Oecologia
DOI 10.1007/s00442-015-3508-y
COMMUNITY ECOLOGY - ORIGINAL RESEARCH
Intraspecific differences in plant chemotype determine
the structure of arthropod food webs
János Bálint1 · Sharon E. Zytynska2 · Rozália Veronika Salamon3 ·
Mohsen Mehrparvar4 · Wolfgang W. Weisser2 · Oswald J. Schmitz5 · Klára Benedek1 ·
Adalbert Balog1 Received: 16 April 2015 / Accepted: 4 November 2015
© Springer-Verlag Berlin Heidelberg 2015
Abstract It is becoming increasingly appreciated that
the structure and functioning of ecological food webs are
controlled by the nature and level of plant chemicals. It is
hypothesized that intraspecific variation in plant chemical
resistance, in which individuals of a host-plant population exhibit genetic differences in their chemical contents
(called ‘plant chemotypes’), may be an important determinant of variation in food web structure and functioning.
We evaluated this hypothesis using field assessments and
plant chemical assays in the tansy plant Tanacetum vulgare
Communicated by Nina Farwig.
Electronic supplementary material The online version of this
article (doi:10.1007/s00442-015-3508-y) contains supplementary
material, which is available to authorized users.
* Klára Benedek
[email protected]
* Adalbert Balog
[email protected]
1
Department of Horticulture, Faculty of Technical and Human
Science, Sapientia University, Sighisoara Street 1C,
Tirgu‑Mures, Romania
2
Terrestrial Ecology Research Group, Department of Ecology
and Ecosystem Management, Centre for Food and Life
Sciences Weihenstephan, Technische Universität München,
Freising‑Weihenstephan, Germany
3
Department of Food Science, Faculty of Technical Science,
Sapientia University, Miercurea Ciuc, Romania
4
Department of Biodiversity, Institute of Science and High
Technology and Environmental Sciences, Graduate
University of Advanced Technology, Kerman, Iran
5
School of Forestry and Environmental Studies, Yale
University, 370 Prospect Street, New Haven, Connecticut,
USA
L. (Asteraceae). We examined food webs in which chemotypes of tansy plants are the resource for two specialized
aphids, their predators and mutualistic ants. The density of
the ant-tended aphid Metopeurum fuscoviride was significantly higher on particular chemotypes (borneol) than others. Clear chemotype preferences between predators were
also detected. Aphid specialist seven-spotted ladybird beetles (Coccinella septempunctata) were more often found on
camphor plants, while significantly higher numbers of the
polyphagous nursery web spider (Pisaura mirabilis) were
observed on borneol plants. The analysis of plant chemotype effects on the arthropod community clearly demonstrates a range of possible outcomes between plant-aphidpredator networks. The findings help to offer a deeper
insight into how one important factor—plant chemical content—influences which species coexist within a food web
on a particular host plant and the nature of their trophic
linkages.
Keywords Aphids Ants · Bottom-up effects ·
Interactions · Predation · Top-down effects
Introduction
Conceptions of what determines the trophic structure and
functioning of ecological food webs increasingly recognize that nutrients, plant defence, herbivory and predation play interdependent roles (Van der Putten et al.
2001; Poehlman et al. 2008; Schmitz 2008; Mooney et al.
2010; Burghardt and Schmitz 2015). Such understanding derives largely from studies examining interspecific
variation in plant-herbivore-predator interactions (Hare
1992; Poehlman et al. 2008; Schmitz 2010; Mooney
et al. 2010; Burghardt and Schmitz 2015). There is also
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Oecologia
growing appreciation that genotypic and phenotypic differences within species can be important in explaining
trophic structure and functioning (Johnson and Agrawal
2005; Crutsinger et al. 2006; Johnson 2008; Whitham
et al. 2012; Barbour et al. 2015). But, in many cases, the
mechanism determining such variation remains elusive
(Barbour et al. 2015).
A candidate mechanism in food chains involving arthropod-plant interactions is intraspecific trait variation in the
nature and concentration of plant chemical defences (Linhart et al. 2005; Gols et al. 2008; Johnson and Agrawal
2005; Johnson 2008; Poehlman et al. 2008; Burghardt and
Schmitz 2015). At an interspecific level, plant defensive
chemicals are known to influence the trophic structure of
food chains by changing how herbivores mediate bottomup effects of plants on predators and top-down effects of
predators on plants, or through changes in plant traits,
such as release of volatile attractants, that alter the direct
effect of plants on predators (Hare 1992; Roth et al. 1997;
Harvey et al. 2003, 2007; Baldwin 2006; Gols et al. 2008;
Dicke and Baldwin 2010). It therefore stands to reason
that if intraspecific differences in chemical defence mediate the magnitude of interspecific variation, then we may
improve our understanding of changes in food chain structure through explicit consideration of intraspecific variation
in defence expression.
