ORIGINAL PAPER

Similar responses of insect herbivores to leaf fluctuatingasymmetry

Tatiana Cornelissen • Peter Stiling

Received: 11 August 2010 / Accepted: 25 November 2010 / Published online: 7 December 2010! Springer Science+Business Media B.V. 2010

Abstract Fluctuating asymmetry (FA) represents small,random variation from symmetry and it has been used as an

indicator of plant quality and susceptibility to herbivory. In

this study, the effects of FA on the responses of distinctherbivore species belonging to several guilds were exam-

ined along an environmental gradient in south Florida. This

approach was chosen because it relies on a multi-speciesapproach to the study of fluctuating asymmetry and pat-

terns of herbivory between and within plants along an

environmental gradient of salinity and plant stress. Toexamine differences in FA between and within plant

communities, seven plant species were investigated. Four

of these plants were coastal species and three speciesoccurred in upland communities. Levels of FA were

assessed before herbivory and plants were followed for the

whole herbivory season in 2006. Coastal plants exhibitedsignificantly higher salt concentration, higher percentage of

asymmetric leaves and higher asymmetry levels than

upland plants. Herbivore abundance varied widely amongstthe seven species studied, but quantitative syntheses of our

results indicated significant and positive responses of insect

herbivores to leaf asymmetry: insects were 25.11% moreabundant on more asymmetric plants and stronger effects

of asymmetry were observed for leaf miners compared to

gall-formers. As demonstrated by other recent studies, FAmight be used as a reliable stress indicator, leading to

similar responses of insect herbivores to variation in leaf

symmetry.

Keywords Fluctuating asymmetry ! Plant stress ! Leafminers ! Gall-formers ! Herbivory ! Coastal-uplandcomparisons

Introduction

Fluctuating asymmetry (FA) represents small, random

variation from symmetry in otherwise bilaterally symmet-

rical characters and it is a widely used measure of devel-opmental instability in plants and animals (Diaz et al.

2004). Developmental instability (DI), which has been

promoted as an indicator of environmental and/or geneticstress (Polak 2003), is the inability of a genotype to con-

sistently produce the same phenotype in a particular envi-ronment (Moller and Swaddle 1997). FA is a type of DI

and it occurs when otherwise bilateral traits show small

variations in the size of the two sides of a character arounda bilateral symmetric axis. Because the development of

both sides of bilaterally symmetric traits is controlled by

the same genes, the degree of FA represents developmentalperturbations and reflects interference in the control of

development processes when growing under given envi-

ronmental conditions.Significant increases in FA have been found for several

species of plants and animals in response to various

stresses (but see Bjorksten et al. 2000 and review by

Handling Editor: Stanislav Gorb.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11829-010-9116-1) contains supplementarymaterial, which is available to authorized users.

T. Cornelissen (&)Department of Bioengineering, Ecosystem BioengineeringBuilding, Campus Tancredo Neves, Universidade Federal de SaoJoao Del Rei, Sao Joao Del Rei, MG 36301-360, Brazile-mail: tatiana@ufsj.edu.br; tgcornel@mail.usf.edu

P. StilingDepartment of Biology SCA 110, University of South Florida,4202 E Fowler Av, Tampa, FL 33620-5150, USA

123

Arthropod-Plant Interactions (2011) 5:59–69

DOI 10.1007/s11829-010-9116-1

Graham et al. 2010), including environmental factors, such

as climatic conditions (Wilsey et al. 1998; Valkama andKozlov 2001), pollution (Kozlov et al. 1996), urbanization

(Weller and Ganzhorn 2004), deficiencies or excess of

nutrients (Moller 1995), temperature and radiation (Jen-tzsch et al. 2003; Hosken et al. 2000), salt stress (Roy and

Stanton 1999), and intra- and inter-specific interactions

such as competition (Rettig et al. 1997), predation andparasitism (Reimchen 1997; Hunt and Allen 1998). FA is

also affected by genetic factors, such as mutation(McKenzie and Clarke 1988), inbreeding (Wakefield et al.

1993, Sherry and Lord 1996), and interspecific hybridisa-

tion (Wilsey et al. 1998; Hochwender and Fritz 1999).Because FA constitutes a quick, simple, and inexpensive

measure, it has frequently been used for monitoring levels

of ecological stress, either abiotic, biotic or both (Hodar2002).

Fluctuating asymmetry is generally estimated by mea-

suring the average deviations from an axis of symmetry,with the key hypothesis being that organisms that develop

in a stress-free environment are better able to buffer or

correct most of the random errors in development (Free-man et al. 2004). As stress increases, either the ability of

the development program to resist perturbations or the

resilience of the system to recover from perturbations isdegraded, resulting in developmental errors that can be

measured as deviations from symmetry (Freeman et al.

