Acta Oecologica 25 (2004) 151157

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Original article

Sex, secondary compounds and asymmetry.

Effects on plantherbivore interaction in a dioecious shrub
Mara-Jos Bauelos *, Marta Sierra, Jos-Ramn Obeso
Department Biologa de Organismos y Sistemas, University of Oviedo, 33071 Oviedo, Spain
Received 11 July 2003; accepted 9 January 2004
Available online 19 March 2004

Abstract
We analysed the links between herbivory, anthraquinone content and developmental instability of leaves in Rhamnus alpinus, taking into
account possible effects of sexual dimorphism. The amount of leaf loss caused by herbivores averaged 3%, rarely exceeding 25%. Leaf losses
were evenly distributed in the shrubs, with highest variability among leaves of the same shoot, thus hiding possible shrub, sex or population
effects. This pattern of herbivory implies a shifting of caterpillars from one leaf to another before consuming all readily available material. We
suggest that this behaviour might be triggered by a short-term change in leaf palatability by means of an increase in the production of secondary
compounds. Supporting this hypothesis, we have found a higher anthraquinone content in damaged leaves compared with undamaged ones.
The leaves of male plants exhibited a higher concentration of anthraquinones than those of females, which contrasts with classic hypotheses.
We relate this to the lower rate of biomass increase in males, which should allow them to allocate more resources to defence. Leaves showed
fluctuating asymmetry (FA), but we did not find any relationship between the degree of asymmetry and sex, herbivory or anthraquinone content
at any level considered. Therefore, FA cannot be considered as an indicator of susceptibility to damage by herbivores or of the ability to induce
the production of defensive compounds in R. alpinus.
2004 Elsevier SAS. All rights reserved.
Keywords: Herbivory; Anthraquinones; Developmental stability; Sexual dimorphism

1. Introduction
Plants have evolved multiple mechanisms for production
of toxins and structural defences against herbivores (Karban
and Myers, 1989; Bennett and Wallsgrove, 1994; Obeso,
1997). These mechanisms are present throughout both the
terrestrial and marine realms (Hay, 1991). Some deterring
structures and secondary compounds are produced as direct
consequence of the activity of herbivores, in which case they
are termed induced defences (Myers and Bazely, 1991;
Agrawal, 2002). The ability to produce these defences can be
related to the capability of individual plants to cope with the
environment, and thus might be estimated from developmental stability of individuals. Several characteristics, including
fluctuating asymmetry (FA), have been proposed as indicators of such developmental stability. FA represents subtle
random deviations from bilateral symmetry in a bilaterally
* Corresponding author. Present address: Botanical Section, Department
of Ecology, Royal Veterinary and Agricultural University, Rolighedsvej 21,
1958 Frederiksberg C, Denmark.
E-mail address: mjbm@kvl.dk (M.-J. Bauelos).
2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.actao.2004.01.001

symmetrical morphological character, which can be the result of stress during development (Van Valen, 1962; Palmer
and Strobeck, 1986; Bjorksten et al., 2000). In plants, the FA
of leaves increases with both genetic and environmental
stressing factors such as competition, predation or pollution
(for a review see Mller and Shykoff, 1999). Our study
species, Rhamnus alpinus, a dioecious shrub, lives on rocky
limestone-derived soils, and therefore, is particularly prone
to suffer water stress. Developmental stress can lead to
changes in chemical or physical characteristics of leaves that
might subsequently alter their quality for the herbivores.
Therefore, FA can be a useful indicator of stress and, at the
same time may indicate susceptibility to damage from herbivores due to the covariation with the production of protective
secondary compounds (Mller and de Lope, 1998).
Females of R. alpinus invest more into reproduction than
males (Bauelos and Obeso, 2004), as expected in dioecious
species (Delph, 1999; Obeso, 2002; but see Marion and
Houle, 1996). When resources are limiting, higher reproductive investment should lead to lower vegetative growth or
survival in females (Cipollini and Whigham, 1994). Female
shrubs of R. alpinus show indeed lower vegetative growth