To this end, we report on an analysis of intraspecific
variation in chemical defence of the herb tansy, Tanacetum
vulgare, and its implications for associated arthropod food
web structure. Tansy exhibits substantial intraspecific variation in essential oil content, in particular monoterpenes and
sesquiterpenes that are emitted as volatiles. The chemicals
are distributed throughout each plant but are predominantly
present in leaves and young stems (Holopainen 1989; Keskitalo et al. 1998; Rohloff et al. 2004). Intraspecific differences in chemical form and concentration have been
described in terms of plant chemotype, where a chemotype
is based on the dominant compound in the chemical profile
(Holopainen 1989; Wolf et al. 2012). Breeding experiments
as well as studies using molecular markers have shown that
the chemical oil produced by a particular tansy chemotype
has a genetic basis (Holopainen et al. 1987a, b; Holopainen
1989; Keskitalo et al. 1998).
Tansy chemotype could determine arthropod community structure. It is well known that predatory arthropods and parasitoids use volatiles emitted by plants as
signals to locate plants carrying their prey or host, which
in turn can determine food web structure through predator top-down control of herbivores (Baldwin 2006; Soler
et al. 2007; Bruinsma et al. 2009). To date, more than
25 species from various taxa of predaceous arthropods
(including those studied in the present work), parasitic
nematodes, and insectivorous birds (Mantyla et al. 2004;
13
Rasmann et al. 2005; Runyon et al. 2006; Baldwin 2006;
Soler et al. 2007; Bruinsma et al. 2009; Benedek et al.
2015) are known to be attracted to plant volatiles and
potentially contribute to plant indirect resistance (Mumm
and Dicke 2010; Dicke and Baldwin 2010). Furthermore,
variability in individual expression of these volatiles may
explain variability in the insect food web composition
associated with different varieties of a plant (Bukovinszky
et al. 2008).
We tested whether chemical content of individual tansy
chemotypes does indeed influence associated food web
structure under field conditions. Using systematic field surveys, we examined relations between arthropod species and
tansy chemical contents. We asked the following questions:
1. Does essential oil composition of tansy vary between
plants?
2. Are there differences in arthropod abundances across
different plant chemotypes?
3. Are different characteristics of these food webs (species composition and food web linkages) affected by
tansy chemical content?
Materials and methods
Study system
Tansy is a perennial herbaceous composite from Europe
and Asia that preferentially grows in disturbed, welldrained, poor soils (Keskitalo et al. 1998). It often forms
isolated patches alongside river valleys, railway tracks and
on wastelands. Single plants comprise a ‘genetically identical’ genet with up to 50 flowering ramets (shoots) (but usually far fewer). Genets propagate clonally underground via
stolons.
Two dominant and specialized aphid species feed on
tansy: Macrosiphoniella tanacetaria (Kaltenbach) and
Metopeurum fuscoviride Stroyan. M. tanacetaria is not
attended by ants and forms small colonies mainly on the
top of ramets (shoots), while M. fuscoviride is an obligatory ant-tended aphid that feeds in more compact and often
large colonies on leaves and near the apex of ramets. The
black garden ant, Lasius niger (L.) commonly tends M.
fuscoviride aphids (Flatt and Weisser 2000). Many predators attack both aphid species, the most important being
the aphid specialist ladybird beetles, e.g. the seven-spotted
ladybird (Coccinella septempunctata); predation from the
generalist nursery web spiders (Pisaura mirabilis) and minute pirate bugs (Orius spp.) can also be observed (Benedek
et al. 2015). The attending ants help to defend the aphids
from the predators (Mehrparvar et al. 2013; Benedek et al.
2015).
Oecologia
Field assessment of arthropods on tansy
The 2-year (2011 and 2012) field study was conducted in
three sites along a 110-km transect in Transylvania, Europe.
Each site was between 2 and 3 km long and the sites were
separated by 50 km (Supplementary material, Fig. 1). Site
1 contained several thousand tansy plants while sites 2 and
3 each contained several hundred plants. Sites were not
isolated from each other (principal railway-linked sites),
therefore exchange of tansy material, i.e. pollen exchange
between sites, was possible. There were no obvious environmental and climate differences between sites—both soil
and climatic conditions were similar. The soil was well
drained, poor clay in all sites. The climate was temperate
specific for the sub-Carpathian region, with abundant precipitation in spring and fall, a relatively warm summer, and
low to very low temperatures (to −20 °C) in winter.
In total 100 plants were randomly selected for the study
(50 plants in site 1, 25 plants in sites 2 and 3). The distance
between the selected plant individuals within each site was
100–110 m to ensure that separate genotypes were selected.
