Functional Ecology 2013
doi: 10.1111/1365-2435.12058
Herbivory mediated by coupling between
biomechanical traits of plants and grasshoppers
bastien Ibanez*,1, Sandra Lavorel2, Sara Puijalon3 and Marco Moretti1
Se
1
Community Ecology Research Unit, Swiss Federal Research Institute WSL, CH-6500, Bellinzona, Switzerland;
Joseph Fourier B.P.53, 38041, Grenoble CEDEX 9,
Laboratoire d’Ecologie Alpine UMR CNRS 5553 Universite
de Lyon, UMR 5023 ‘Ecologie des Hydrosystemes Naturels et Anthropises’ Universite
Lyon 1,
France; and 3Universite
CNRS, ENTPE, 69622, Villeurbanne, France
2
Summary
1. Despite their potential to provide a mechanistic understanding of ecosystem processes, the
functional traits that govern interaction networks remain poorly understood. We investigated
the extent to which biomechanical traits are related to consumption in a plant–grasshopper
herbivory network.
2. Using a choice experiment, we assessed the feeding patterns of 26 grasshopper species for
24 common plant species from subalpine grasslands. We quantified shear and punch toughness
for each plant species, while grasshopper incisive and molar strengths were estimated by a lever
mechanics model, following the measurement of mandibular traits.
3. Models incorporating co-phylogenetic effects showed that the ratio between the grasshopper
incisive strength and plant toughness, that reflects the cutting effort, is correlated with the mass
of plant eaten. Moreover, a strong relationship between the incisive strength of the grasshoppers and the weighed mean toughness of the plants they eat was found.
4. Our results suggest that biomechanical constraints imposed by plants influence the evolution
of grasshoppers’ mandibular traits. Such scaling relationships offer promising avenues towards
the understanding of trait – function links in interaction networks.
Key-words: cafeteria experiment, functional trait, incisive and molar strength, interaction
network, phylogenetic model, shear and punch toughness
Introduction
Complex networks of interacting species have been
increasingly studied during the past 10 years (Proulx,
Promislow & Phillips 2005), but the mechanisms that rule
interactions in species-rich assemblages remain underexplored (Vazquez et al. 2009). Functional traits have been
successfully used to explain patterns of interaction between
species. For example, Petchey et al. (2008) showed that
body size and foraging behaviour influence interaction
probabilities in trophic networks. Ibanez (2012) demonstrated that the combination of the morphology of the
proboscis of insects with the morphology of the nectar
holders of flowers governs interactions between plants and
pollinators, while Honek et al. (2007) showed that carabid
beetles select seed sizes according to their own body size.
These pioneering studies have paved the way towards a
better understanding of the mechanisms that rule interactions in species-rich assemblages.
*Correspondence author. E-mail: [email protected]
In the case of herbivory, it has been demonstrated that
feeding patterns are influenced by several plant traits, such
as nitrogen content (Perez-Harguindeguy et al. 2003;
Loranger et al. 2012), constitutive and inducible secondary
compounds (Mole & Joern 1994) and structural traits such
as spinescence, pubescence, sclerophylly and abrasiveness
(Dıaz et al. 2007; Hanley et al. 2007; Reynolds, Keeping &
Meyer 2009; Keathley & Potter 2011) or a combination of
these traits (Perez-Harguindeguy et al. 2003; Loranger
et al. 2012). For instance, Peeters, Sanson & Read (2007)
showed that the abundance of leaf chewers on bushes was
negatively correlated with leaf tissue toughness, that is, a
measurement of the energy required to fracture the leaf
(Aranwela, Sanson & Read 1999). This suggests that
toughness worked as a barrier trait (Santamarıa &
Rodrıguez-Girones 2007) preventing insects from chewing
the leaves. In turn, the diet of herbivores appears to be
related to several traits, such as the craniodental anatomy
of grazing ruminants (Codron et al. 2008), the mandible
shape of grasshoppers (Isely 1944; Patterson 1984; Bernays
1991) and the size of their heads (Bernays & Hamai 1987).
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society
2 S. Ibanez et al.
In his pioneering studies, Patterson (1983, 1984) measured
several functional traits of the grasshoppers’ mandibles
and showed they were correlated with their diet (either
grass, forb or mixed feeders), thus comparing continuous
morphological traits to discrete diet attributes. Despite
such evidence, no study to date has explored how quantitative information on functional traits of both guilds can
be combined to understand the mechanisms that govern
interactions in species-rich assemblages of plants and
herbivores.
In this study, we attempt to fill this gap by analysing the
consumption behaviour of generalist herbivore grasshoppers (Caelifera) and bush crickets (Ensifera), here used as
model organisms (hereafter referred to as ‘grasshoppers’
for simplicity). Grasshoppers can be very abundant in
open plant communities, consuming up to 30% of the
plant biomass in mountain grasslands (Blumer & Diemer
1996). Our study took place in the same type of ecosystem,
in subalpine grasslands of the central French Alps. Grasshoppers are typical chewing insects whose mandibles are
composed of an incisive and a molar region (Isely 1944;
Clissold 2007). Grass-feeder mandibles have long blunt
incisor cusps and parallel molar ridges, while forb-feeder
mandibles have short sharp incisor cusps and several
molar cusps instead of ridges (Smith & Capinera 2005;
Clissold 2007). The incisive region of both mandibles acts
as scissors cutting plant fragments that will subsequently
be handled by the molar region which chews these fragments (Clissold 2007). We propose to refine the coarse
approach which contrasts the discrete mandibular traits of
grass vs. forb-feeding species, by considering quantitative
relationships among the biomechanical traits of plants and
those of grasshoppers to explain their interactions.
The main purpose of our study was to evaluate the link
between the mandibular strength of different grasshopper
species relative to the toughness of their preferred plants.
To do this, it was necessary to quantify plant consumption
by grasshoppers in standardised conditions, to avoid the
confounding effects on plant abundance and plant architecture occurring in the field. For this purpose, we
designed a ‘cafeteria’ choice experiment (Cornelissen et al.