2004). FA has been widely used to compare populationssubjected or not to a number of environmental stressors

such as temperature, nutrition, radiation, chemicals, popu-

lation density, noise, parasites, light conditions, fire, pre-dation risk, and habitat structure (Moller and Swaddle

1997, reviewed in Hoffman and Woods 2003). The work-

ing hypothesis in such comparisons is that DI, and its mosteasily observable outcome, FA, will be higher in the more

stressed populations compared to the control or unstressed

populations (Leamy and Klingenberg 2005). FA measureshave also been used to detect and measure salinity stress in

plants (e.g., Anne et al. 1998, Roy and Stanton 1999;

Sinclair and Hoffmann 2003). Moller and VanDongen(2003), for example, demonstrated that Ulmus glabraleaves that developed on plants to which a saline-solution

was added became significantly more asymmetric withtime than plants that were treated with water only.

Although FA has been related to salinity and/or water

stress in several natural and cultivated plant systems, noneof these studies related variation in levels of FA among

plants under different salinity conditions with herbivory

levels experienced in natural communities.In addition to being an indicator of plant stress, some

studies have shown correlations between FA and insect

herbivore attack, suggesting leaf FA may also serve as anindicator of susceptibility to herbivory. According to this

hypothesis, plants with more asymmetric leaves or higher

levels of leaf asymmetry would be more attacked onaverage than more symmetric plants. Differences in nutri-

tional quality and/or secondary chemistry between sym-

metric and asymmetric leaves have been suggested aspotential explanations for higher herbivore abundance or

higher herbivory levels in more asymmetrical plants or

leaves (see Sakai and Shimamoto 1965; Lempa et al. 2000;Telhado et al. 2010). Moller (1995) suggested that foliar

asymmetry in elm trees directly influenced the abundanceof the leaf-mining beetle Rhynchaenus rufus. Other cor-

relative evidence that FA might be associated with higher

risks of herbivory comes from studies with larvae of thegeometrid Epirrita autumnata (Lempa et al. 2000), as

mountain birch trees with higher FA levels were more

heavily attacked by E. autumnata than trees exhibitinglower FA levels. For oaks, previous studies conducted by

our research group with leaf miners and gall-formers sug-

gested that leaf fluctuating asymmetry was used by herbi-vores as a predictor of plant quality, positively influencing

insect abundance (e.g., Cornelissen et al. 2003; Cornelissen

and Stiling 2005, 2010).In this study, we used a multi-species approach to the

study of fluctuating asymmetry and patterns of herbivory,

both between and within plants along an environmentalgradient in Florida. The role of environmental stress,

although important in trophic dynamic theory, has received

considerably less attention than the role of nutrients (Moonand Stiling 2004). Stress, most frequently in the form of

salinity or drought stress, has been shown in a number of

studies to significantly impact top-down and bottom-upeffects on herbivore abundance and population dynamics

(e.g., Waring and Cobb 1992, reviewed by Huberty and

Denno 2004; Kanaga et al. 2009). Coastal-upland transi-tions provide valuable environmental gradients because in

these habitats there are often gradients of both nutrients and

environmental stress (Adam 1990). Plants growing closerto the shore may experience soil pore water salinities that

are 15–40% greater than salinity levels experienced by

upland plants (Moon and Stiling 2004) and we used thisnatural gradient in salinity levels to examine the role of salt

stress in the expression of leaf fluctuating asymmetry for

several plant species.This study examined the relationship between leaf

fluctuating asymmetry and herbivory by several insect

guilds on 7 plant species that occur along a salinity gradientfrom coastal to upland habitats in Tampa Bay, FL. Spe-

cifically, the following hypotheses were tested:

1. Fluctuating Asymmetry on coastal and upland systemscoastal systems are subject to higher levels of salinity,

nutrient and/or water stress than upland systems andplants growing on coastal areas should exhibit higher

60 T. Cornelissen, P. Stiling

123

levels of FA and higher herbivory levels than plants

growing in upland areas;

2. Fluctuating Asymmetry, plant stress and herbivoryfluctuating asymmetry in otherwise symmetrical bilat-

eral traits is a surrogate of plant stress and asymmet-

rical leaves should differ in nutritional quality andherbivore susceptibility compared to symmetrical

leaves;

3. Fluctuating Asymmetry between individual plants andlevels of herbivory if FA in leaves predicts plant

susceptibility to herbivores, plants with more asym-

metric leaves or higher degrees of asymmetry shouldbe subject to higher levels of herbivory than individual

plants with a lower incidence of foliar asymmetry.