All plants were labelled individually at the start of the
assessment, and the same plants were examined during the
entire study. In both years, weekly censuses of the marked
plants were carried out, between 7 and 11 a.m. Sampling
began in May when the aphids first appeared and continued
until the end of September when aphids had disappeared
from the plants. Aphids and other arthropods observed
on the marked plants were counted. M. tanacetaria and
M. fuscoviride were counted as individuals, except when
the colony size of M. fuscoviride was large. In this case,
all aphids on a 5-cm section of the shoot within the aphid
colony were selected and all aphids counted. Total aphid
number on a ramet was then estimated by multiplying that
count with total colony length (in centimetres) along the
ramet (e.g. 80 aphids in a 5-cm2 area, colony length 15 cm,
total 240 aphids). The aphid species were distinguished by
their body colour: M. tanacetaria is green and M. fuscoviride is black. After counting the aphids, a 10-min visual
scan was made of all aphid colonies on a plant. We counted
all ant workers and all aphid predators that were observed
attending the aphid colony or were less than 1 cm away
from an aphid.
We quantify food web linkages by noting ants that were
actively tending aphids, and any interactions between
ants and a predator, between a predator and an aphid, and
between predators.
Volatile extraction from plants and quantitative
analysis of volatiles
In June of year 2, live plant material (100 g; fresh leaves
and stems) was collected from unoccupied ramets of each
marked plant and stored at −20 °C. These samples were
used to measure and compare, using gas chromatography,
the main spectrum of volatile compounds in each individual plant. Standards of pure volatile compounds (camphor,
borneol, cineol, piperiton and α-thujone and β-thujone)
were obtained from the Roth Laboratory (Canada) to create
chromatograms for comparison.
Plant material from an individual plant was placed into a
round-bottom Clevenger micro-distillation flask containing
50 ml water. Flasks were heated at 250 W for 4 h. A 6-ml
aliquot of water and essential oil was then extracted with
n-hexane (4 ml) using a mixer-settler method (András et al.
2009). Samples were vortex mixed (IKA Vortex Genius 3)
for 30 min. The resulting emulsion was separated by centrifuging (Hettich Universal 32) at 1200 r.p.m. for 5 min.
The separated organic phase was stored in 4-ml glass vials
over anhydrous sodium sulphate at 4 °C until it was subjected to gas chromatographic analysis. Extracted essential
oils were analysed using a Varian CP 3380 gas chromatography coupled to a flame ionization detector [CP-Sil 88
(100 m × 0.25 mm) silica capillary column]. The system
was operated at hydrogen gas pressure of 235 kPa, with
2 μl in the injection probe at a temperature program of
270 °C. The starting temperature varied from 50 to 210 °C
with a 5 °C min−1 gradient, and then a constant temperature was applied during the 55-min analysis for each sample (András et al. 2009; Kapás et al. 2011).
The volatile components (the percentage values based
on compound quantity from each plants) were identified
by comparing their retention times with the standard chromatogram (camphor, borneol, cineol, piperiton, α-thujone
and β-thujone). Individual plants were identified to chemotype based on their volatile compound quantity. Plants were
designated as pure chemotypes when a single chemical
made up 90 % or more of the total quantity of compounds
detected e.g. ‘thujone pure’. Hybrid chemotypes were designated as those plants in which two volatiles together made
up more than 90 % of the bouquet i.e. ‘thujone hybrid’. In
all these cases, the proportion of the dominant volatile was
at least three times higher than the next most abundant volatile compound. When more than two compounds made up
>90 %, the mixed chemotype was designated by the dominant compound present, e.g. ‘thujone mixed’.
Field data analyses
We quantified food web structure in terms of species composition and abundance. Our analysis began by testing for
inter-annual differences in abundance of each arthropod
species by constructing principal response curves (Brink
and Braak 1999), which account for population dynamics through the season. Mean number of arthropods per
individual plant per sampling date was considered. This
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Oecologia
Fig. 1 Principal components
analysis (PCA) covariance plot
of chemotype profiles by the
most dominant chemotypes
analysis revealed that M. fuscoviride, ants and predators
were consistently present together from 22 June until 22
August in the first year, and from 20 June until 18 August
in the second year. Therefore only the nine weekly sampling dates between these start and end dates were considered for further data analyses. We tested for a year effect by
comparing the mean number of arthropods on each plant
using a repeated measures analysis on weekly sampling
dates between years. No significant temporal difference
was detected (multivariate ANOVA; MANOVA). Therefore
we pooled data for both years in all subsequent analyses.
We used two generalized linear modelling approaches
with quasi-Poisson error distributions to analyse the data
further. The first model tested for the effect of discrete
chemotype (considered pure or hybrid as defined above) on
arthropod abundance, using each species group as a response
variable, and site, plant chemotype and arthropod species
groups as explanatory variables. The second model tested for
the effect of plant chemotype profile (considering all volatiles according to their relative proportion in a tansy plant)
on different attributes of the food web. This was done by first
subjecting data on the relative amount of each chemical per
plant (%) to a principal components analysis (PCA) to identify the proportion of variation in each PCA axis (1–3) that
was explained by each chemical (α-thujone, β-thujone, camphor, borneol, cineol and piperiton) (Fig. 1). We then used
the average counts of each invertebrate grouping as response
variables, and used site, PCA axis 1 (PCA1), PCA2 and
PCA3 scores, each chemical and species group as independent variables. PCA covariance analyses were run using community analysis package 4 (Pisces Conservation). All other
statistics were run in R Studio version 0.97.314 using R version 3.0.1 (R Core Team 2013).