1999; Perez-Harguindeguy et al. 2003), in which 26 native
grasshopper species were given the opportunity to feed on
a variety of 24 common plant species from subalpine
grasslands in the Central French Alps. We then measured
plant and grasshopper biomechanical traits to quantify
their relationships and identify the mechanisms that drive
plant choice by these herbivores, also accounting for
phylogenetic effects.
municipality of Villar d’Arene in the Central French Alps near
Lautaret Pass (06°24′ 22″E, 45°02′ 05′′N). They were brought
into a cage (L = 8 m, l = 3 m, H = 2 m) covered by an insectproof mesh (Boddingtons, Leipzig, Germany), containing eighteen 40-cm diameter pots with intact monoliths from various
local plant communities. Because grasshoppers are capable of
learning (Dukas & Bernays 2000), we forced them to encounter
all the plant species that occur in the study area, during their last
developmental stages for at least 15 days before the cafeteria
experiment. The day before each cafeteria experiment session,
fresh and intact leaves of 24 plant species (see Appendix S2), representative of the communities in the study area, were collected
in the field and immediately hydrated for one night in dark and
cool conditions (4°C). These plant species were either dominant
or subdominant in the natural communities in the study region,
and their functional traits cover the functional range of those
communities (Lavorel et al. 2011). For the cafeteria experiment,
we offered whole leaves for consumption rather than standardising a leaf area (e.g. 1 cm2 for each plant species, Perez-Harguindeguy et al. 2003) because preliminary tests showed that leaf
fragments dried out too quickly and became unpalatable and difficult to measure. Leaves were randomly placed each time into a
40 cm 9 20 cm 9 4 cm box closed with a cover, and they were
hydrated by a 2 mL snap-cap vial of water, to prevent desiccation, at room temperature. Adult grasshoppers were collected
from the greenhouse, starved for one night (which we expect is
often the case in nature, at least in subalpine environments) and
then introduced into the box. Each cafeteria session was conducted with a single individual. After each session, an entirely
new set of leaves was placed in the box. For each of the 26 tested
species, we assessed the feeding patterns of five females and five
males in a single session, for a total of 260 sessions. The sessions
were conducted over 26 days, with 10 sessions in 10 different
boxes per day. Each session lasted 5 h, at the end of which each
leaf was checked for herbivory signs, and, if any, the proportion
of the area eaten was estimated visually. The eaten leaf mass was
estimated by multiplying the proportion of the leaf eaten by the
mean area of nine leaves of the plant species measured before the
experiment and by the leaf mass per area of each plant species.
We found that visual estimation was more reliable than image
analysis because preliminary investigations showed that the variations in leaf area due to changes in the water content in leaves
during the experiment were of the same order of magnitude as
the area eaten. The leaf mass of each plant species eaten by the
five grasshopper individuals of each species and sex was summed.
The final data set used in the analysis consisted of 52 vectors of
plant consumption, one for each grasshopper species and sex.
Hereafter, the set of grasshopper species that consumed a given
plant species will be referred to as ‘consumers’. A ‘consumption
probability’ of 1 will refer to when a consumption event between
a grasshopper and a plant was recorded, and of 0 if not. ‘Consumption intensity’ will refer to the dry mass of plant consumed
by a grasshopper species when the corresponding consumption
probability equals 1. The cafeteria experiment with 24 plant species was preferred to paired comparisons which required too
many tests (276 paired tests discriminating the 24 plant species
for each grasshopper species). It should be pointed out that the
physiological status of the grasshoppers changed during the cafeteria experiment given that they were feeding during the selection
process.
Material and methods
GRASSHOPPER BIOMECHANICAL MODEL AND
CAFETERIA CHOICE EXPERIMENT
MEASUREMENTS
In June 2010 and 2011, juvenile grasshoppers belonging to 26
species (see Appendix S1 in Supporting Information) were collected in subalpine grasslands on the southern aspect of the
To estimate the incisive and molar strength of the grasshoppers, we
used a biomechanical model based on a third-order lever (Clissold
2007) represented in Figs 1 and 2, and described by the formula:
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
Biomechanical traits mediate herbivory 3
F ¼ FA LA =L
where F is the incisive or molar strength, FA the adductor muscle
strength, LA the adductor muscle lever, L the incisive (LI) or
molar lever (LM). Because the angle of attachment alpha varies
during the movement of the mandibles and its measurement needs
3D imaging (Paul & Gronenberg 1999), we therefore assumed, for
simplicity, that alpha was constant across all species. This hypothesis is relaxed in Appendix S3, in which the thickness of the plants
is taken into account. We estimated FA, that cannot be measured
based on photographs, with two different proxies: (i) the mandible
section area A (Fig. 1) and (ii) the jaw area, estimated as the product of head height and head width. Both proxies are expected to
be correlated with the volume of the adductor muscle that occupies most of the head of the insects (Paul & Gronenberg 1999).
These two different proxies were used to improve the robustness
of the analysis. The estimated incisive FI and molar FM strengths
are defined as follows:
incisive strength (and molar strength respectively) is distributed all
along the incisive region length (resp. the molar area) during the
cutting process (resp. the chewing process). The estimated FI and
FM are not equal to the real incisive and molar strengths, which
cannot be directly measured. They rather correspond to relative
indexes that can be used for comparisons among different grasshopper species.
The body size of the grasshoppers was estimated by the sum of
the head length, the pronotum length and the posterior femur
length. Mandibular traits were measured on all grasshopper individuals, which were placed in plastic tubes filled with 70% ethanol
immediately after the cafeteria choice experiment. Because grasshopper mandibles are asymmetrical, all the traits used in the biomechanical model (Fig. 1) were measured on photographs of the
left mandible (Patterson 1983), previously dissected from each
individual and photographed with a digital camera (Eurocam,
Euromex, Arnhem, Holland). Each photo was calibrated manually
and analysed with the free software JMICROVISION (RODUIT 2011).