Study systems

Of the seven plant species studied, 4 species (Coccolobauvifera, Iva frutescens, Baccharis halimifolia, and Avi-cennia germinans) grow in coastal areas 30 m or less fromshore, whereas 3 species (Myrica cerifera, Persea borbo-nia, and Quercus geminata) grow in upland hammocks at

least 50 m from shore. All of these plant species supportmultiple species and guilds of herbivores, such as leaf

miners, gall formers, stem borers and leaf chewers (Plate 1in Supplementary material).

Coastal species

The sea grape, Coccoloba uvifera (Polygonaceae) is a salt-

tolerant dioecious tree that can grow in any coastal habitatfrom beach dunes to upland hammocks. In Florida, C.uvifera occurs in coastal areas in the Gulf of Mexico and in

the Atlantic coast (Wunderlin and Hansen 2004). Its leaves,which can be up to 25 cm wide, are frequently attacked by

two species of lepidopteran leaf miners (Nepticulidae) that

create serpentine mines. These two types of mines areeasily distinguished by their width, as the mines created by

morphospecies 1 do not exceed 0.5 mm in width (except at

the oval-shaped terminal chamber), whereas mines createdby morphospecies 2 are typically 2–3 mm wide, and no not

end in a widened chamber. A single leaf can support up to

100 mines, although we have noticed variable densitiesamong plants in the study site.

Iva frutescens (Asteraceae), also known as marsh elder,

is a perennial, succulent herb or woody shrub up to 12 feettall. It occurs on beach dunes and salt marshes on coastal

counties in Florida. In our study sites, the leaf miner moth

Bucculatrix sp. (Lepidoptera: Bucculatricidae) is com-monly found on the midvein of Iva leaves. The adults

emerge in April and May and lay eggs on upper sides of

fully-grown leaves and newly emerged larvae feed as

miners, creating a characteristic thin, serpentine blackmine. Another very common herbivore on I. frutescens is

an undescribed eriophyid mite (Acari: Eriophyidae) that

creates numerous leaf galls, with densities reaching up to100 galls on a single leaf. Galls are white and glabrous.

The dioecious shrub Baccharis halimifolia (Asteraceae)

can tolerate a wide range of salinities and can be foundthroughout the coastal gradient In Florida. The most

common herbivores on B. halimifolia at our site are the leafbeetle Trirhabda baccharidis (Coleoptera: Chrysomelidae),

the gall-making fly Neolasioptera lathami (Diptera: Cec-idomyiidae), and the leaf mining flies Liriomyza trifolii andAmauromyza maculosa (Diptera: Agromyzidae).

The black mangrove Avicennia germinans (Avicennia-

ceae) grows in coastal tidal areas throughout the tropics,and it is found on the west gulf coast of Florida from Cedar

Key to Key West in our study sites, Avicennia leaves are

attacked by the agromyzid fly Phytoliriomyza sp. (Diptera:Agromyzidae) whose larvae create distinct linear mines on

the adaxial surface of new leaves. The larva forms a narrow

linear mine in the first instar, later producing a largeirregular blotchy mine with conspicuous black frass. Cec-idomyia avicenniae (Diptera: Cecidomyiidae) induces

spheroid galls on the abaxial and adaxial leaf surfaces ofthis host plant (Gagne 2004) and galls are yellow–green,

glabrous, and one-chambered. Leaves are also damaged by

larvae of the weevil Stenobaris avicenniae (Coleoptera:Curculionidae) and by an unidentified pyralid moth

(Pyralidae).

Upland species

The wax myrtle, Myrica cerifera (Myricaceae) is a com-mon dioecious plant of upland hammocks of coastal hab-

itats in Florida. The most common herbivores of this

species are two species of lepidopteran leaf miners: Nep-ticula myricafoliella (Nepticulidae), which creates a ser-

pentine mine, and an unidentified species, which creates a

blotch-shaped mine. Wax myrtle leaves are also frequentlyattacked by an unknown species of leaf-tier lepidopteran

(Hesperiidae).

Another common plant of upland hammocks is Red Bay(Persea borbonia). This species experiences significant

chewing damage caused by katydids (Microcentrotus sp.),bush crickets, and lepidopteran larvae. Another commonherbivore of P. borbonia is the leaf-galling homopteran

Trioza magnoliae (Psyllidae), which creates large galls

usually on the leaf margins, in which nymphs feed (John-son and Lyon 1988). A single leaf can have as many as 8

galls, and as many as 80% of the leaves may contain galls.