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M.-J. Bauelos et al. / Acta Oecologica 25 (2004) 151157

rates than males (Bauelos and Obeso, 2004), and slower

growing species (Coley et al., 1985) or sexes within species
(Jing and Coley, 1990; gren et al., 1999) are expected to
invest more in defence (but see Almeida-Cortez et al., 1999).
However, higher female reproductive investment could be
traded off against defence as well as against vegetative
growth. In addition, total biomass production (reproductive
plus vegetative biomass) is greater in females than in males,
although vegetative growth in females is slower (see De
Jong, 1995). Therefore, there are two reasons to expect that
males may be better defended than females: they have lower
accrual of total biomass (De Jong, 1995) and they may have
more resources available for investment in defence.
We analysed the total anthraquinone content in leaves of
R. alpinus. Anthraquinones are phenolic substances frequently found in Rhamnaceae (Sydiskis et al., 1991; Voss and
Brennecke, 1991; Izhaki, 2002). They have a strong purgative effect mainly due to peristalsis stimulation (Samuelson,
1992; Hilker and Koepf, 1994), and are known to deter
insects and birds (Trial and Dimond, 1979).
If FA indicates higher susceptibility to herbivory damage,
it should be higher in the sex more susceptible to herbivory.
Inter-sexual differences in FA (Bortolotti and Gabrielson,
1995; Sodhi, 1996), and the relationship between FA and
sexual selection have frequently been studied in animals
(Palmer and Strobeck, 1986; Mller and Thornhill, 1998).
However, to the best of our knowledge there is no information about sex and FA in plants.
Here we elucidate potential links among herbivory, developmental instability (measured as FA) and anthraquinone
content of leaves, taking into account possible effects of the
sex of the plant. We address the following questions:
(1) How is the intensity of herbivory distributed across
populations, sexes, shrubs and leaves in R. alpinus?
(2) Does FA correlate with susceptibility to herbivory?
(3) Are there evidences that anthraquinones have a defensive function against herbivores?
(4) Does sexual dimorphism in reproductive investment
lead to a sexual dimorphism in the plantherbivore
interaction? More specifically, we test the prediction
that the sex with lower rate of total biomass increase
(i.e. males) should show: (a) a higher concentration of
defensive compounds to avoid losses of more valuable
biomass, (b) lower herbivory damage, and (c) lower
levels of FA, related to susceptibility by herbivores.

open in late spring (AprilMay); the young leaves unfold

during the flowering season, which extends for 23 weeks.
The caterpillars of butterflies (mainly Geometridae and
Pieridae) are the major consumers of the foliage, but we
occasionally observed ungulates (mainly cattle) browsing on
R. alpinus as well.
We studied two populations, each including at least
100 reproductive individuals. Both were located at 1250 m
a.s.l. in the Cantabrian Range (Somiedo Natural Park, Asturias, NW Spain), where R. alpinus grows in calcareous
areas with rather open vegetation, sharing the habitat with
other woody species such as hazel trees (Corylus avellana),
whitebeams (Sorbus aria) or hawthorns (Crataegus monogyna).
2.2. Quantitative assessment of herbivory
We compared herbivory between sexes, populations,
shrubs and shoots within shrubs in September 1998 at the end
of the reproductive season. We randomly selected five shrubs
of similar size per sex at the two study sites, tagging
10 shoots per shrub; the shoots had 510 leaves. The level of
herbivory on each leaf was estimated visually. We defined six
levels of damage, i.e. no damage, 15% of foliar area removed, 610%, 1125%, 2650%, 5175% and more than
75% of foliar area removed, respectively. We coded these
damage levels from 0 to 6 for the statistical analyses. The
estimates were repeated in the same individuals in 1999,
following the same scheme. We compared these estimates
with the results of a previous analysis conducted with digital
images of leaves (n = 200 leaves for each analysis: two sites,
two sexes, five shrubs per sex, 10 shoots per branch, one leaf
per shoot). The estimates did not differ significantly
(t398 = 1.07; P = 0.28), showing mean damage values of 3.9%
(0.6 S.E.) for the field estimates, and 3.2% (0.4 S.E.,
n = 200 leaves) for the digital images. Therefore, we considered both procedures as equivalent estimates of the level of
herbivory, and used visual estimates for non-destructive
analyses of herbivory and digital images for the simultaneous
analysis of FA and herbivory, as described below.
We estimated the hierarchical distribution of variation in
the herbivory level among sites, shrubs and shoots by performing a nested ANOVA for both sexes using arcsine transformed data (x = arcsine x).
2.3. Herbivory and leaf asymmetry

2. Methods
2.1. Study species and sites
R. alpinus (Rhamnaceae), the Alpine Buckthorn, is a
dioecious broad-leaved shrub up to 4 m tall, which occurs in
rocky calcareous slopes in the mountains of southern and
southern-central Europe and northern Africa (Tutin et al.,
1968). It is a deciduous shrub with wintering buds, which

We tested whether leaf asymmetry was related to herbivory damage and if the relationship was sex-specific, by
randomly selecting 10 shrubs per sex (10 leaves from each
shrub, Ntotal = 40 individuals, 400 leaves) in September 1998.
We dried the leaves compressed between sheets of filter
paper, digitised them, and measured the total leaf area, the
area of holes in the leaf (area eaten by herbivores), and foliar
asymmetry to the nearest 0.01 mm with an image-analysis
system. Besides, we obtained a mean value of damage per