FI FA LA =LI 1=RI
FM FA LA =LM 1=RM
where RI is the incisive region length and RM is the molar region
area. The standardisation by RI and RM is crucial because the
PLANT BIOMECHANICAL TRAITS
Shearing and punching tests were used to measure the specific
work required to punch and to shear leaves (Aranwela, Sanson
& Read 1999; Sanson et al. 2001). Shearing tests measure the
force required to cut the leaf lamina with a razor blade (Ang,
Lucas & Tan 2008). Punching tests (‘punch and die’ tests) measure the force required to punch a hole through the leaf lamina
(Aranwela, Sanson & Read 1999; Sanson et al. 2001). All traits
were measured on mature leaves, within 48 h after plant collection in the field. For each species, each test was undertaken on
10 randomly chosen leaves. Punching and shearing tests were
performed on a universal testing machine (Instron 5942, Canton,
MA, USA).
PUNCHING TEST
Fig. 1. Morphology of a grasshopper mandible, showing the incisive region (of strength FI and length RI), the molar region (of
strength FM and area Rm, in light grey). The incisive (LI), molar
(LM) and adductor muscle (LA) levers are measured from the axis
of rotation defined as the line between the attachment points to
the head (dark grey). The mandible section area (A, light grey) is
used as a proxy for the adductor muscle strength (FA).
We built a device consisting of a flat-ended cylindrical steel rod
(punch, 20 mm diameter) mounted onto the moving head of the
testing machine and a stationary base with a sharp-edged hole
with a 01 mm clearance (following Aranwela, Sanson & Read
1999; Sanson et al. 2001). The punch was set to go through the
hole without any friction. The punch moved downward at a constant speed of 10 mm s1. The leaves were positioned to avoid primary and secondary veins where possible. However, the grasses’
toughness we actually measured might have been slightly higher
than the grasses’ toughness experienced by Gomphocerinae species
because Gomphocerinae species cut across veins during their first
bites and then cut down the leaves along lines of least resistance
(Clissold 2007). Leaf thickness was measured with a digital thickness gauge (001 mm), avoiding major veins. The load applied to
the leaf (N) and the displacement (m) were recorded simultaneously with a frequency of 10 Hz. The total work to punch
(Wpunch) in Joules corresponds to the area under the force–displacement curve (Aranwela, Sanson & Read 1999; Read & Sanson
2003). Wpunch was used to calculate:
1. the work to punch: WP = Wpunch/A (J m2)
2. the specific work to punch (work to punch per unit of leaf
thickness): SWP = (Wpunch/A)/T (J m2 m1)
with A being the area of the punch (m2) and T the leaf thickness
(m).
Fig. 2. Biomechanical third-order lever model using traits measured in Fig. 1. Incisive and molar strengths are estimated as:
FI ~ A LA/LI 1/RI and FM ~ A LA/LM 1/RM (see methods for
details).
SHEARING TEST
Shearing tests were conducted with a leaf-cutting device following
Ang, Lucas & Tan (2008). A single stainless steel blade of a
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
4 S. Ibanez et al.
straight razor (Dovo, Solinge, Germany) was mounted on the
moving head of the testing machine with an approach angle of
20°. The leaf was positioned on two supports (with a 15 mm span),
placed so that the blade was equidistant from the two supports.
The blade was moved downward at a constant speed of
10 mm s1, shearing the leaf into halves. The cut position was perpendicular to the midrib and equidistant between the base and
apex of the leaves. To avoid the midrib, different parts of the
leaves were cut for different species (entire leaf, half leaf or leaf
strip bordering the central vein) according to leaf morphology.
The width and cross section area sheared were measured by making a thin cut adjacent to the shearing plane. Images of the cuts
were taken using a binocular microscope and a digital camera.
They were then analysed with IMAGEJ 102 to calculate width and
area. As for Wpunch, the total work to shear (Wshear, J) was used to
calculate (Aranwela, Sanson & Read 1999; Read & Sanson 2003):
1. the work to shear per unit leaf width: WS = Wshear/w (J m1)
2. the specific work to shear per unit leaf cross section area:
SWS = (Wshear/C) (J m2)
with w being the leaf width (m) and C the leaf cross section area
(m2).
When grasshoppers cut and chew leaves, they can cause crack
opening (the toughness of which is measured by tearing tests),
in-plane shear (punching tests) and out-of-plane shear (shearing
tests, Clissold 2007), but it is currently unknown which types of
fracture are involved during the cutting and chewing processes.
Also, it is unclear whether we should consider the material
toughness (specific work to shear and punch, calculated based
on the standardisation per unit of leaf thickness) or the structural toughness (no standardisation). During the analysis, we
will therefore use the four toughness measures: WP, SWP, WS,
and SWS.
PLANT BIOCHEMICAL TRAITS
In order to compare biomechanical traits with biochemical traits
assumed to contribute to plant selection by grasshoppers, we measured leaf dry matter content (LDMC), leaf nitrogen content
(LNC), specific leaf area (SLA) and the carbon/nitrogen ratio,
using standardised protocols (Cornelissen et al. 2003).
DATA ANALYSIS
The data for females and males of each species were analysed
separately because sexual dimorphism might lead to differences
in biomechanical traits and also because females might have
consumed different plants depending on their reproductive status. Prior to the entire analysis, all traits were log(x + 1) transformed. We used the free R statistical software (R Development
Core Team 2011) throughout the analysis, which was divided
into the three following steps: (i) an analysis of the correlation
between plant (resp. grasshopper) traits and the mean traits of
their consumers (resp. the plants they eat), as well as two statistical models that link plant consumption to biomechanical traits:
(ii) a linear model including co-phylogenetic effects, to account
for the non-independence of the species and (iii) a Bayesian
model including zero-inflation to test whether biomechanical
traits influence consumption probability and/or consumption
intensity. In the statistical models, we used the ratio of the
grasshopper incisive FI (or molar FM) strength divided by one
of the four plant toughness measures (work to shear WS, specific work to shear SWS, work to punch WP and specific work
to punch SWP). To confer the same weight to plant and grasshopper trait variations in the grasshopper strength to plant
toughness ratios, the variables were scaled to a variance equal
to 1 prior to the analysis.