Food web construction and its parameters
We tested pure and hybrid chemotypes to determine
the direction and strength of correlation between M.
13
fuscoviride aphids and ants, M. fuscoviride aphids and
predators, ants and predators and between predators (i.e.
spiders and Orius, spiders and ladybirds). We assumed that
a significant negative correlation reveals a strong predatory
effect; a significant positive correlation reveals mutualism.
All observed interactions during the nine sampling
dates/year when all arthropods were present were grouped
according to the plant chemotypes on which they were
observed. These data included 334 observed interactions
between ants and M. fuscoviride aphids, 13 ant-M. tanacetaria aphid interactions, 218 predator-M. fuscoviride
aphid interactions, 16 predator-M. tanacetaria aphid interactions, 49 ant-predator and 20 predator–predator interactions. Based on these observed interactions, and estimated
correlations between arthropod species, we constructed
food web networks using CoSBiLab software (Jordán et al.
2012). We calculated the following indices for each chemotype-based food web:
1. Web degree, which represents linkage density (links
per species) that considers the number of species and
number of links between them. Both metrics can be
simply derived by counting the species that interact
and the number of links (interactions) between them.
If, for example ladybird predation on M. fuscoviride
aphids was observed several times, this was considered
as one interaction. This parameter indicates the complexity of the web. This is the most local index of food
web topology (Barabási and Albert 1999; Dunne et al.
2002).
2. Bottom-up (Kbu,i) and top-down (Ktd,i) effects are indices which emphasize vertical interactions and indicate
a strong interference between trophic groups (Jordán
et al. 2012). Large top-down effects require weak interference, while large bottom-up effects require both
weak interference and strong prey dependence (Dunne
et al. 2002). These indices can be calculated as: Oecologia
Kbu,i =
n
c=1
1
dc (1 + Kbc )
For node i, i.e. species i, Kbu,I quantifies the bottom-up effect of species I where n is the number of
predators eating species i, dc is the number of prey
of its cth predator and Kbc is the bottom-up keystone
index of the cth predator.
Ktd,i =
m
e=1
1
fe (1 + Kte )
Ktd,I quantifies the top-down effect of species i.
Here, m is the number of prey eaten by species i, fe is
the number of predators of its eth prey and Kte is the
top-down keystone index of the eth prey. Kbc (bottomup) and Kte (top-down) keystone indices are computed
as the sum of the dominant (key) predators/plants,
respectively, and dominant herbivore/plant averaged
over the other species from the food web.
The calculated values for the food web indices were
tested for normality of errors and homogeneity of variance.
Bottom-up and top-down indices were considered as separate variables for each individual plant per sampling date.
Effects of chemotype on food web bottom-up and topdown indices for each sampling period were tested using
repeated-measures MANOVA.
Model fit with observed field interactions was compared
using χ2-tests on the differences between the covariance
matrices, and by the root mean square error of approximation. The statistical analyses were performed in R version
2.8.0. and the package bipartite for food web analyses and
SEM (R Core Team 2013).
Results
Classification of tansy by essential oils, arthropod
densities on different tansy chemotypes
Ninety-five of the 100 plants examined could be assigned
to either a pure or hybrid chemotype. Fifty plants were pure
chemotypes. Of these, 25 were camphor (camphor pure),
seven borneol (borneol pure) and 18 thujone (thujone pure)
chemotypes. Another 44 plants were considered hybrid
chemotypes of which 15 were dominated by camphor (camphor hybrid), four dominated by borneol (borneol hybrid),
25 dominated by thujone (thujone hybrid). Only one plant
was assigned as mixed thujone (thujone mix) (Supplementary material, Fig. 1). When thujone was detected in both
pure and hybrid plants, this was β-thujone. We used only
these 95 plants for further analyses because our confidence
in assigning them to chemotype was high, which was not
the case for the five plants that were excluded from further
analysis.
All three sites contained multiple chemotypes, although
the borneol chemotype did not occur at site 2. Sites 1 and 2
were dominated by camphor and thujone chemotypes and site
3 was dominated by borneol and thujone chemotypes (Fig. 1).
We detected a significant effect of site and chemotype on
most arthropod abundances, except those of Orius (Table 1).
For example, significantly more M. fuscoviride aphids were
found on borneol plants than on other chemotypes throughout the nine census dates in both years, and abundances of
M. tanacetaria aphids were lower on thujone plants (Table 1).
This aphid species inhabited the top of the plants in small colonies where predation by ants and all predators was observed.