M.-J. Bauelos et al. / Acta Oecologica 25 (2004) 151157

shrub (Total Proportion Damaged, hereafter TPD), with the

equation:
n

Di

TPDshrub = i =n0

(1)

Ti

i=0

where Di and Ti are the damaged area and the total projected
area of each leaf, respectively (Williams and Abbott, 1991).
Foliar asymmetry was recorded for two morphological
leaf traits, as recommended by Mller (1995), i.e. leaf width
and inter-rib distance (Fig. 1). We defined leaf width as the
distance from the mid-rib to the edge of the leaf at its widest
point. The endpoint of the line from the edge of the leaf to the
mid-rib was the same for the right and the left sides. We
defined the inter-rib distance as the distance between each of
the four longest, central side-ribs. These measures were
taken in the right and left sides of the leaves. We considered
absolute asymmetry in leaf width as the absolute value of the
difference between the leaf width of the right and the left
sides of a particular leaf. Absolute asymmetry of the inter-rib
distance was defined as the mean absolute value of the
difference between the three right and the three left inter-rib
distances of a particular leaf (Fig. 1).
In order to control for measurement error (ME) in FA a
subsample of 50 leaves was re-measured at least 1 week later
without reference to the previous measurements. Then we
checked the significance of FA relative to ME with a two-way
mixed model ANOVA for each trait, with the factors Individual leaf (random) and Side (left or right), and considering
the two measurements as replicates (Palmer and Strobeck,
2003). The significance of the interaction term (Individual
leaf Side) indicated that between-sides variation (FA) was
significantly greater than that expected due to ME for both
traits (F49,100 = 14.0; P < 0.001 for leaf width, and
F49,100 = 6.48; P < 0.001 for inter-rib distance). We did not
carry out two independent scans to digitalise the leaves after
repositioning them, incurring therefore a possible source of
error due to the exact position and orientation of the leaf in
the scanning surface. In order to minimise that potential

153

error, leaves were always positioned in the centre of the

scanning surface, where the boundary distortion was minimal.
For both leaf characters measured, we tested if the distribution of FA did not deviate from a normal distribution
(KolmogorovSmirnov test) with a mean value of zero (ttest; Palmer and Strobeck, 1986), to discard asymmetries
different from FA (directional asymmetry, antisymmetry).
For these tests we used the signed left minus right character
values.
Then we calculated a composite FA index (CFA hereafter)
for each leaf, defined as the sum of the standardised FA
magnitude of each trait obtained by dividing each FA value
by the average absolute FA value in the population, so that
both traits contribute equally to the final score (Leung et al.,
2000). To correct for multiplicative error, which can arise
when growth involves the addition of tissue to that which is
already present as in the case of leaf growth (Graham et al.,
2003), we took logarithms of the right and left measures
before computing the composite index:
k

CFAi =

j=1

(loga

log b) ij

avg log a log b

where i is each individual leaf and j represents each trait

measured.
Then we tested for differences in asymmetry (CFA) between sexes (fixed factor), populations and shrubs (random
factors) with a nested ANOVA, considering leaf size as a
covariate. We removed sequentially non-significant interactions and main effects (P > 0.2) to generate the simplest
explanatory model.
The relationship between asymmetry (CFA) and the extent of herbivory (proportion of area eaten) was studied for
both sexes. To discard shrub effects (Van Noordwijk and de
Jong, 1986), we performed individual correlations for each
shrub. A similar analysis was performed between mean CFA
value per shrub and the TPD.

Fig. 1. Measures of leaf asymmetry for the two traits considered. We defined leaf width as the distance from the mid-rib to the edge of the leaf at its widest site.
Inter-rib was the mean distance between each of the four longest, central side-ribs. We defined absolute asymmetry as the absolute value of the difference
between the right and the left sides for each particular measure.

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M.-J. Bauelos et al. / Acta Oecologica 25 (2004) 151157

Table 1
Levels of variation in herbivory extent (arcsin-transformed) on leaves of R. alpinus in males and females based on nested ANOVAs
Source of variation

Sex
Population
Sex population
Shrub (sex population)
Shoot (shrub (sex
population))
Error
a

df
MS

1
1
1
16

2.59
21.86
0.96
19.38

2.70
1.13
0.05
2.46

180

7.87

2.88

1233 a

2.73

1998
P

1999
P

MS

0.34
0.30
0.82
<0.01

% Explained
variance
0
0
0
65.5

0.02
12.29
3.36
25.68

0.01
0.48
0.13
3.87

0.94
0.49
0.72
<0.01

% Explained
variance
0
0
0
76.9

<0.01

8.29

6.63

2.41

<0.01

4.4

26.16

2.75

18.7

Degrees of freedom for the error term in 1999 were 1028, due to the different number of leaves produced per branch each year.