CORRELATIONS BETWEEN INTERACTION NICHES AND
TRAIT VALUES
For each plant species, we calculated the mean incisive and molar
strength of its consumers, weighed by the mass eaten by each of
them. This quantity is interpreted as an ‘interaction niche’ value
and corresponds to the Community Weighed Mean value (CWM,
Lavorel et al. 2008) of the incisive or molar strength used to eat a
unit of leaf mass of that species. The CWMs were calculated
following (Lavorel et al. 2008):
CWM ¼
n
X
pi traiti
i¼1
where n is the total number of grasshopper species eating the particular plant (consumers), pi the relative abundance of consumer i
and traiti the trait value of consumer i. Similarly, we calculated
the weighed mean toughness of the plants eaten by each sex of the
grasshopper species. We then used linear models to compare (i)
the shear and punch toughness of the plants to the mean incisive
and molar strengths of their consumers, and (ii) the incisive and
molar strengths of the grasshoppers to the mean shear and punch
toughness of the plants they ate.
CO-PHYLOGENETIC LINEAR MODEL
As closely related grasshopper species tend to have similar diets
(Joern 1979), plant and grasshopper species are not independent
statistical units because of their phylogenetic relationships, which
must be taken into account in all comparative analyses (Felsenstein 1985). Moreover, the functional feeding types of the mandibles have a very strong phylogenetic signal. Gomphocerinae all
have grass-feeding mandibles, Catantopinae and Calliptaminae all
have forb-feeding mandibles in, while Ensifera all have omnivorous mandibles. Therefore, the discrete functional feeding types of
the mandibles were considered through the phylogenetic relationships. The methods we used to obtain grasshopper and plant phylogenies are described in Appendix S1 and S2. We evaluated the
strength of the phylogenetic signal of the two phylogenies in the
interaction network with the method developed by Ives & Godfray (2006) and implemented in the ‘pblm’ function of the ‘picante’ R package (Kembel et al. 2010). This method uses a linear
model approach to fit the phylogenetic variance – covariance
matrix and covariables to the interaction network. The code of
the pblm function allows covariables (e.g. traits) that are specific
to each species of each guild (e.g. toughness of the plants; strength
of the grasshoppers’ mandibles), but we needed to use covariables
specific to each plant–grasshopper pair. For this purpose, we modified the code of the pblm function accordingly (R code available
upon request). Our dataset is zero-inflated because most of the
possible interactions between plants and grasshoppers were not
observed. We therefore restricted the analysis to the nonzero components of the dataset, which has a normal distribution after logtransformation, and adapted the pblm code in this way.
MCMC GENERALISED LINEAR MIXED MODELS
To take zero-inflation into account, which is typical of interaction
web data, and to analyse the whole data set simultaneously, we
used the Monte Carlo Markov Chain generalised linear mixed
model function of the package MCMCGLMM (Hadfield 2010)
with uninformative priors. Our data were zero-inflated log-normal, but the MCMCGLMM function only supports zero-inflated
Poisson data, so we truncated our data to the nearest integer to fit
this restriction. Zero-inflated models include a 0/1 component (in
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
Biomechanical traits mediate herbivory 5
our case, this reflects the presence/absence of a given plant–grasshopper interaction) and a nonzero component (given that the
interaction is present, it reflects how much of the plant has been
eaten). We used the shear and punch ratios as fixed effects, while
plant and grasshopper species, as well as sex and year of the
choice experiment (2010 or 2011), were all included as random
factors. The model tests their influence on the 0/1 component, that
is, whether grasshoppers were physically able to cut and chew
plants or not, and on the nonzero component that reflects whether
grasshoppers ate plants that require less time and energy to be cut
and chewed. Because the effect of only one phylogeny can be
introduced into the model at once, we restricted the analysis to
the grasshoppers’ phylogenetic effects because the plants’ phylogeny showed only a small signal in the co-phylogenetic model (see
Results).
Results
The number of plant species consumed per grasshopper
species by sex, varied from 3 to 14 from the 24 available,
with a median of 75. All plants were consumed by at least
4 of the 26 grasshopper species (both sexes being considered separately), with a maximum of 32 and a median of
17 grasshopper-and-sex. A visual representation of the
interaction network between grasshoppers and plants is
provided Fig. 3 (for the raw values see Table S4).
BIOMECHANICAL TRAITS VALUES
The four plant biomechanical traits (WS, SWS, WP and
SWP) were highly correlated with each other (Table 1A,
Spearman’s q, 066 < q < 085), as found previously (Read
& Sanson 2003). The four biomechanical traits were also
positively correlated with LDMC, LNC and the C/N ratio
(040 < q < 074), and negatively correlated with leaf
thickness (067 < q < 009). The measured plant trait
values are available in Table S5.
Concerning grasshopper traits (Table 1B), there was a
negative correlation between incisive and molar strength.
There was no correlation between size and incisive
strength, but a positive correlation between size and molar
strength, and between size and adductor muscle strength
proxies. Also, we found a negative correlation between
absolute incisive and molar strength on one hand, and
adductor muscle strength proxies on the other hand.
The effective force applied by the mandibles depends on
the angle ‘a’ between the adductor muscle and the adductor lever (Paul & Gronenberg 1999; Clissold 2007), but we
considered a to be constant and equal to 90°. This assumption is potentially critical because small grasshoppers must
open their mandibles wider than large grasshoppers when
they cut thick leaves, so that a varies between species.
When the strength of grasshoppers was estimated taking
into account plant thickness, which modifies the angle
between the adductor strength and the adductor lever (see
Appendix S3), it was highly correlated (q > 09) with the
measure of grasshopper strength without the effect of plant
thickness, except for unrealistic plant thickness values
(>3 mm), so that the grasshoppers’ strength did not
strongly depend on the leaves’ thickness (see Appendix
S3). Moreover, we found no link between the leaves’ thickness and any size-related trait of the grasshoppers (details
not shown). This contrasts with Vincent’s (2006) intraspecific level study, where a positive correlation was found
between the head width of Romalea microptera and the
thickness of the plants they ate.