The sand live oak Quercus geminata (Fagaceae) is acommon tree in upland hammocks and sandhills in Florida

Fluctuating asymmetry in coastal and upland communities 61

123

and reaches up to 2 m in our study site. Stilbosis qua-dripustulatus (Lepidoptera: Cosmopterygidae) is a univol-tine species whose larvae create mines on the adaxial

surfaces of Q. geminata. Adults emerge in early summer

and oviposition occurs approximately in late May or earlyJune. Larvae take from 60 to 90 days to complete their 5

instars and mines may reach 3.0 cm in length. Leaves are

also frequently damaged by chewing insects such as theeastern buck moth Hemileuca maia (Lepidoptera: Satur-

niidae), and at least 4 cynipid species (Hymenoptera:Cynipidae) of galling insects are commonly observed on

sand live oak leaves and stems: Andricus quercusfoliatus,Disholcaspis quercussuccinipes, Callirrhytis quercusbata-toides, and Belonocnema quercusvirens. A. quercusfoliatusis the most common gall and induces white flower-like

galls on sand live oak stems.

Methods

This study was conducted on the coast of the Gulf of

Mexico at Fort DeSoto Park (N 27" 37.245’, W 82"43.614’) in Pinellas County, Florida, between 2005 and

2006. Between 15 and 20 individual plants of each of the 7

host species were marked and followed over the herbivoryseason of 2006.

Differences in plant quality in coastal and uplandsystems

To examine differences in soil salinity and soil nutrientsbetween coastal an upland communities, soil samples from

each individual plant were collected in 2005 and analyzed

for Ec (Electro Conductivity) and percentage of organicmatter, all determined at the UF/IFAS Extension Soil

Testing Laboratory (Gainesville, FL). Ec is a measure of

soluble salts within the soil and as the concentration ofsoluble salts increases, the Ec of the soil extract increases.

To examine differences in nutritional quality and pat-

terns of asymmetry on all plant species from coastal andupland communities, 10 mature undamaged leaves of each

plant species were collected on 10 individual plants to

quantify foliar nitrogen content at the beginning of eachherbivory season. Leaves were ground in a Wiley mill and

analyzed for foliar nitrogen (expressed as percentage of dry

mass) using a CE Instruments NC2100 CN Analyzer (CEElantech Incorporated, Lakewood, New Jersey, USA).

The working hypothesis was that plants from coastal

communities would exhibit higher soil Ec and lowersoil organic matter content than plants from upland

communities.

Fluctuating asymmetry, plant stress and herbivory

To examine the relationship between FA and leaf qualityand to examine the frequency of occurrence of asymmetric

leaves and levels of leaf asymmetry on each plant, 20

leaves were sampled from each individual plant betweenMarch and April of 2006 (n = approximately 4,200

leaves). Because herbivores themselves may act as plant

stressors, these leaves were sampled before the beginningof mine initiation, gall formation and before leaves were

damaged by free-feeding herbivores. To quantify leaf FA,

widths of all leaves were measured on both the right andthe left side, from the leaf edge to the midrib, at the middle

point of the leaf, which usually coincides with the widest

part of the leaf. These distances were measured afterphotographing each leaf with a digital camera at a standard

distance of 30 cm in the laboratory and analyzing leaf

length, leaf area, and right and left widths using the soft-ware UTHSCSA Image Tool (University of Texas, USA).

All the digital pictures were calibrated to the nearest

0.01 mm before measurements were taken. Absoluteasymmetry in leaf width was defined as the unsigned dif-

ference between right width (RW) and left width (LW) of a

particular leaf as FAwidth = |RW - LW|. The absolutevalue of right-left traits is a good estimator of variance in

FA among leaves assuming that there is no directional

asymmetry (consistently larger left or right side) or anti-symmetry (consistent lack of symmetry, but in no partic-

ular direction).

To examine differences in nutritional quality betweensymmetric and asymmetric leaves, 20 symmetric and 20

asymmetric leaves sampled from each plant species

(n = 280 leaves) were analyzed for foliar nitrogen content,as described before. To categorize the leaves into either

symmetric or asymmetric, we followed Cornelissen and

Stiling (2005) and Telhado et al. (2010). If the differencebetween right and left widths (RW-LW) was different from

zero (either positive or negative) leaves were categorized

as asymmetric. On the other hand, if the difference betweenright and left widths was equal to zero leaves were cate-

gorized as symmetric. However, because we used only 2

decimal places when categorizing FA values and calcu-lating FA indices, leaves with FA measurements ranging

between 0.001 and 0.09 mm were rounded to zero and

categorized as symmetric, in a more conservativeapproach. Leaves yielding absolute FA values equal or

greater than 0.1 mm (either positive or negative) were all

classified as asymmetric.The working hypotheses were that plants from coastal

communities should exhibit higher levels of asymmetryand/or higher percentage of asymmetric leaves than plants

from upland communities and asymmetric leaves should

62 T. Cornelissen, P. Stiling

123

exhibit higher nutritional quality for herbivores than sym-

metric leaves.