2.4. Herbivory and anthraquinone content

We analysed the relationship between herbivory and anthraquinone content in the same shrubs as those analysed for
foliar asymmetry. To test whether anthraquinone content
varies between leaves with and without herbivore damage,
we collected for each sex leaves with more than 10% of total
area damaged from 10 individuals, and undamaged leaves
from 10 other individuals, simultaneously to the collection of
leaves for asymmetry analysis. We collected enough material
to obtain a final dry mass of 3 g per shrub (2530 leaves per
sample). Samples were oven-dried and milled, and chemical
analysis was performed following the method of Van den
Berg and Labadie (1985).
We compared anthraquinone concentration between undamaged and damaged leaves in both sexes with a factorial
ANCOVA (leaf damage and sex as fixed factors, and locality
as random factor). We considered mean herbivory levels per
shrub (TDP, see above) as a covariate, to avoid confounding
the damage effect on individual leaves with plant identity
effects. We removed sequentially non-significant interactions and main effects (P > 0.2) to generate the simplest
explanatory model.
Finally, we analysed the relationship between anthraquinone and mean asymmetry (CFA) per shrub, considering only those shrubs from which we collected damaged
leaves.
3. Results
3.1. How much do herbivores eat?
The degree of herbivory averaged 3% of leaf area
(2.6% 0.5 S.E. for males and 3.1% 0.5 for females,
n = 200 per sex) and rarely exceeded 25%. There were no
differences between sexes in the amount of damage, and
most variability was found among shrubs, accounting for
65.5% of explained variance, followed by variability among
leaves within the same shoot, which accounted for 26%
(Table 1).
3.2. Herbivory and leaf asymmetry
The frequency distributions of asymmetry showed
roughly normal distribution (KolmogorovSmirnov test:

P > 0.2 in all data sets) with a mean value of zero (t-test:
P > 0.5 and power > 0.99 in all data sets). Therefore, following Palmer and Strobeck (1986), we considered that both leaf
characters exhibited FA (Table 2).
No significant differences were found in leaf asymmetry
between sexes (power of the test = 0.99 selecting medium
size effects) or among shrubs (power = 0.71) (Table 3).
We found no relationship between CFA and herbivory,
neither considering individual leaves (r = 0.06, n = 200,
P = 0.18, power = 0.99), nor mean values per shrub (r =
0.23, n = 40, P = 0.15, power = 0.62). Similar results occurred when males and females were analysed separately,
and when leaves from each shrub were analysed separately
(P > 0.1 in all cases, though the power was quite low
0.23due to the small sample size).
Table 2
Foliar FA in R. alpinus (means S.E.), estimated from measures of leaf
width and inter-rib distance, as mean values of the signed difference between
left and right sides of a leaf (RL leaf width), and as absolute values of the
difference between left and right sides of a leaf (absolute leaf FA) (n = 200).
Combining both traits (leaf width and inter-rib distance), we defined the
CFA index for each leaf as the sum of the standardised FA magnitude of each
trait. See Section 2 for further details
Measure
Leaf width
Leaf width (mm)
RL leaf width (mm)
Absolute leaf FA
Inter-rib distance
Inter-rib distance (mm)
RL leaf width (mm)
Absolute leaf FA
CFA

Males

Females

18.15 0.34
0.08 0.16
1.59 0.12

19.18 0.3
0.09 0.13
1.44 0.08

6.44 1.13
0.1 0.06
0.7 0.05
2.04 0.09

6.49 0.12
0.03 0.06
0.72 0.04
1.95 0.07

Table 3
Effect of sex (fixed factor) and shrub (random factor) on leaf asymmetry
(CFA) in R. alpinus, tested with ANCOVA. Leaf area was considered as
covariate. Here we show the simplest model after backwards stepwise
removal of the two-way interaction and the population effect (random),
which were non-significant (P > 0.2)
Source of variance
Leaf area (covariate)
Sex
Shrub (sex)
Error