There was a significant positive effect of the borneol hybrid
chemotype on the number of ants observed (Table 1). Three
predators were often observed on plants: the seven-spotted
ladybird (C. septempunctata), the nursery web spider P. mirabilis and several species of minute pirate bugs (Orius spp.).
The numbers of the seven-spotted ladybird was lower on thujone pure chemotypes, and more nursery web spiders were
observed on borneol pure and borneol hybrid plants than on
other chemotypes (Table 1). The abundance of M. fuscoviride
aphids was positively associated with ants and spiders, but
negatively associated with the other aphid species and Orius
(Table 1). M. tanacetaria was negatively associated with M.
fuscoviride and positively associated with spider abundance.
Ant abundance was positively associated with M. fuscoviride
and negatively associated with M. tanacetaria and Orius.
Ladybird abundance was only influenced by M. fuscoviride
abundance. Therefore, both aphid species and Orius had
positive associations with spiders, while negative associations
between ants and spiders and Orius were detected (Table 1).
PCA showed that variation in chemical profile among
individual plants is primarily explained by the relative
amount of camphor and β-thujone in the plants and following this, the amount of borneol then α-thujone (Fig. 1;
Table 2). Consistent with the discrete chemotype analysis,
borneol had a significant positive effect on M. fuscoviride
density (PCA2; Fig. 2a; Table 3). The abundance of ladybirds was to some extent positively influenced by β-thujone
and camphor (PCA1) concentrations and negatively influenced by α-thujone (PCA3). The abundance of spiders was
also influenced by PCA1 and PCA2, explained by a higher
abundance of spiders on plants containing more β-thujone
or borneol and less camphor (Fig. 2b; Table 3).
Interactions, food webs and metrics
There were high positive associations for all cases
between ants and M. fuscoviride aphids (camphor pure
r = 0.72, p < 0.05; thujone pure r = 0.59, p < 0.05; borneol pure r = 0.60, p < 0.05; camphor hybrid r = 0.65,
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Oecologia
Table 1 The effect of discrete chemotype grouping on different aspects of the community, using each species group as a response variable, and
site, discrete chemotype and species groups as explanatory variables with interactions between species and chemotype
Variables
ME aphid
MA aphid
Lasius
Ladybird
Site
F2,83 = 10.80***
F2,83 = 7.82***
F6,77 = 2.78* (↑BP F6,77 = 3.56**
t = 1.50†)
(↓BTP t = 2.07*)
(↓TP t = 2.53**)
F2,83 = 3.38*
F6,77 = 3.45**
(↑BH t = 2.12*)
F2,83 = 26.94*** F2,83 = 72.27***
Chemotype
ME aphid
MA aphid
↓ F1,76 = 12.96***
↓ F1,76 = 31.50*** ↑ F1,76 = 33.24*** ↓ F1,76 = 3.24†
F1,75 = 0.27 NS
↑ F1,75 = 39.60*** ↓ F1,75 = 4.82*
Lasius
Ladybird
F6,77 = 7.57***
(↓TP t = 2.02*)
F1,75 = 0.06 NS
F1,74 = 0.07 NS
↓ F1,74 = 3.93†
F1,74 = 0.12 NS
Orius
Chemotype × ME
aphids
↑ F1,72 = 4.74*
F1,72 = 0.39 NS
–
Chemotype × MA
aphid
–
Chemotype × Lasius
–
F6,66 = 5.23***
(TP t = 1.80†)
Chemotype × Ladybird
–
F6,60 = 3.31**
(BTP t = 1.89†)
(TP t = 2.16*)
F6,54 = 2.51*
(BH t = 1.86†)
(TP t = 1.91†)
Chemotype × Spider
–
–
–
F6,54 = 3.17**
Chemotype × Orius
F6,66 = 2.15†
(BP t = 2.20*)
(CP t = 2.39*)
–
–
–
Spider
F1,74 = 0.14 NS
↑ F1,73 = 14.16*** ↑ F1,73 = 32.54*** ↓F1,73 = 7.08*
Spider
F6,77 = 14.84***
(↑BP t = 1.67†)
(↑BH t = 1.78†)
Orius
F2,83 = 2.09 NS
F6,77 = 1.61 NS
↑ F1,76 = 6.22*
↓ F1,76 = 8.25**
F1,74 = 2.37 NS
↓ F1,74 = 14.02***
↑ F1,75 = 4.62*
F1,73 = 0.01 NS
F1,73 = 0.07 NS
F1,75 = 0.69 NS
F1,73 = 0.15 NS
↑ F1,72 = 24.88***
↓ F1,72 = 9.63**
F1,72 = 0.02 NS
F6,66 = 3.41**
↑ F1,72 = 26.90***
–
–
F6,60 = 4.51***
(CP t = 2.27*)
(TP t = 2.71**)
F6,60 = 3.04*
(CP t = 2.31*)
–
–
–
–
F6,66 = 3.42**
F6,66 = 3.82**
–
F6,66 = 3.02*
(BP t = 2.96***)
(BTP t = 2.01*)
–
–
Minimum adequate model results from generalized linear models with quasi-Poisson error distribution. Arrows before value show the direction