Fluctuating asymmetry between individual plants

and levels of herbivory

To examine the relationship between plant fluctuating

asymmetry and herbivory between-individual plants, weused data on asymmetry from the 20 leaves collected and

digitized from each individual plant to calculate two indi-ces of FA (Palmer and Strobeck 1986):

Index 1 "P

Ri # Lij jN

Index 2 "

P Ri#Lij j$Ri % Li&=2

! "

N

where Ri is the value of the right side, Li is the value of

the left side and N is the number of measurements taken.Index 1 is the absolute fluctuating asymmetry and is the

most intuitive asymmetry measurement (Roy and Stanton

1999). Index 2 is size-scaled, calculated as the absolutevalue of right (Ri) minus left (Li) sides divided by the

average (Ri ? Li)/2, to correct for the fact that asym-

metry may be size-dependent, i.e., larger leaves mightexhibit higher levels of FA. According to Palmer (1996),

an important consideration in asymmetry studies is

measurement error, since errors may look like asymme-try, requiring that either the symmetry differences mea-

sured are larger than the measurement error, or that

subsequent measurements taken on the same leaf arehighly correlated. We estimated measurement error by re-

measuring 10% of all leaves collected 10 days after the

first measurements were taken and the two measurementswere correlated using an Index of repeatability (Falconer

1981).

The FA indices were then correlated with the density ofleaf miners and gall formers recorded on each individual

plant. All tagged plants were monitored for the occurrence

of herbivores in 2006. The abundance of each herbivorewas assessed by recording the number of mines, leaf galls,

and galled leaves in 200 leaves randomly selected for

counts on each individual plant, and the number of stemgalls seen on the associated stems while counting these 200

leaves. Counts were performed by the end of May,

beginning of August and end of October.The working hypothesis was that plants with higher

degrees of asymmetry should exhibit higher herbivory rates

than plants with lower degrees of asymmetry.

Data analysis

Leaf characters demonstrate FA if signed right-minus-leftvalues are normally distributed with a mean value of zero,

reflecting randomly directed deviations from the optimal

symmetrical phenotype. One sample t tests and Lilliefor’stests were used to test whether mean values of signed right-

minus-left values differed significantly from zero for all

plants species studied. We estimated measurement error byre-measuring approximately 10% of all leaves collected

and the two measurements for this group of leaves were

correlated using an Index of repeatability (Falconer 1981).All the variables analysed were firstly submitted to

Lilliefor’s test for data normality and transformations were

employed to stabilize variances and normalize the data,when necessary. However, for the sake of clarity, figure

axes and means (?1 SEM) show untransformed data.

Differences in salinity and soil organic matter betweencoastal and upland plants were examined using a hierar-

chical nested design. We used habitat type (upland vs.

coastal) as the main factor, host plants on each habitat asthe nested factor (seven plant species) and replicate indi-

vidual plants for each species as the residual (at least 15

individual plants of each species). Differences in nitrogencontent between symmetric and asymmetric leaves were

examined using a Two-Way ANOVA with leaf types

(symmetric and asymmetric) and plant species (one toseven) as factors. To examine the relationship between

plant fluctuating asymmetry and herbivory between-indi-viduals, we regressed the size-corrected FA index (FAIndex 2) calculated for each individual plant (n = 15 for

most species) with the density of each herbivore species (8

leaf miners and 4 gall-formers). All statistical analyseswere conducted with Systat 9.0.

To provide a general understanding of the relationship

between asymmetry and herbivory rates at the community-and habitat-level, and to examine the true effect of asym-

metry on insect herbivores, we calculated Pearson product-

moment correlation coefficients r as a measure of effectsize, further weighed by their sample sizes (number of

replicates for each comparison). At the end, all individual

correlation coefficients were z-transformed according tothe formula:

z " 1

2ln

1% r

1# r

# $

and the asymptotic variance of z was calculated as

vz "1

n# 3

Fluctuating asymmetry in coastal and upland communities 63

123

where n is the sample size. Fisher’s Z-transform ranges

from -# to ?#, where negative values of z represent anegative effect, positive values of z represent a positive

effect and z = 0 represents no effect (Rosenberg et al.