CORRELATIONS BETWEEN INTERACTION NICHE AND
TRAIT VALUES
The mean incisive strength of consumers (estimated either
with the mandible area proxy or with the jaw area proxy)
of a given plant species was positively correlated with the
four biomechanical measures of that species (P < 001 in
all cases, 0387 < R2 < 0767, Table 2A and Fig. 4a). In
contrast, the mean molar strength of consumers of a plant
species was significantly negatively correlated (0266 <
R2 < 0375, Table 2A) with the four toughness measures
of that species but only when the jaw area proxy was used.
In a similar way, the mean toughness of the plant species
eaten (estimated with the four measures) by a particular
grasshopper was positively correlated with its incisive
strength, regardless of the proxy used (P < 0001 in all
cases, 0235 < R2 < 0355, Table 2B and Fig. 4b). In contrast, the mean toughness of the plant species eaten by a
particular grasshopper was negatively correlated with its
molar strength (0101 < R2 < 0252, Table 2A). The specific work to shear led to the less significant results, while
the work to punch gave the strongest correlations with the
grasshopper traits (Table 2). This suggests that in-plane
shearing was more significant than out-of-plane shearing
and that the structural toughness was the critical trait.
The correlation between the biomechanical traits of plants
and grasshoppers can be observed even when single taxonomic groups are considered. For example, the toughness of
dicots ranged from low to medium (green and blue points in
Fig. 4a), and it was correlated with the mean incisive
strength of their consumers (Fig. 4a). Similarly, the incisive
strength of the grass-feeding subfamily Gomphocerinae ranged from medium to high (green symbols in Fig. 4b), and it
was correlated with the mean toughness of the plants eaten.
Several specific examples also illustrate this pattern. The men
of Chorthippus biguttulus and Myrmeleottetix maculatus
(squares no. 7 and no. 17 in Fig. 4b) had a lower incisive
strength than the other Gomphocerinae, and they chose the
softest graminoids (Trisetum flavescens and Festuca rubra) as
well as the forb Centaureau uniflora (Fig. 3), which resulted
in a softer diet (Fig. 4b). From the plants’ side, the toughest
legume Trifolium alpinum (point no. 21 in Fig. 4a) was eaten
by many grass-feeding Gomphocerinae and by none of the
forb-feeding Catantopinae (Fig. 3), whereas the second softest grass Trisetum flavescens (point no. 23 in Fig. 4a) was
chosen by many Tettigoniinae as well as by relatively weak
Gomphocerinae, as mentioned above.
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
6 S. Ibanez et al.
Herbs
Legumes
Heracleum sphondylium
Laserpitium latifolium
Hypochaeris maculata
Lathyrus pratensis
Rhinanthus alectorolephus
Geum montanum
Centaurea uniflora
Plantago media
Helianthemum grandiflorum
Lotus corniculata
Vaccinium myrtillus
Trifolium alpinum
Onobrychis montana
Trifolium montanum
Bromus erectus
Anthoxanthum odoratum
Carex sempervirens
Festuca laevigata
Dactylis glomerata
Trisetum flavescens
Tettigoniinae
Festuca paniculata
Catantopinae
Poa pratensis
Calliptaminae
Festuca rubra
Oedipodinae
Sesleria caerulea
Gomphocerinae
Arcyptera fusca_F
Arcyptera fusca_M
Euthystira brachyptera_F
Euthystira brachyptera_M
Chorthippus apricarius_F
Chorthippus apricarius_M
Chorthippus biguttulus_F
Chorthippus biguttulus_M
Chorthippus parallelus_F
Chorthippus parallelus_M
Chorthippus scalaris_F
Chorthippus scalaris_M
Euchorthippus declivus_F
Euchorthippus declivus_M
Gomphocerus sibiricus_F
Gomphocerus sibiricus_M
Myrmeleotettix maculatus_F
Myrmeleotettix maculatus_M
Omocestus haemorrhoidalis_F
Omocestus haemorrhoidalis_M
Omocestus viridulus_F
Omocestus viridulus_M
Stenobothrus lineatus_F
Stenobothrus lineatus_M
Stenobothrus nigromaculatus_F
Stenobothrus nigromaculatus_M
Stethophyma grossum_F
Stethophyma grossum_M
Calliptamus italicus_F
Calliptamus italicus_M
Calliptamus siciliae_F
Calliptamus siciliae_M
Bohemanella frigida_F
Bohemanella frigida_M
Miramella alpina_F
Miramella alpina_M
Podisma pedestris_F
Podisma pedestris_M
Decticus verrucivorus_F
Decticus verrucivorus_M
Metrioptera bicolor_F
Metrioptera bicolor_M
Metrioptera brachyptera_F
Metrioptera brachyptera_M
Anonconotus alpinus_F
Anonconotus alpinus_M
Platycleis albopunctata_F
Platycleis albopunctata_M
Tettigonia cantans_F
Tettigonia cantans_M
Tettigonia viridissima_F
Tettigonia viridissima_M
Other forbs
Fig. 3. Visualisation of the interaction network between plants and grasshoppers. The size of each black square is proportional to the relative mass of each plant species eaten by the grasshopper species. The absence of squares means that no consumption event was recorded.
After the grasshoppers species names, F means females and M means males. The vertical (resp. horizontal) bars separate distinct
taxonomic groups of plants (resp. grasshoppers).
CO-PHYLOGENETIC LINEAR MODEL
The phylogenetic signal of the plants was equal to
dp = 011, and the phylogenetic signal of the grasshopper
was di = 081 (d = 0 corresponds to the absence of phylogenetic signal, d = 1 to a Brownian motion model, and
0 < d < 1 to stabilizing selection, Ives & Godfray 2006).