df
1
1
38
359

MS
9.02
0.63
1.30
1.39

F
6.49
0.48
0.94

P
0.01 *
0.49
0.57

M.-J. Bauelos et al. / Acta Oecologica 25 (2004) 151157

Table 4
Effect of sex, leaf damage (fixed factors) on anthraquinone concentration in
leaves of R. alpinus, tested with a factorial ANCOVA. Shrub mean herbivory
level (TDP, see text for further explanation) was considered as covariate.
Here we show the model after backwards stepwise removal of the two- and
three-way interactions, as well as the population effect (random), which
were non-significant (P > 0.2)
Source of variance
Shrub TDP (covariate)
Leaf damage
Sex
Error

df
1
1
1
36

MS
114
6154
3099
875

F
0.32
17.62
8.87

P
0.57
<0.01 **
<0.01 **

3.3. Herbivory and anthraquinone content

Damaged leaves showed higher anthraquinone concentrations with an additional and independent effect of sex, i.e.
males showing a higher concentration than females (Table 4,
Fig. 2). The effect of TDP as covariate was not significant
suggesting that the observed damage effect on leaves was
independent of the mean damage level in the whole shrub.
Selecting medium size effects, the power of the test was 0.33,
so these results must be taken with caution.
No relationship was detected between anthraquinone content and mean asymmetry per shrub (r = 0.25, n = 20,
P = 0.27, power = 0.38).

4. Discussion
R. alpinus showed levels of leaf herbivory consistent with
the usual range for trees and shrubs in temperate areas, which
normally does not exceed 15% (Seastedt et al., 1983; Alonso
and Herrera, 1996; Jackson et al., 1999; but see the extremely
higher level found in Kaitaniemi et al., 1999). Level of
herbivore damage varied within shrubs and among leaves on
the same shoot, suggesting that caterpillars shift from one
leaf to another before consuming all the food readily avail-

155

able. The higher anthraquinone content found in damaged

leaves might indicate that this behaviour is due to changes in
leaf palatability, i.e. an induced defence. Herbivory can induce the production of secondary defence compounds not
only in the damaged leaf, but also in nearby leaves that share
a common vascular connection (see revision in Karban and
Baldwin, 1997).
The magnitude of FA in the leaves of R. alpinus was
similar to that reported for other plant species (Mller and
Eriksson, 1994; Mller and Shykoff, 1999). However, we
found no relationship between herbivore damage and FA. At
least three possible interpretations might account for this lack
of relationship: (1) FA is not related to herbivore defence. In
fact, although our method did not allow measuring anthraquinone content in individual leaves, the shrub anthraquinone content was not related to the shrub-averaged
values of FA. Therefore, FA was not an indicator of susceptibility to herbivory at shrub level. (2) A second hypothesis
relating FA and herbivory considers that herbivory itself
might be the stress factor causing an increase of foliar FA
under certain conditions (see review in Mller, 1996; Mller
and de Lope, 1998), as has been reported for Salix borealis
(Zvereva et al., 1997a). Our results suggest that leaves did not
suffer modification of their symmetry within the level of
herbivory found in our study sites. Experiments that manipulate herbivory levels would permit testing this hypothesis. (3)
Current herbivory may only affect the FA of future leaf
generations (Zvereva et al., 1997b). As in the previous point,
this hypothesis should also be tested with experiments manipulating the level of herbivory.
We found sexual dimorphism in the content of anthraquinone in R. alpinus leaves. Males were better protected, possibly because they expended fewer resources on
reproduction and therefore could allocate more to defence.
An alternative interpretation might be that males had a lower
biomass production rate when considering both reproductive
and vegetative structures, leading to a higher content of

Fig. 2. Anthraquinone content (microgram anthraquinone per gram dry weight) in undamaged and damaged leaves of males and females of R. alpinus (n = 5 per
sex and treatment in each population).

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M.-J. Bauelos et al. / Acta Oecologica 25 (2004) 151157

secondary compounds, as found in R. alpinus. However,

higher levels of anthraquinones in males did not translate into
less herbivore damage to this sex.

5. Conclusions
The leaves damaged by herbivores had higher anthraquinone content, possibly reflecting an induced effect.
The leaves of males contained more anthraquinone, which
might relate to their lower intrinsic rate of biomass increase.
However, no relationship between sex and herbivory was
detected. FA also differed not between sexes, nor was it
related to herbivory at leaf or shrub levels, or to anthraquinone content at the shrub level. Apparently, in R. alpinus FA is not a good indicator for susceptibility to herbivory.

Acknowledgements
We thank Olga Sez and Mario Quevedo for field assistance, and Esteban Cabal for technical assistance. Mario
Quevedo and Johannes Kollmann revised previous drafts of
the manuscript. This research was founded by projects PB941538 (DGICYT) and REC98-01 (FICYT) and by a grant to
MJB (FICYT, FC97-BECA-029).

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