2000). To estimate the cumulative effect size (E??) for the15 replicates per plant species, effect sizes were combined

using a weighted average (Rosenberg et al. 2000) and toexamine the effects of herbivore guild (leaf miners vs. gall-

formers) and plant habitat (coastal vs. upland) in shaping

responses to leaf asymmetry, we used a mixed-modeleffect. We calculated the total heterogeneity (QT) as well as

heterogeneity within (QW) and between groups (QB) and

the significance of these statistics was evaluated using aChi-square distribution. Effect sizes were considered sig-

nificant if bootstrapped (999 iterations) 95% confidence

intervals did not overlap zero.

Results

Tests of fluctuating asymmetry

All plants studied exhibited true patterns of fluctuating

asymmetry, as demonstrated by the normal distribution of

the signed values of RW-LW (Lilliefor’s tests, allP[ 0.05),which did not deviate significantly from a mean of zero.

These results indicate there was no directional asymmetry or

antisymmetry in the populations studied. The mean repeat-ability of FAmeasurements was high for all species (Index of

repeatability = 0.916–0.984, P\ 0.0001) indicating small

measurement errors and the reliability of FA measurements.

Differences between coastal and upland systems

Coastal species exhibited significantly higher Ec values than

upland species (coastal: 3.27 ± 0.23 ds/m, upland:

0.23 ± 0.03 ds/m, F1,99 = 154.71, P\ 0.0001) and there

was significant variation in salinity levels among plantswithin habitats (F6,94 = 46.16, P\ 0.0001). We also

observed a salinity gradient, with salt concentration

decreasing from A. germinans to Q. geminata (Fig. 1).Organic matter followed an opposite trend, with soils from

coastal communities exhibiting significantly lower concen-

tration than soils from upland communities (coastal plants:3.32 ± 0.607, upland plants: 11.40 ± 1.70, F1,99 = 20.9,

P\ 0.001) and there was also variation in organic matterlevels among plants within habitats (F6,94 = 7.78,

P\ 0.001). There was no significant interaction, however,

between habitat type and plant species (P[ 0.05).Nitrogen concentration varied among studied plants

(Fig. 2) and Iva exhibited the highest nitrogen content

among all plants. Coastal plants tended to exhibit highernitrogen content than upland plants (average foliar nitrogen

in coastal plants: 1.95 ± 0.11, upland plants: 1.76 ± 0.06),

but differences were not statistically significant (habitateffect: F1,62 = 2.18, P[ 0.05).

Fluctuating asymmetry, plant stress and herbivory

Coastal species exhibited a significantly higher percentage

of asymmetric leaves (69% ± 2.1) than upland species(55.3% ± 1.0; F1,110 = 24.16, P\ 0.0001) and asymme-

try levels were significantly higher for coastal species

(mean FA Index 1: 1.243 ± 0.143) compared to uplandspecies (mean FA Index 1: 0.646 ± 0.36; F1,110 = 11.41,

P = 0.001). The coastal plant Coccoloba uvifera exhibited

the highest FA value amongst all plants studied (mean FAIndex 1: 2.982 ± 0.29), whereas the upland plant Myricacerifera exhibited the lowest FA value (mean FA Index 1:

0.480 ± 0.041).Asymmetric leaves from coastal and upland species

exhibited higher nitrogen content than symmetric leaves

Fig. 1 Salinity gradientexhibited by plants from coastalto upland communities. Salinitywas measured in terms ofElectro conductivity (Ec) and ispresented as mean (± 1 SE)values for each plant species. 1Avicennia germinans(Ec = 4.21 ± 0.23), 2Coccoloba uvifera(Ec = 4.01 ± 0.44), 3 Ivafrutescens (Ec = 2.26 ± 0.39),4 Baccharis halimifolia(Ec = 1.74 ± 0.30), 5 Myricacerifera (Ec = 0.304 ± 0.072),6 Persea borbonia(Ec = 0.265 ± 0.044), and 7Quercus geminata(Ec = 0.123 ± 0.025)

64 T. Cornelissen, P. Stiling

123

(Two-Way ANOVA, F1,62 = 9.03, P = 0.044; Fig. 2) and

significant differences in nitrogen on both leaf types wasobserved among species (Two-Way ANOVA,

F6,62 = 20.58, P\ 0.001). There was no significant inter-

action between leaf type (symmetric vs. asymmetric) andplant species (F6,62 = 0.396, P = 0.849).