of the main effects: upwards arrow indicates a positive relationship and downwards arrow indicates a negative relationship. Parentheses show
the chemotype driving the significant chemotype effect, with post hoc test result given. - shows where a term was not retained in the minimum
adequate model. Empty cells indicate the term was not used, i.e. when the response and explanatory variables were the same
ME Metopeurum fuscoviride, MA Macrosiphoniella tanacetaria, BP borneol pure, BH borneol hybrid, BTP β-thujone pure, CP camphor pure,
TP thujone pure
* p < 0.05, ** p < 0.01, *** p < 0.001; † p < 0.1, NS not significant
Table 2 Variance component analysis on principal components analysis (PCA) scores, showing the relevant chemicals associated with
each axis
Chemical
PCA1 (55.8 %)
PCA2 (28.5 %)
PCA3 (13.0 %)
α-Thujone
β-Thujone
Camphor
Borneol
Cineol
3.18
51.98
42.78
0.00
2.05
0.00
9.33
0.37
81.61
0.00
83.03
0.00
0.08
8.01
8.88
Piperiton
0.00
8.70
0.00
Values represent axis loadings. Variance component analyses run
using restricted maximum likelihood estimation. Bold values represents dominant chemicals
13
p < 0.05; thujone hybrid r = 0.61, p < 0.01; borneol hybrid
r = −0.74, p < 0.01). Negative associations were detected
between M. fuscoviride and the seven-spotted ladybird
beetles on camphor pure (r = −0.42, p < 0.05), camphor
hybrid (r = −0.87, p < 0.01) and thujone hybrid plants
(r = −0.61, p < 0.01). The nursery web spider density was
negatively correlated with the density/abundance of M. fuscoviride only on borneol plants (borneol pure r = −0.67,
p < 0.01; borneol hybrid r = −0.47, p < 0.05). A negative
association between the number of ladybirds and spiders
(r = −0.406, p < 0.001) was observed.
Analyses revealed that different food webs exist on different tansy chemotypes. Dominance of ladybirds on camphor and of nursery web spiders on borneol chemotypes
Oecologia
Fig. 2 Effect of plant chemotype a % borneol on Metopeurum fuscoviride (ME) aphid
number and b chemotype
profile on spider number, with
PCA axis 2 (black solid line and
solid circles) and PCA axis 1
(red solid triangles and dashed
line) (Color figure online)
Table 3 The effect of plant chemotype profile (continuous using
PCA) on different aspects of the community, using each species
group as a response variable, and site, the first three PCA axes scores
and species groups as explanatory variables with interactions between
species and PCA axes
ME aphid
MA aphid
Lasius
Ladybird
Spider
Orius
F2.79 = 11.11***
–
F2.78 = 2.89†
–
F2.79 = 3.48*
F2.79 = 15.06***
F2.79 = 48.47***
PCA2
↓ F1,78 = 9.34**
↑ F1,77 = 7.86**
↑ F1,78 = 9.40**
F1,77 = 1.15 NS
↑ F1,78 = 2.84†
–
F2.79 = 0.51 NS
–
↓ F1,78 = 7.46**
↓
↑ F1,78 = 3.55†
F1,77 = 20.33***
PCA3
–
–
–
↓
Site
PCA1
ME aphids
MA aphids
Lasius
Ladybird
↓
↓ F1,77 = 9.62**
↑
F1,76 = 41.63***
F1,76 = 12.84***
↑
F1,76 = 47.13***
F1,75 = 0.31 NS
↑ F1,75 = 6.17*
–
F1,77 = 15.64***
↓ F1,76 = 5.55*
↑ F1,76 = 5.92*
↑ F1,77 = 4.82*
F1,75 = 0.32 NS
↑ F1,75 = 4.80*
F1,76 = 0.84 NS
F1,74 = 0.60 NS
↓ F1,75 = 5.05*
↓ F1,73 = 3.96†
F1,74 = 1.32 NS
F1,74 = 0.08 NS
↓ F1,75 = 4.41*
F1,74 = 0.01 NS
F1,74 = 0.01 NS
F1,74 = 1.67 NS
F1,73 = 0.49 NS
F1,73 = 0.80 NS
F1,73 = 0.75 NS
Orius
F1,73 = 4.49*
F1,72 = 0.56 NS
↓ F1,72 = 6.63*
F1,72 = 0.01 NS
Two-way interactions
PCA2 × Lasius
F1,72 = 9.92**
PCA1 × MA
F1,71 = 32.13***
PCA2 × Orius
F1,71 = 5.45*
PCA1 × Ladybird
F1,70 = 5.11*
Spider
↑
↑ F1,73 = 28.00***
F1,72 = 28.95***
PCA2 × ME
F1,69 = 18.99***
PCA2 × MA
F1,68 = 4.66*
PCA2 × Orius
F1,67 = 5.88*
See Table 2 for variance components for the different chemicals on each PCA axis. Minimum adequate model results from generalized linear
models with quasi-Poisson error distribution. Arrows before value show the direction of main effects: upwards arrow indicates a positive relationship and downwards arrow indicates a negative relationship. Parentheses show the chemotype driving the significant chemotype effect, with
post hoc test result given. - shows where a term was not retained in the minimum adequate model. Empty cells indicate the term was not used,