The grasshopper incisive strength to plant toughness ratios
had a positive significant effect on the plant mass eaten by
grasshoppers, while the grasshopper molar strength to
plant toughness ratios had no effect (Table 3A).
hopper will consume a given plant, independently of the
consumption intensity. In contrast, six ratios of eight
(those calculated with either SWS, WP or SWP) had a significant positive effect on the leaf mass consumed
(Table 3B). Hence, grasshoppers may evaluate the plant
biomechanical properties with their first bites and then
decide how much they will eat. Concerning the grasshopper molar strength to plant toughness ratios, none of the
tested ratios had an effect on consumption probability,
while two ratios of eight had a significant positive effect on
the leaf mass consumed and one a significant negative
effect (Table 3B), the others being non-significant.
MCMC GENERALISED LINEAR MIXED MODELS
Concerning the grasshopper incisive strength to plant
toughness ratios, only two ratios of the eight tested (those
calculated with WS) had an effect on consumption probability (Table 3B), that is, on the probability that a grass-
Discussion
The three analytical approaches we used (simple regression,
co-phylogenetic linear model, MCMC generalised linear mixed model) converge towards a strong positive
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
Biomechanical traits mediate herbivory 7
Table 1. Correlations (Spearman’s q) among (A) plant traits and (B) grasshopper traits. All traits were log(x + 1) transformed
(A) Plant traits
I. Work to shear
II. Specific work to shear
III. Work to punch
IV. Specific work to punch
V. Leaf thickness
VI. Leaf dry matter content (LDMC)
VII. Carbon: Nitrogen ratio (C:N)
VIII. Leaf Nitrogen Content (LNC)
IX. Specific Leaf Area (SLA)
(B) Grasshopper traits
I. Incisive strength (mandibular section proxy)
II. Incisive strength (jaw area proxy)
III. Molar strength (mandibular section proxy)
IV. Molar strength (jaw area proxy)
V. Mandible bottom area
VI. Jaw area (head length x head height)
VII. Size (head + pronotum + femur)
VIII. Incisive lever/incisive length
IX. Molar lever/molar area
I
II
III
IV
V
VI
VII
VIII
IX
1
082
081
066
009
062
051
049
044
082
1
085
083
041
074
05
049
01
081
085
1
091
031
065
058
058
015
066
083
091
1
067
067
042
04
008
009
041
031
067
1
044
003
006
04
062
074
065
067
044
1
055
051
005
051
05
058
042
003
055
1
099
02
049
049
058
04
006
051
099
1
022
044
01
015
008
04
005
02
022
1
1
086
046
063
045
02
001
039
071
086
1
058
056
024
02
013
05
066
046
058
1
092
026
032
051
066
067
063
056
092
1
008
03
04
063
074
045
024
026
008
1
089
087
061
051
02
02
032
03
089
1
09
073
039
001
013
051
04
087
09
1
086
023
039
05
066
063
061
073
086
1
01
071
066
067
074
051
039
023
01
1
Table 2. Simple regressions between (A) the plant trait values and the mean trait values of their consumers and (B) the grasshoppers trait
values and the mean trait values of the plants they eat
Plant traits
(A) Mean trait values of the consumers
Work to shear
Specific work to shear
Work to punch
Specific work to punch
Incisive strength (mandibular section proxy)
Incisive strength (jaw area proxy)
Molar strength (mandibular section proxy)
Molar strength (jaw area proxy)
(+) 0515***
(+) 0387**
() 0104 ns
() 0266 **
(+) 0658***
(+) 045***
() 014 ns
() 038**
(+) 0767***
(+) 0549***
() 0106 ns
() 0313**
(+) 0696***
(+) 0514***
() 0155 ns
() 0375**
Mean trait values of the plants eaten
(B) Grasshopper traits
Work to shear
Specific work to shear
Work to punch
Specific work to punch
Incisive strength (mandibular section proxy)
Incisive strength (jaw area proxy)
Molar strength (mandibular section proxy)
Molar strength (jaw area proxy)
(+) 033***
(+) 0284***
() 0159**
() 0252***
(+) 0283***
(+) 0235***
() 0135**
() 0225***
(+) 0355***
(+) 0269***
() 0101*
() 0198***
(+) 0321 ***
(+) 0274***
() 0137**
() 0227***
LDMC, Leaf Dry Matter Content; LNC, Leaf Nitrogen Content; C:N, Carbon:Nitrogen ratio.
The sign in parenthesis indicates whether the relationship is positive or negative, the algebric value corresponds to the R2 of the regression,
and the stars indicate the P-value of the corresponding test as follows : ***means P < 0001, **means P < 001, *means P < 005 and ns
means non-significant.
The bold values correspond to the regressions presented in Fig. 3.
relationship between the incisive strength of the grasshoppers and the toughness of the plants they eat (Fig. 4). Concerning the relationship between their molar strength and
plant toughness, the simple regressions, when significant,
highlighted a negative relationship (Table 2) that reflects
the negative correlation between incisive and molar strength
(Table 1B). Future studies might explore whether there is a
trade-off between incisive and molar strength in the mandibles of grasshoppers or whether the biomechanical model
we used to estimate the molar strength led to a poor estimate because the molar ridges and cusps were not taken
into account. The co-phylogenetic linear model found no
significant relationship between molar strength and plant
toughness (Table 3A), while the MCMC generalised linear
mixed model found a positive relationship in only two cases
of eight, the other cases being non-significant (Table 3B).
Taken all together, these results suggest that only the incisive strength of the grasshoppers was linked to the toughness of the plant they ate, but not their molar strength.
By quantifying the biomechanical traits of both grasshoppers and plants, we were able to analyse linkages
between traits that are mechanistically related between
consumers and plants: strength vs toughness, respectively.