Fluctuating asymmetry between individual plantsand levels of herbivory

Herbivore abundance varied widely among the seven spe-

cies studied. For leaf miners on coastal plants, variation in

fluctuating asymmetry explained 48.8% of the variation ofserpentine mines on C. uvifera, 41.6% of the variation of

serpentine mines on Iva, 34.2% of the variation in the

abundance of blotchy mines on B. halimifolia and 27.4% ofthe variation in the abundance of linear mines on A. ger-minans (Fig. 3). Variation on serpentine mines on B. ha-limifolia was influenced by variation in the levels of FA butthe relationship was only marginally significant (r2 =

0.203, P = 0.09). For upland plants, on the other hand,

fluctuating asymmetry did not explain variation in leafminer abundance for oak or red bay leaves (Fig. 3), but

explained 26.5% on the variation of serpentine mines in

wax myrtle.For gall-formers, variation in plant fluctuating asym-

metry positively influenced the abundance of leaf galls on

P.borbonia (r2 = 0.259, P = 0.05) and Andricus stemgalls on Q. geminata (r2 = 0.277, P = 0.04). No signifi-

cant relationship was observed between variation in FA

and galls on coastal plants such as Iva and Baccharis(Fig. 4).

General responses of insect herbivores to leaf

asymmetry

Quantitative synthesis of our results indicated significant

and positive relationships between insect herbivores and

leaf asymmetry. For all insect species combined, herbi-vores were 25.11% more abundant on more asymmetric

plants (E ?? = 0.2511, bootstrapped CI = 0.1596–0.341,

df = 11). When herbivores were partitioned into feedingguilds, stronger and significant effects of leaf asymmetry

were observed for leaf miners, which increased by almost

30% on more asymmetric plants (E ?=0.2984, CI =0.063–0.536, df = 7) and effects of asymmetry on gall-

formers were positive but not significant (E ?=0.1518,

CI = -0.02–0.28, df = 3). When plants were categorizedinto habitat, we observed stronger effects of asymmetry on

coastal herbivores (E? = 0.283, CI = 0.24–0.54, df = 6)

compared to inland herbivores (E? = 0.202, CI = 0,15–0.56, df = 4; QB = 3.52, df = 1, P\ 0.05).

Discussion

The concept of environmental stress has unknown impli-cations for organism functioning and population dynamics

(Escos et al. 2000). Plants in coastal, saline and infertile

environments might be adapted to low levels of resourcesand water and might show adaptive mechanisms to deal

with stress. Our results have shown, however, that the

coastal plants studied do show signs of environmentalstress, measured as fluctuating asymmetry. Environmental

stresses, such as water and nutrient deficiency, high salinity

and shade, have been shown to increase plant develop-mental instability (Roy and Stanton 1999), which may

serve as a potential tool to evaluate individual responses to

environmental gradients (Inbar and Kark 2007). Amongstall the plants studied, Iva and Coccoloba exhibited the

highest values of FA, and inland plants, such as Myrica,exhibited lower values of FA. These results indicate thatcoastal plants are stressed and this can result in higher

levels of herbivory. Insect herbivores might be directly

affected by these coastal-inland gradients or indirectlyaffected by changes in leaf quality, resulting in variable

herbivory rates. In fact, plants in coastal habitats supported

twice as many leaf miners as inland plants (ANCOVA,F1,124 = 9.85, P = 0.002) and four times more gall-

formers than inland plants (ANCOVA, F1,57 = 27.72,

P = 0.0001).Lempa et al. (2000) and Cornelissen and Stiling (2005)

suggested links between leaf asymmetry and biochemical

pathways on host plants for the synthesis of defensivecompounds, and that this might influence the differential

distribution of sessile insects on symmetric and asymmetric

Fol

iar

nitr

ogen

(%

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

521 73 6

Symmetric leaves

Asymmetric leaves

4

UplandCoastal

Fig. 2 Variation in foliar nitrogen (%) between coastal and inlandplants and symmetric and asymmetric leaves. Bars representmean ± SE and numbers on the x-axis represent each plant speciesstudied as described on Fig. 1