i.e. when the response and explanatory variables were the same. For other abbreviations, see Tables 1 and 2
* p < 0.05, ** p < 0.01, *** p < 0.001; † p < 0.1
was clearly observed, while no predator dominance
occurred on thujone plants (Fig. 3a–c). Path analyses
revealed that significant trophic relations exist between
the arthropod species in all tansy chemotypes. Our model
predicted relations between ladybirds and M. tanacetaria
on borneol plants, but statistics revealed no significant
effects (Fig. 3b). Ants protected M. fuscoviride colonies
on all pure and hybrid chemotypes. Intra-guild predation
13
Oecologia
Fig. 3 Food web on tansy pure camphor pure (a), borneol pure (b),
thujone pure plants (c). Bold arrow represents mutualistic relations
between Lasius niger and M. fuscoviride aphids. Hybrid chemotypes
Table 4 Food web parameters
on tansy pure and hybrid
chemotypes
had the same food webs as the appropriate dominant plants, thus they
are not repeated again in figures. Numbers represent χ2-values for
significant path coefficients. *p < 0.05, **p < 0.01, ***p < 0.001
Pure chemotypes
Camphor pure
Borneol pure
Thujone pure
Number of species
No. links between species
7
13
0.92 NS
7
13
1.12 NS
7
12
0.92 NS
Bottom-up index (Kbu,i)
Top-down index (Ktd,i)
0.65 NS
↑ 1.15**
0.63 NS
Hybrid chemotypes
Camphor hybrid
Borneol hybrid
Thujone hybrid
Number of species
No. links between species (L)
Bottom-up index (Kbu,i)
7
13
0.95 NS
7
13
1.04 NS
7
12
0.94 NS
Top-down index (Ktd,i)
0.73 NS
↑ 0.95*
0.73 NS
Bottom-up (bu) and top-down (td) indices (K) were considered as separate variables for each individual
plant and values were compared between chemotypes with multivariate ANOVA. Upwards arrow shows a
significantly higher influence
* p ≤ 0.05, ** p ≤ 0.01
was observed only on borneol plants, where nursery web
spiders preyed upon Orius and ladybirds (Fig. 3b). There
was no observed ant predation of M. tanacetaria aphids on
borneol (Fig. 3b).
The number of species used for food web analyses
was seven for all pure and hybrid chemotypes. The number of links between species did not differ considerably.
There were 13 links for camphor and borneol plants
(both pure and hybrid) and 12 for thujone plants (both
pure and hybrid). No significant differences of bottomup effects were detected between pure chemotypes,
or between hybrid chemotypes (Table 4). There was,
however, a significantly higher top-down influence on
13
borneol pure and borneol hybrid plants, while no differences in top-down effects on camphor and thujone plants
were observed (Table 4).
Discussion
This study demonstrates that variation in chemical defence
exists within the tansy species T. vulgare, with the plants in
our study area dominated by β-thujone, camphor and borneol chemotypes. Moreover, there is considerable spatial
variation in the composition of different chemotypes (i.e.
β-thujone and camphor, β-thujone and borneol, camphor
Oecologia
and borneol) across sites (Supplementary material, Fig. 1).
A previous study has also shown that genetic differences
also exist between M. fuscoviride aphids on different tansy
chemotypes (Benedek et al. 2015).
The ant-tended aphid M. fuscoviride was dominant in
borneol and borneol-hybrid plants. Ants had clear protective effects on M. fuscoviride aphids on all plants irrespective of their chemotype. Consequently, M. fuscoviride
predators were uniformly influenced by ants on all plants.
Previous studies suggested that the population growth and
colony persistence of M. fuscoviride in the presence of ants
are much greater than in the absence of ants (Stadler and
Dixon 2008). M. tanacetaria aphid colonies were relatively
unstable and disappeared rapidly from all plants. As M.
tanacetaria is not ant-tended it may be preyed on by both
ants and predators, therefore high predator pressure may
lead to its rapid extinction from plants. Species-species
interactions revealed that ant abundance was positively correlated with M. fuscoviride and negatively influenced by
spiders (also described by Mestre et al. 2014) and Orius.