Previous studies focused on categorical plant and
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
Mean incisive strength index of the consumers
(a) Plants niche
0·80
7
Non−leguminous dicots
Legumes
Graminoids
5 20
0·75
2
3
21
18
0·70
1 Anthoxanthum_odoratum
2 Bromus_erectus
3 Carex_sempervirens
6
5 Dactylis_glomerata
0·65
23
24
6 Festuca_laevigata
1
7 Festuca_paniculata
8
8 Festuca_rubra
22
9 Geum_montanum
0·60
10 Helianthemum_grandiflorum
11 Heracleum_sphondylium
12 Hypochaeris_maculata
19
0·55
4 Centaurea_uniflora
13 Laserpitium_latifolium
417
15
14 Lathyrus_pratensis
15 Lotus_corniculata
16
11
16 Onobrychis_montana
17 Plantago_media
18 Poa_pratensis
0·50
12
10
19 Rhinanthus_alectorolephus
20 Sesleria_caerulea
9
13
0·45
21 Trifolium_alpinum
22 Trifolium_montanum
14
23 Trisetum_flavescens
24 Vaccinium_myrtillus
5·5
6·0
6·5
7·0
Mean work to punch the plants consumed log(J m–2)
8 S. Ibanez et al.
(b) Grasshoppers niche
7·5
7·0
23
Calliptaminae
Catantopinae
14
Gomphocerinae
Oedipodinae
Tettigoniinae
Females14
23
Males
8
2
9
11
19
6
618
18
12
20
17
21
6·0
4
1
19
7 20
24
21 3 3
16
4 10
15
26
2526
25
1
9
15
5·5
13
17
2
8
13
12
22
24
6·5
22
11
7
10
16
5
1 Anonconotus_alpinus
2 Arcyptera_fusca
3 Bohemanella_frigida
4 Calliptamus_italicus
5 Calliptamus_siciliae
6 Chorthippus_apricarius
7 Chorthippus_biguttulus
8 Chorthippus_parallelus
9 Chorthippus_scalaris
10 Decticus_verrucivorus
11 Euchorthippus_declivus
12 Euthystira_brachyptera
13 Gomphocerus_sibiricus
14 Metrioptera_bicolor
15 Metrioptera_brachyptera
16 Miramella_alpina
17 Myrmeleotettix_maculatus
18 Omocestus_haemorrhoidalis
19 Omocestus_viridulus
20 Platycleis_albopunctata
21 Podisma_pedestris
22 Stenobothrus_lineatus
23 Stenobothrus_nigromaculatus
24 Stethophyma_grossum
25 Tettigonia_cantans
26 Tettigonia_viridissima
5
7·5
0·4
Plant work to punch log(J m–2)
0·6
0·8
1·0
1·2
1·4
Grasshopper incisive strength index
(c) Pairwise interaction intensities
Plant work to punch log(J m–2)
7·5
7·0
6·5
6·0
5·5
0·4
0·6
0·8
1·0
1·2
1·4
Grasshopper incisive strength index
Fig. 4. Correlations between (a) the plants’ work to punch and the mean incisive strength index of the plants’ consumers and (b) the incisive strength index of the grasshoppers and the mean work to punch the plants they eat. (c) represents all pairwise herbivory events in
function of the grasshoppers’ incisive strength index and of the plants’ work to punch. The size of the circles is proportional to the relative
mass of plant eaten by each grasshopper species.
grasshopper groups rather than on such quantitative traits,
showing that the mandibles of grasshoppers are specialised
to feed either on grasses or on forbs (Isely 1944; Patterson
1984; Bernays 1991). Our quantitative approach allowed
us to investigate the variability of the biomechanical traits
within each mandible type and each plant type. For example, we showed that grass-feeding species having a relatively strong incisive strength tended to eat relatively
tough plants and that dicotyledonous species having a high
toughness tended to be eaten by stronger grasshoppers
(Fig. 4). While previous studies did not include phylogenetic effects into their analyses (Patterson 1983, 1984),
we showed that the link between grasshoppers’ incisive
strength and plants’ toughness remained even after incorporating these. From a methodological point of view, the
quantification of both strength and toughness enabled the
computation of the strength/toughness ratio which appears
to be very useful in complex statistical models. Ratios
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
Biomechanical traits mediate herbivory 9
Table 3. Linear models based on (A) Monte Carlo Markov Chain and (B) Generalised Least Squares. The models combine the following
statistical properties : phylogenetic effects of either both phylogenies simultaneously (B) or the grasshopper’s phylogeny alone (A), random
effects accounting for plant and grasshopper species identity and sampling year (A), independent parametrisation accounting for the eating
probability and the amount eaten (zero-inflated poisson model, A), and effect of the leaf nitrogen content (A and B, the results are not
shown because this covariate never had a significant effect)
(A) Co-phylogenetic linear model
Work to shear
Specific work to shear
Work to punch
Specific work to punch
Incisive strength (mandibular section proxy)
Incisive strength (jaw area proxy)
Molar strength (mandibular section proxy)
Molar strength (jaw area proxy)
(+)*
(+)*
(+)ns
(+)ns
(+)*
(+)*
(+)ns
()ns
(+)*
(+)*
(+)ns
()ns
(+)*
(+)*
(+)ns
()ns
Work to shear
Specific work to shear
Work to punch
Specific work to
punch
(B) MCMC Generalized linear mixed
model
Eating
probability
Amount
eaten
Eating
probability
Amount
eaten
Eating
probability
Amount
eaten
Eating
probability
Amount
eaten
Incisive strength (mandibular section
proxy)
Incisive strength (jaw area proxy)
Molar strength (mandibular section
proxy)
Molar strength (jaw area proxy)
(+)**
(+)ns
(+)ns
(+)**
(+)ns
(+)**
(+)ns
(+)**
(+)*
()ns
(+)ns
(+)**
(+)ns
()ns
(+)**
(+)ns
(+)ns
()ns
(+)**
(+)ns
()ns
()ns
(+)**
(+)ns
()ns
(+)*
(+)ns
()**
()ns
()**
()ns
()**
The sign in parenthesis indicates whether the relationship is positive or negative, and the stars indicate the P-value of the corresponding
test as follows: **means P < 0.01, *means P < 0.05 and ns means non-significant.
between the functional traits of interacting species have
also been useful to predict interactions in food webs
(Petchey et al. 2008).
DO BIOMECHANICAL TRAITS WORK AS BARRIER OR
COMPLEMENTARITY TRAITS?