Fluctuating asymmetry in coastal and upland communities 65

123

Min

e de

nsity

0

5

10

15

20

25M

ine

dens

ity

0

2

4

6

8

10

12

14

16

18

20

22

FA INDEX 2

Min

e de

nsity

0

5

10

15

20

25

Min

e de

nsity

0

2

4

6

8

10

12

FA INDEX 2

r2 =0.416p=0.009

r2 =0.265p=0.049

r2 =0.11p=0.71

r2 =0.06p=0.36

Min

e de

nsity

6

8

10

12

14

16

18

20

22

24

26

28

Min

e de

nsity

0

10

20

30

40

50

Min

e de

snity

0

2

4

6

8

10

0.0 0.4 0.8 1.2 1.6 0.5 1.0 1.5 2.0

0.4 0.8 1.2 1.6 2.0 2.40.0 0.5 1.0 1.5 2.0 2.5 3.0

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 0.6 0.8 1.0 1.2

0.0 0.2 0.4 0.6 0.8 1.0 1.20.2 0.4 0.6 0.8 1.0 1.2 1.4

Min

e de

nsity

0

20

40

60

80

100

120

r2=0.203p=0.09

r2=0.342p=0.02

r2=0.488p=0.03

r2=0.274p=0.01

Fig. 3 Leaf miner responses tovariation on plant fluctuatingasymmetry as measured by FAIndex 2 (size-scaled). Figuresshow untransformed axes

66 T. Cornelissen, P. Stiling

123

plants. In this study, we have demonstrated not only that

coastal plants are under more stressful conditions that can

be assessed using FA, but also that asymmetric leavesrepresent high-quality tissue for endophagous insects, at

least in terms of the amount of available nitrogen. We did

not address changes in leaf quality that were related tosecondary chemistry, but our results indicated that one of

the primary factors that influence herbivore abundance and

survivorship—foliar nitrogen—was higher on asymmetricleaves, suggesting that insect herbivores might be selecting

stressed plants based upon higher availability of nitrogen.

Increased herbivore density with increased levels of plantstress measured as fluctuating asymmetry has been previ-

ously demonstrated for several insects species on unrelated

plants (see Moller 1995; Lempa et al. 2000; Cornelissenet al. 2003; Cornelissen and Stiling 2005; Perez-Contreras

et al. 2008).

In our study, different herbivores within the same guildresponded differently to leaf asymmetry, but leaf miners

exhibited stronger and more consistent responses than gall-

formers. Although both guilds are endophagous and bothbenefit from increased plant quality, leaf miners are more

constrained by female choices than are gall-formers. The

former guild usually develops in the same leaf as ovipo-

sition occurs (Connor and Taverner 1997, Sinclair and

Hughes 2010), whereas gall-formers are also constrained tointernal feeding, but with the possibility to change aspects

of leaf quality to benefit the growing larvae. Levels of plant

fluctuating asymmetry are related to tissue quality andovipositing females may use variation in plant morphology

as a cue for between- and/or within-plant selection for

oviposition. Higher asymmetry may indicate low levels ofdefensive compounds and/or higher levels of available

nitrogen as seen in tobacco (Sakai and Shimamoto 1965),

birchs (Lempa et al. 2000), oaks (Cornelissen and Stiling2005) and pines (Perez-Contreras et al. 2008). For the

processionary moth (Thaumetopoea pityocampa), for

example, Perez-Contreras et al. (2008), found that asym-metric leaves of Pinus exhibited lower levels of limonene,

a compound previously shown to reduce the processionary

moth oviposition. Although herbivores responded differ-ently to plant asymmetry, quantitative syntheses of our

community responses do indicate a positive relationship

between leaf asymmetry and herbivory rates suffered byplants on both coastal and inland habitats. As demonstrated

by other recent studies (e.g., Puerta-Pinheiro et al. 2008;

Ste

m g

all d

ensi

ty

0

10

20

30

40

FA INDEX 2

Ste

m g

all d

ensi

ty0

20

40

60

80

100

120

FA INDEX 2

Num

ber

galle

d le

aves

0

20

40

60

80

100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.4 0.6 0.8 1.0 1.2 1.40.0 0.4 0.8 1.2 1.6 2.0

0.5 1.0 1.5 2.0

Leaf

gal

l den

sity

0

5

10

15

20

25

30

r2 =0.259p=0.05

r2 =0.277p=0.04

r2=0.03

p=0.50r2 =0.013p=0.69

Fig. 4 Gall-former responses tovariation on plant fluctuatingasymmetry as measured by FAIndex 2 (size-scaled). Figuresshow untransformed axes

Fluctuating asymmetry in coastal and upland communities 67

123

Perez-Contreras et al. 2008), FA might be used as a reliable

stress indicator.

Acknowledgments This research was supported by the NationalScience Foundation (NSF grant DEB 03-15190) and T. Cornelissenwas partially supported by the Brazilian National Research CouncilCNPq through a graduate fellowship (grant number 200064/01-0). Wethank Andrey Castro and Tere Albarracin for their invaluable help inthe field and Sylvia Luckanewick for help in laboratory analyses andleaf measurements.

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