance in
turkey oak leaves and development times do not exceed Melanoplus differentialis. Ecology 65:836–843
10 days. These mines are unlikely to be strongly affected Louda SM, Collinge SK (1992) Plant resistance to insect herbivores:
by variations in plant quality due to their fast development a field test of the environmental stress hypothesis. Ecology
rates and the fact that they create limited depth mines in 73:153–169
young leaves with higher nitrogen content and smaller Martel J, Lempa K, Haukioja E (1999) Effects of stress and rapid
growth on fluctuating asymmetry and insect damage in birch
concentration of defensive chemicals. Brachys and leaves. Oikos 86:208–216
Stilbosis mines may be more likely to be affected by Mattson WJ, Haack RA (1987) The role of drought stress in
host quality, having long developmental times, full depth provoking outbreaks of phytophagous insects. In: Barbosa P,
mines, and a higher likelihood to be affected by spatial and Schultz J (eds) Insect outbreaks: ecological and evolutionary
perspectives. Academic, Orlando, pp 365–407
seasonal variation in host quality. McClure MS (1980) Foliar nitrogen: a basis for host suitability for
elongate hemlock scale, Fiorinia externa (Homoptera: Diaspi-
Acknowledgements This research was supported by DEB 00- didae). Ecology 61:72–79
89226 and we are grateful to the staff at the USF Botanical Garden Moller AP (1995) Leaf-mining insects and fluctuating asymmetry in
and to A.L. Castro for providing the drawings of the oak leaves. T.C. Ulmus glabra leaves.J Anim Ecol 64:697–707
also acknowledges the Brazilian National Research Council CNPq Moller AP, Swaddle JP (1997) Asymmetry, developmental stability
(process number 200064/01-0) for a Ph.D scholarship at USF. and evolution. Oxford University Press, Oxford
Mopper S, Beck M, Simberloff D, Stiling P (1995) Local adaptation
and agents of selection in a mobile insect. Evolution 49:810–
815
References Palmer AR (1996) Waltzing with asymmetry. Bioscience 46:518–
532
Alados CL, Navarro T, Escos J, Cabezudo B, Emlen JM (2001) Palmer RA, Strobeck C (1986) Fluctuating asymmetry: measure-
Translational and fluctuating asymmetry as tools to detect stress ment, analysis, and patterns. Annu Rev Ecol Syst 17:391–421
in stress-adapted and nonadapted plants. Int J Plant Sci Price PW, Fernandes GW, Waring GL (1987) Adaptive nature of
162:607–616 insect galls. Environ Entomol 16:15–24
Auerbach M, Simberloff D (1988) Rapid leaf-miner colonization of Quinn GP, Keough MJ (2002) Experimental design and data
introduced trees and shifts in sources of herbivores mortality. analysis for biologists. Cambridge University Press, Cambridge
Oikos 52:41–50 Roy BA, Stanton ML (1999) Asymmetry of wild mustard, Sinapis
Bultman TL, Faeth SH (1987) Impact of irrigation and experimental arvensis (Brassicaceae), in response to severe physiological
drought stress on leaf-mining insects on Emory oak. Oikos stresses. J Evol Biol 12:440–449
48:5–10 Sakai KI, Shimamoto Y (1965) Developmental instability in leaves
Clancy KM, Price PW (1987) Rapid herbivore growth enhances and flowers of Nicotiana tabacum. Genetics 51:801–813
enemy attack: sub lethal plant defenses remain a paradox. Shorthouse JD, Rohfritsch O (1992) Biology of insect-induced galls.
Ecology 68:736–738 Oxford University Press, New York
Connor EF, Taverner MP (1997) The evolution and adaptive Simberloff D, Stiling P (1987) Larval dispersion and survivorship in
significance of the leaf-mining habit. Oikos 79:6–25 a leaf-mining moth. Ecology 68:1647–1657
DeBruyn L, Scheirs J, Verhagen R (2002) Nutrient stress, host plant Waddell KJ, Fox CW, White KD, Mousseau TA (2001) Leaf
quality and herbivore performance of a leaf-mining fly on abscission phenology of a scrub oak: consequences for growth
grass. Oecologia 130:594–599 and survivorship of a leaf-mining beetle. Oecologia 127:251–
Falconer DS (1981) Introduction to quantitative genetics, 2nd edn. 258
Longman, New York White TCR (1984) The abundance of invertebrate herbivory in
Forkner R, Hunter MD (2000) What goes up must come down? relation to the availability of nitrogen in stressed food plants.
Nutrient addition and predation pressure on oak herbivores. Oecologia 63:90–105
Ecology 81:1588–1600 Wiggins DA (1997) Fluctuating asymmetry in Colophospermum
Hagerman AN (1987) Radial diffusion method for determining mopane leaves and oviposition preference in an African silk
tannin in plant extracts. J Chem Ecol 13:437–449 moth Imbrasia belina. Oikos 79:484–488
Hartley SE (1998) The chemical composition of plant galls: are Wilkinson L (1999) SYSTAT: the system for statistics. Version 9.0.
levels of nutrients and secondary compounds controlled by the SYSTAT, Evanston
gall-former? Oecologia 113:492–501 Zvereva E, Kozlov M, Haukioja E (1997) Stress responses of Salix
Hawkins BA, Cornell HV, Hochberg ME (1997) Predators, borealis to pollution and defoliation. J Appl Ecol 34:1387–
parasitoids, and pathogens as mortality agents in phytophagous 1396
insect populations. Ecology 78:2145–2152

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