The variation in the associations of ants, aphids and
predators within food webs is complex, but some of that
complexity can be explained by tansy chemotypes supporting different food webs according to their chemical
compositions. Food web analyses revealed that the aphid
specialist ladybirds were significantly and predominantly
associated with camphor pure and camphor hybrid, and
negatively associated with thujone hybrid (if the chemical
was α-thujone) plants. Significantly higher numbers of the
polyphagous nursery web spider were observed on borneol
pure and borneol hybrid plants. While predator variation by
chemotypes could mean spatial dispersion and separation
due to interspecific competition (Begon et al. 2006), our
study offers a new, alternative mechanism that plant volatile
chemical composition mediates animal community composition. The predators may occupy different plants to partition prey resources based on plant chemical composition
rather than by classic resource-partitioning mechanisms.
Therefore, the differences in M. fuscoviride aphid densities
and their higher abundance on borneol plants most probably are due to differences in predator densities. In particular, different predators may be attracted by different volatile
compounds, i.e. the attraction of spiders to borneol. Further,
the association of ants with borneol plants could either be
a result of the higher number of M. fuscoviride aphids or,
alternatively, a direct effect of plant chemotype on ants,
which then tend the M. fuscoviride aphids more efficiently
on borneol plants. Experiments assessing the preference of
ants and aphids for different plant chemotypes are required
to determine the direction of this effect. Since there is still
some confounding effect of site and chemotype, despite the
use of appropriate statistics, it cannot be excluded that geographical differences among the three sites might contribute
to differences in arthropod abundances. However, our previous research where tansy chemotypes were replanted in
a common field also shows the same effects of borneol on
spiders (Benedek et al. 2015). Thus, higher M. fuscoviride
abundances on borneol plants may also be the consequence
of a lower predation rate by the generalist nursery web spiders, which is also suggested by our modelling approaches.
The attraction of predators to specific volatiles is also
supported by the results of the present study. Separately
assessing individual tansy plants, e.g. camphor plants
found in sites dominated by thujone (site 2) and borneol
plants dominated by camphor (site 1) (Supplementary
material, Fig. 1) reveals similar patterns [spiders related to
borneol and ladybirds to camphor and β-thujone (PCA1)].
Both pure and hybrid borneol plants are, nevertheless, certainly capable of attracting higher numbers of spiders, and
the same effect of camphor and β-thujone plants on ladybird numbers was also observed (PCA1). Previous studies
on thyme demonstrated a similar role of chemotype, where
the negative effects of spider and coccinellid predators
on aphids varied by plant chemical content (Linhart et al.
2005). Aphid (Aphis serpylli) densities on thyme plants
containing carvacrol, geraniol and thymol monoterpenes
were reduced by arthropod predators. Aphids on plants
with linalol, however, were not affected by predators, perhaps because predators showed chemotype-specific behaviour and avoided linalol-containing plants (Linhart et al.
2005). Host plants may also have trait-mediated effects on
predators, such that particular chemical signals from plants
can indicate higher prey quality and abundance that may
have fitness effects on predators [i.e. increase fitness by
feeding on prey on a particular tansy chemotype (Huang
et al. 2010; Snoeren et al. 2010; Pierre et al. 2011)]. The
higher M. fuscoviride density on borneol plants probably
resulted from intraguild predator interactions that weakened top-down effects; specifically here it appears that the
generalist nursery web spider may have preyed upon other
predators (ladybird beetles and Orius) (Table 4).
The degree of top-down control can also be modulated
if predators like ladybirds and spiders are differentially
attracted by tansy volatile signals to locate plants carrying their prey. By partitioning host plants based on their
volatiles some predators (like spiders and lady beetles in
this study) can avoid intra-guild predation on tansy. Such
resource partition by predators may be a consequence of
variability in tansy inducible resistance causing variability in the predator species’ preferred prey being associated
with particular varieties of a plant. Or, some predators like
the generalist nursery web spider may be less affected by
plant chemicals than specialist ladybirds with high attraction to camphor, as suggested by our food web modelling.
In conclusion, our study demonstrates a range of possible
community-level outcomes between plant-aphid-predator
13
Oecologia
networks that can be influenced by plant chemical content.
Our work points to hypotheses that explain the mechanisms
underlying species’ presence and mediating interactions
(intra-guild effects, resource partitioning based on chemotype) that are not considered in classic analyses of species
interactions. These hypotheses should help to motivate
more experimental work that will resolve these mechanisms through the manipulation of chemotype abundances
within local sites.
Acknowledgments This research was founded by the Institute of Research Programs of the Sapientia University, grant no.
1/13/05.01.2012. The authors declare no conflict of interest.
Author contribution statement W. W. W. and A. B. conceived the
experiments. A. B., K. B. and J. B. designed the experiments. A. B.,
K. B., J. B., R. V. S. and M. M. performed the experiments. S. E. Z.,
W. W. W., O. J. S. and A. B. analysed the data. A. B., W. W. W. and
O. J. S. wrote the manuscript. S. E. Z. provided editorial advice.
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