The toughness of plants is a physical barrier that is
expected to work following a threshold rule, according to
which grasshoppers eat plants that they are able to cut
(Seath 1977; Lucas 2004). If the threshold rule severely
constrained our choice experiment, we would observe a
triangular distribution in Fig. 4c (see Stang, Klinkhamer
& Van der Meijden 2007 for a triangular distribution of
the interactions between plants and pollinators in the presence of barrier traits). However, even the grasshoppers
with the lowest incisive strength consumed some tough
plants (Fig. 4c), although in smaller amounts, indicating
that they were physically able to cut such plants. Similarly,
even the toughest plant Festuca paniculata was eaten by
relatively weak grasshoppers. Another line of evidence
comes from the results of the zero-inflated model, in which
the grasshopper strength to plant toughness ratios had no
influence on the consumption probability (the presence vs
absence of circles in Fig. 4c) but significantly affected the
consumption intensity (the magnitude of the circles in
Fig. 4c). Therefore, our data show that the threshold rule,
according to which grasshoppers eat plants that they are
able to cut, did not severely constrain plant choice during
the experiment.
The match between the toughness of the grasshoppers’
diet and their incisive strength indicates that the bio-
mechanical traits worked as complementarity traits rather
than barrier traits. This is intriguing because most often
complementarity traits are either nutritional or signal traits
(Santamarıa & Rodrıguez-Girones 2007), and species may
benefit from interacting with species whose traits match
their optimal requirements (e.g. Behmer 2008 in the case
of nutritional requirements). In the case of biomechanical
traits, strong grasshoppers did not benefit more from eating tougher plants than weak grasshoppers. In fact, grasshoppers reared on tough food have limited growth (Miura
& Ohsaki 2004; Clissold et al. 2009), and toughness is generally associated with lower nutritional quality (Santos
et al. 2012). As a consequence, the match between the
toughness of the grasshoppers’ diet and their incisive
strength cannot be explained by biomechanical traits
alone.
THE INTERPLAY BETWEEN BIOCHEMICAL AND
BIOMECHANICAL TRAITS
The interplay between biochemical and biomechanical
traits could explain the match we found between the toughness of the grasshoppers’ diet and their incisive strength.
Because plant toughness limits food ingestion, nutrient
absorption and ultimately growth (Miura & Ohsaki 2004;
Clissold et al. 2009), one could expect grasshoppers to
avoid tough plant species, which would lead to a pattern
very different from our observations. However, plant macronutrient and secondary compound contents also impose
their own selection pressures (Behmer, Simpson & Raubenheimer 2002; Behmer 2008), and the interplay between biochemical and biomechanical traits may allow many
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology
10 S. Ibanez et al.
herbivory niches that correspond to each grasshopper species. Because of the selection pressures exerted by biochemical traits, some grasshopper species have to deal with
tougher food than other species. It is therefore likely that
the mandibles and head muscles of grasshoppers have
evolved following the biomechanical constraints of their
diet. Although correlation studies cannot provide positive
proofs, we believe the observed pattern corresponds to
evolved host preferences. For example, we observed no
correlation between the incisive strength of the grasshoppers and their size (Table 1) and a strong negative correlation between the adductor muscle proxies and the ratio
between the incisive lever and the incisive region length (i.
e. LA/LI 1/RI, Table 1). Given that size was correlated with
the adductor muscle proxies, these findings indicate that
small species have evolved efficient mandible shapes to
compensate for their relatively small adductor muscle
strength. The present work focused on adults, but it would
be interesting to test if this pattern holds for nymphs.
Moreover, the positive link between the mass eaten and the
incisive strength to plant toughness ratio (Table 3B) suggests that it is less costly for a relatively strong grasshopper
to eat tough food than for a weaker grasshopper. Reciprocally, from the plants’ side, toughness is also considered to
be subject to selection pressures from herbivores (Coley &
Barone 1996), but many other ecological factors influence
the evolution of tough leaves (Turner 1994).
Conclusion
Scaling relationships between functional traits of species
that form interaction networks is a widespread pattern
generally involving the species’ size (Honek et al. 2007;
Petchey et al. 2008; Ibanez 2012). We applied scaling theory to a new dimension involving force rather than size
and found scaling between the toughness of plant leaves
and the strength of grasshoppers’ mandibles, respectively.
Only a few studies have investigated how biomechanical
traits influence interactions between plants and herbivores
(Pennings et al. 1998; Peeters, Sanson & Read 2007; Clissold et al. 2009; Nogueira, Peracchi & Monteiro 2009).
Biomechanical traits are often ignored and under-explored
because they require specific skills, time and equipment to
be measured. To our knowledge, this is the first study that
takes plant and herbivore quantitative biomechanical traits
into consideration simultaneously, from an experimental
set of species co-occurring in natural communities. The
close match we found between the incisive strength of 26
grasshopper species and the toughness of their diet, chosen
among 24 plant species, suggests that the influence of
biomechanical traits is potentially widespread in plant–
grasshopper interactions.
Acknowledgements
We are grateful to Marjorie Bison, Quentin Duparc and Carole Bengasini
for their help during the choice experiments and measurements of plant
mechanical properties, to Matthew Helmus for his suggestions to modify
the pblm function, to Felix Vallier for his help in constructing the equipment for the biomechanical measurements, to Sebastien Lavergne for his
advice to build the plant phylogeny, to Franck Quaine for his advice on the
mandibular biomechanics and to Curtis Gautschi for his help with language. This research was conducted at the Station Alpine Joseph Fourier
(UMS CNRS 3370, France) and on the long-term research site Zone Atelier
Alpes (ZAA), a member of the ILTER-Europe network. ZAA publication
no. 26.
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Received 30 September 2012; accepted 13 December 2012
Handling Editor: Art Woods
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Appendix S1. Insect phylogeny.
Appendix S2. Plant phylogeny.
Appendix S3. Influence of plant thickness on the incisive strength
of grasshoppers.
Table S1. Interaction network raw data.
Table S2. Plant trait values.
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology